Folding and interaction studies of
subunits in protein complexes
Ximena Aguilar Iturri
Umeå University
Department of Chemistry
901 87 Umeå, Sweden
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7459-795-0
Cover: Protein Human Med25-ACID PDB-code 2XNF
Electronic version available at: http://umu.diva-portal.org/
Printed by: VMC-KBC UMeå
Umeå, Sweden 2014
Dedicado a toda mi familia en Bolivia, en especial a Mami Olga, mejor
amiga Mary, mi ‘gemela’ Claudia y los mellizos Olga Jr y Rodrigo.
Suma yatekanani
“地図がなくても迷うことはないが、目的地がなければ迷ってしまう。”
Takemoto Yuta, Honey & Clover
Table of Contents
Table of Contents
i
Abstract
iii
List of papers
v
Abbreviations
vii
1. Introduction
1
1.1 Protein folding
1
1.1.1 Macromolecular crowding
3
1.2 Protein-protein interactions
4
1.2.1 Homo-oligomer: cpn10
5
1.2.2 Hetero-protein complex: Mediator
6
1.3 Mediator and transcription regulation
8
1.4 Molecular recognition - Coupled folding and binding
10
1.4.1 Med25
11
1.4.2 The plant-specific transcription factor Dreb2a
12
1.4.3 The human herpes simplex virus 1 VP16 protein
13
2. Aims
15
3. Methods
17
3.1 Protein stability, folding and binding
17
3.2 CD spectroscopy
19
3.3 Fluorescence spectroscopy
19
3.4 Nuclear Magnetic Resonance
20
3.4.1 Chemical shift perturbation
21
3.5 Surface plasmon resonance
22
3.6 Isothermal titration calorimetry
22
3.7 Cross-linking
23
3.8 GST pull-downs
23
4. Results
24
4.1 Summary of papers
24
4.1.1 Macromolecular crowding stabilizes cpn10 monomers
24
4.1.2 Conformational changes upon interaction between Med25 and Dreb2a
27
4.1.3 Similar binding affinities but different binding energetics
5. Discussion
29
33
5.1 Protein folding studies in cell-like environment
33
5.2 Interaction between transcriptional regulatory proteins
34
5.2.1 Small contacts might end up in great connections
34
6. Conclusions
37
7. Acknowledgements
38
8. References
44
i
ii
Abstract
Proteins function as worker molecules in the cell and their natural
environment is crowded. How they fold in a cell-like environment and how
they recognize their interacting partners in such conditions, are questions
that underlie the work of this thesis.
Two distinct subjects were investigated using a combination of biochemicaland biophysical methods. First, the unfolding/dissociation of a heptameric
protein (cpn10) in the presence of the crowding agent Ficoll 70. Ficoll 70 was
used to mimic the crowded environment in the cell and it has been used
previously to study macromolecular crowding effects, or excluded volume
effects, in protein folding studies. Second, the conformational changes upon
interaction between the Mediator subunit Med25 and the transcription
factor Dreb2a from Arabidopsis thaliana. Mediator is a transcriptional coregulator complex which is conserved from yeast to humans. The molecular
mechanisms of its action are however not entirely understood. It has been
proposed that the Mediator complex conveys regulatory signals from
promoter-bound transcription factors (activators/repressors) to the RNA
polymerase II machinery through conformational rearrangements.
The results from the folding study showed that cpn10 was stabilized in the
presence of Ficoll 70 during thermal- and chemical induced unfolding
(GuHCl). The thermal transition midpoint increased by 4°C, and the
chemical midpoint by 0.5 M GuHCl as compared to buffer conditions. Also
the heptamer-monomer dissociation was affected in the presence of Ficoll
70, the transition midpoint was lower in Ficoll 70 (3.1 µM) compared to in
buffer (8.1 µM) thus indicating tighter binding in crowded conditions. The
coupled unfolding/dissociation free energy for the heptamer increased by
about 36 kJ/mol in Ficoll. Altogether, the results revealed that the stability
effect on cpn10 due to macromolecular crowding was larger in the individual
monomers (33%) than at the monomer-monomer interfaces (8%).
The results from the interaction study indicated conformational changes
upon interaction between the A. thaliana Med25 ACtivator Interaction
Domain (ACID) and Dreb2a. Structural changes were probed to originate
from unstructured Dreb2a and not from the Med25-ACID. Human Med25ACID was also found to interact with the plant-specific Dreb2a, even though
the ACIDs from human and A. thaliana share low sequence homology.
Moreover, the human Med25-interacting transcription factor VP16 was
found to interact with A. thaliana Med25. Finally, NMR, ITC and pull-down
experiments showed that the unrelated transcription factors Dreb2a and
iii
VP16 interact with overlapping regions in the ACIDs of A. thaliana and
human Med25.
The results presented in this thesis contribute to previous reports in two
different aspects. Firstly, they lend support to the findings that the
intracellular environment affects the biophysical properties of proteins. It
will therefore be important to continue comparing results between in vitro
and cell-like conditions to measure the magnitude of such effects and to
improve the understanding of protein folding and thereby misfolding of
proteins in cells. Better knowledge of protein misfolding mechanisms is
critical since they are associated to several neurodegenerative diseases such
as Alzheimer’s and Parkinson's. Secondly, our results substantiate the notion
that transcription factors are able to bind multiple targets and that they gain
structure upon binding. They also show that subunits of the conserved
Mediator complex, despite low sequence homologies, retain a conserved
structure and function when comparing evolutionary diverged species.
Keywords: Macromolecular crowding, cpn10, Mediator, Med25, Dreb2a,
VP16, conformational changes, NMR, ITC.
iv
List of papers
I)
Ximena Aguilar, Christoph F. Weise, Tobias Sparrman,
Magnus
Wolf-Watz,
and
Pernilla
Wittung-Stafshede.
Macromolecular Crowding Extended to a Heptameric System:
The Co-chaperonin Protein 10. Biochemistry 2011; 50 (14),
3034-3044.
Reprinted with permission from Biochemistry, 2011, 50 (14), 3034-3044.
Copyright 2011 American Chemical Society.
II)
Jeanette Blomberg, Ximena Aguilar, Kristoffer Brännström,
Linn Rautio, Anders Olofsson, Pernilla Wittung-Stafshede, and
Stefan Björklund. Interactions between DNA, transcriptional
regulator Dreb2a and the Med25 mediator subunit from
Arabidopsis thaliana involve conformational changes. Nucleic
Acids Research 2012; 40: 5938 - 5950.
Reprinted with permission from Nucleic Acids Research, 2012, 40 (13), 5938
- 5950. Copyright 2012 Oxford University Press.
III)
Ximena Aguilar, Jeanette Blomberg, Kristoffer Brännström,
Anders Olofsson, Jürgen Schleucher, and Stefan Björklund.
Interaction studies of the Human and Arabidopsis thaliana
Med25-ACID proteins with the Herpes Simplex virus VP16- and
plant-specific Dreb2a transcription factors.
Manuscript.
v
vi
Abbreviations
ACID
aMed25
A. thaliana
CD
Cpn10
CSP
DBD
Dreb2a
Dreb2af.l.
E. coli
F
GST
GuHCl
hMed25
HSQC
ITC
KD
Med25
NMR
NRD
PDB
PIC
Pol II
SDS-PAGE
SEC
SPR
TAD
TF
U
UV
VP16
ACtivator Interaction Domain
Med25 from Arabidopsis thaliana
Arabidopsis thaliana
Circular Dichroism
Co-chaperonin protein 10
Chemical Shift Perturbation
DNA Binding Domain
Dehydration responsive element binding
protein
Dreb2a full-length protein
Escherichia coli
Folded
Glutathione S-transferase
Guanidine Hydrochloride
Med25 from human
Heteronuclear Single Quantum Coherence
Isothermal Titration Calorimetry
Dissociation constant
Mediator subunit 25
Nuclear Magnetic Resonance
Negative Regulatory Domain
Protein Data Bank
Pre-Initiation Complex
RNA polymerase II
Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis
Size Exclusion Chromatography
Surface Plasmon Resonance
Transcriptional Activation Domain
Transcription Factor
Unfolded
Ultraviolet
Virion protein 16
vii
viii
1. Introduction
1.1 Protein folding
Protein folding is the final step in the central biological process where the
genetic information stored in DNA is converted into functional proteins.
Newly translated proteins need to acquire a unique structure in order to be
functional. How this process works and the mechanisms behind it have been
extensively investigated for more than fifty years. The importance to unravel
the molecular mechanisms of protein folding relies on the impact proteins
have for the proper function of the cell. Proteins are actually the hard worker
molecules in living organisms and are involved in all cellular functions. Even
though there is a rigid cellular assistance and control for folding - e.g.
chaperones which are molecular helpers that prevent misfolding1 - still,
polypeptides might escape cellular control to end up as misfolded proteins
which have the potential to form aggregates or amyloid fibrils associated
with neurodegenerative diseases such as Alzheimer’s and Parkinson’s 2,3.
A pioneering scientist in this field was Christian Anfinsen who already in the
1960’s, together with co-workers, showed that proteins can fold
spontaneously and also refold in vitro. Anfinsen’s work suggested that a
protein three-dimensional structure was encoded in its amino acid
sequence4,5. However, this statement gave rise to a paradox noted by Cyrus
Levinthal, who remarked that a polypeptide chain has a large degree of
freedom - depending on the number of amino acids and peptide bonds which means that if the process of folding is random, then the numbers of
possible conformations are massive and it would take a long time for a
protein to fold into the correct conformation6. This was called the Levinthal’s
paradox since it was already known at that time that proteins fold in the
milliseconds timescale. Levinthal therefore proposed that folding occurs
through a series of partially structured intermediate states and other
researchers reported on the existence of transient folding intermediates7-9.
What emerged from these studies was a new way to look at proteins, they are
not static and the transition from the unfolded to the folded state occurs in a
cooperative manner. In the 1990’s, a theoretical concept was introduced to
understand the mechanism of folding; the energy landscape theory10. This
concept explains folding as a result of the nature of a molecule that strives
for low free energy in order to obtain stability11. The energy landscape theory
provides an understanding for the presence of intermediate states. In a
simple way, the landscape might be viewed like a funnel-shaped topology,
where polypeptides strive to reach a low energy state in a downhill manner
1
whereby it folds by adopting different intermediate states until reaching the
minimal energy level, the folded state12,13 (Figure 1A).
Currently, protein folding is studied by applying thermodynamic laws which
state that a system strives to reach a state of minimum energy. One
important driving force for the folding process is the hydrophobic effect that
excludes water molecules from contact with hydrophobic amino acids, and
exposes the hydrophilic amino acids to the aqueous solvent. The energy
diagram in Figure 1B illustrates a folding reaction where the principles of
Gibbs free energy can be applied (the equations are included in section 3.1).
The reaction involves a two-state process where the protein is in equilibrium
between the folded and unfolded state, and there is an energy barrier or
transition state (TS) to pass from one state to another14. This model can only
be applied to proteins that show the two-state transition and as discussed
above, most proteins fold in sequential steps by forming intermediates.
Then, multiple energy barriers are needed to be passed in order to reach the
folded state. That is the reason why the folding process of only certain
proteins that “behave well” has been studied experimentally so far. However,
new methods and approaches are continuously being developed in order to
understand the chemistry and biology of protein folding15-20.
Figure 1. A) Illustration of the free energy landscape for protein folding. Proteins
have a high energy level and a large degree of conformational freedom in the
unfolded state. The folding process includes the formation of intermediates and the
folded state is reached when the degree of conformational freedom and the energy
levels are low. B) Energy diagram of a folding reaction for a two-state process.
Proteins have to pass an energy barrier represented by the transition state (TS) to go
from one state to another.
2
1.1.1 Macromolecular crowding
Almost all our knowledge about protein folding comes from experiments
done in in vitro conditions. The methods that are currently available to study
proteins are insufficient to monitor folding directly inside cells. A convenient
way to study protein folding so far has been to use isolated, purified stable
proteins (model proteins) in dilute buffers in vitro; an environment which is
completely different from the crowded intracellular medium encountered
within cells (Figure 2). Macromolecules (proteins, nucleic acids,
carbohydrates, lipids) occupy a significant fraction of the total volume (10 –
40 %) in the cell and their concentration in the cytoplasm has been
estimated to range from 80-400 mg/ml21-24. This crowded environment
restricts the available space inside the cell and gives rise to an excluded
volume effect termed as ‘macromolecular crowding’25-28. The excluded
volume effect is a consequence of the steric repulsion (a molecule occupies a
space not accessible to another) of macromolecules and has the potential to
influence kinetics and equilibria of cellular reactions 29. For individual
proteins, the predicted effect is a stabilization of the folded state since
compact conformations will be indirectly favored due to unfavorable effects
on the extended unfolded states30-32. On the other hand, an increase in
volume occupancy may also enhance unwanted association processes leading
to aggregate formation33,34.
Figure 2. A) Illustration of a eukaryotic cell depicting subcellular organelles such as
the nucleus, mitochondria, endoplasmic reticulum and the Golgi apparatus (asterisk).
B) A 3D reconstruction from a high resolution electron tomography of the Golgi
apparatus in a mammalian cell. The picture illustrates the high macromolecular
content in the intracellular environment. Reprinted from (Marsh BJ 2005)35
Copyright 2005, Elsevier with permission of the Publisher.
3
Studies of macromolecular crowding effects on protein folding,
conformational changes, thermodynamics, kinetics and dynamics is a
research field that is currently getting increased attention. To address these
questions, one has to perform studies in vivo or in cell-like conditions in
vitro. A majority of the experimental crowding studies in vitro mimic the
crowded environment by using inert, synthetic, sugar-based polymers
termed crowding agents (Ficoll 70 and Dextran)36-40. In such studies, effects
of macromolecular crowding on protein stability with respect to
unfolding37,40-42, on association equilibria and rates36,43,44, and on enzyme
activity45,46 have been reported. Today it is known that the interior cell milieu
affects the biophysical properties of proteins and new approaches to
investigate how they fold and function in their true environment are
continuously being developed.
Part of this thesis (paper I) covers the impact of macromolecular crowding
on the folding, assembly and overall stability of the model homo-oligomer
protein, co-chaperonin protein 10 (cpn10), which is introduced in section
1.2.1.
1.2 Protein-protein interactions
The fate of a protein can take several directions. Some proteins accomplish
their function as monomers, other proteins assemble into complexes, while
the fold and function of other proteins depend on the interaction with their
biological targets47. Interactions between proteins are essential for several
aspects of cellular function, e.g. antigen-antibody binding, signal
transduction, biosynthetic and degradation pathways, and regulation of gene
expression. Studies have shown that the majority of proteins are involved in
complex formation and that a protein on average has about eight interacting
partners48-51. The way in which proteins associate ranges from simple
oligomers to gigantic multi-protein complexes. Oligomers are present as
homo-oligomers composed of several identical polypeptide chains or as
hetero-oligomers which contain non-identical polypeptide chains52. These
oligomeric states are predominant in the cell and studies have showed that a
majority of them are present in cells as homo-oligomers52. Proteins that
associate into large complexes function as protein machines, e.g. RNA
polymerase II (Pol II), the proteasome and Mediator. The capacity of
proteins to form multimeric complexes and their ability to network with
other proteins has most likely been a requirement for the development of
complex cellular processes53.
How oligomers and protein-complexes self-assemble and then physically
interact with several other proteins is not entirely understood. Extensive
4
research has elucidated some aspects that play important roles for the
specific molecular recognition events that are required for interaction
between proteins54-58. Just in terms of chemical properties which are
restricted to the inter- and intra-protein interfaces, the recognition between
the correct partners is a result of several complementary attributes ranging
from the amino acid composition, hydrophobicity, electrostatic interactions,
hydrogen bonding and hydration. All these chemical attributes contribute to
protein-protein recognition and determine the energetics buried at the
protein interfaces, which will be distinct for every individual case. Still, in
common for all kind of interactions is to keep the free energy of binding in
balance in order to lend stability to the complex59-62.
In the following section, two protein complexes that have been part of my
thesis project are introduced: 1) Proteins that associate into a homoheptamer (cpn10) and 2) proteins that assembles into a large hetero-protein
complex (Mediator).
1.2.1 Homo-oligomer: cpn10
Cpn10, also known as heat shock protein 10, is an essential protein found in
the mitochondrial matrix. Cpn10 homologs are present in most organisms6365 and its function is to interact with another oligomer, the cpn60. The cpn60
oligomer forms a chamber, and the cpn10 oligomer operates as the lid of the
chamber in an ATP-dependent chaperonin complex-system that assist in the
folding process of certain proteins65. The cpn60-cpn10 proteins show
conserved structure and function in all organisms and the most widely
studied pair is the bacterial GroEL-GroES of E. coli65,66. In addition to the
chaperonin function, new functions for human cpn10 have been reported,
but they are still poorly understood. For example, cpn10 has been found to
be identical to an immunosuppressive early pregnancy factor67. It has also
been found in red blood cells68, as a protein which is over-expressed in
carcinogenesis69, and it has been formulated as a therapeutic drug for
treatment of rheumatoid arthritis70.
The structure of cpn10 is ring-shaped, and the complex is composed of seven
identical subunits or monomers. Each monomer comprises 101 residues and
each subunit is approximately 10 kDa71. The monomers assemble to form a
70 kDa homo-heptameric protein complex through anti-parallel β-strand
pairing between the N-terminal of one monomer and the C-terminal of the
neighboring monomer (Figure 3). Each monomer unit adopts an irregular βbarrel structure forming two flexible protruding loops72. Studies in vitro
have shown that the monomers are able to fold but their stability in the
monomeric state is low73,74. The overall cpn10 heptamer stability has been
5
shown to be dominated by inter-subunit interactions where the hydrophobic
subunit-interfaces interact non-covalently75. The cpn10 oligomer unfolds
reversibly in vitro and unfolding/refolding pathways for the cpn10
homologous from E. coli and Aquifex aeolicus have been characterized75-82.
Figure 3. Structure of the human cpn10 heptameric protein which is composed of
seven identical monomers. One monomer is highlighted in pink. Subunit-subunit
interfaces between monomers, formed between anti-parallel β-strands, are depicted
in red and blue71.
Studies of oligomer folding are complicated since they involve both,
intramolecular interactions (monomer folding) and intermolecular
interactions (subunit assembly)83,84. For cpn10 folding studies, an apparent
two-state process for unfolding coupled to dissociation can be applied.
Therefore, the cpn10 protein represents an excellent model system for
fundamental folding/assembly studies in cell-like conditions and it was thus
used as model protein in our studies described in paper I.
1.2.2 Hetero-protein complex: Mediator
In addition to homo-oligomers such as the cpn10 described above, many
proteins associate into hetero-protein complexes (e.g. ribosome, proteasome,
Mediator). The Pol II transcription machinery in eukaryotes requires a
Mediator complex for proper transcription regulation (Figure 4, 5). Mediator
is a giant evolutionarily conserved multi-protein complex (~1.2 MDa) and it
was originally identified in Saccharomyces cerevisiae in the 1990’s85,86. It is
organized into four modules (head, middle, tail and kinase) containing all
together between 25 (yeast) and 29 subunits (humans) which share a unified
subunit nomenclature87 (Figure 5). Cross-species comparisons have shown
that almost all yeast Mediator proteins have homologs in the human
complex88. However, it has been found that the Med23, Med25, Med26,
Med28 and Med30 (Figure 5) subunits are unique to higher eukaryotes such
6
as humans and plants89,90. This means that Mediator has diverged over time
and an explanation for the existence of additional subunits in higher
eukaryotes most probably originates from the requirement for the more
complex transcription regulation that is found in multicellular
organisms53,89. During the last decades, several studies have described the
function and location of individual Mediator subunits. It was found that
different subunits are involved in regulating distinct sets of genes by
interacting with diverse transcription factors (TFs). It has also been reported
that some subunits are essential for survival, e.g. Med17 and Med21 which
are required for transcription of all protein-coding genes in yeast91,92. Nonessential subunits have more specialized roles in the regulation of selective
genes. It has also been suggested that subunit composition depends of the
cell-stage; proliferating cells might have the complete Mediator with all the
subunits whereas differentiated cells probably have the Mediator with a
subset of subunits93,94. Other studies have shown that subunits function
individually, e.g. Med12 that might function independently of the rest of
Mediator as a regulator of TGFB signaling in the cytoplasm95.
As exemplified above, the regulatory properties of Mediator are broad and
include participation in transcription initiation and elongation 96-99. In
addition, Mediator has been reported to be involved in mRNA processing
and chromatin architecture modification 100-103. However, this thesis
emphasizes on the role of Mediator when interacting with transcriptional
regulatory proteins; an essential function by which Mediator transmit the
regulatory signals to Pol II machinery. The interaction of a specific Mediator
subunit protein (Med25) and specific TFs (Dehydration responsive element
binding protein (Dreb2a) and VP16 were investigated in papers II and III.
Figure 4. The Mediator complex interacting with Pol II. Cryo-EM structure of
Mediator-pol II complex where pol II structure was docked in the central part.
Reprinted from Roger D. Kornberg 2007104,105 Copyright 2007, with permission from
the National Academy of Sciences U.S.A. and Nature Publishing Group.
7
1.3 Mediator and transcription regulation
Pol II transcribes all protein-coding genes in eukaryotes and this function is
controlled by a range of transcriptional regulatory proteins. There is a group
of general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH)
that assembles and form a pre-initiation complex (PIC) which together with
Pol II and Mediator constitute the transcription machinery (~4 MDa)
(Figure 5) 105-107. Another important group of regulatory proteins are TFs that
activate or repress expression of target genes in response to an
environmental or cellular trigger. A feature of TFs is that they contain one or
more DNA-binding domains (DBD) which bind to specific DNA sequences or
in the promoters of genes. In bacteria, TFs interact directly with RNA
polymerase and a Mediator complex is not present. In eukaryotes, TFs do
not bind directly to Pol II; they control Pol II activity by interacting with
Mediator and other co-factors through their transcriptional activation- or
negative regulatory domains (TAD and NRD)108. The question is why the
transcription machinery needs the large multi-protein complex Mediator
and how exactly Mediator works. To answer these questions many efforts
have been done in several groups to provide a deeper insight into the
molecular mechanism of Mediator function. Mediator appears to function as
a
bridge
between
promoter-bound
transcriptional
regulators
(activators/repressors) and the general Pol II transcription machinery
(Figure 5).
The head module of Mediator interacts directly with Pol II in both human
and yeast cells (Figure 4), and this interaction generates large structural
changes109-111. Mediator binding to Pol II is the most functionally significant
similarity among Mediators from different organisms. How can then
Mediator transmit the regulatory signals from activators and repressors to
Pol II? It has been reported that Mediator structure is dynamic and that it
has a high degree of conformational flexibility112. This might be due to the
high content of intrinsically disordered regions within the Mediator
subunits, regions which have similar alignment among homologs 113, and are
prone to fold and undergo conformational changes upon interaction with
activators, repressors and with other Mediator subunits. Several studies have
shown that the Mediator complex undergoes global structural changes upon
binding to different TFs, and the changes in Mediator were shown to be
different depending on the bound TF112,114,115. The conformational changes in
Mediator triggered by interaction with TFs might expose distinct protein
surfaces or binding modes, which might induce unique protein-protein
interactions between Mediator and the Pol II transcription machinery.
Through this mechanism, Mediator would adopt an ‘active’ structural state
when interacting with transcriptional activators that positively affects the
8
activity of the Pol II machinery. Conversely, interaction with transcriptional
repressor proteins would elicit different conformational changes in Mediator
that would have a negative effect on the Pol II machinery. The
rearrangement of Mediator has also been described by a multiple allosteric
model where a structural shift at one site propagates throughout a proteinprotein network; this process implies coordination among the subunits and
might require dissociation at one site and re-association at another site116.
Consequently, protein-protein interactions underlie the mechanism of action
of Mediator by forming a network that offers communication between
transcriptional regulatory proteins and the Pol II machinery. It is therefore
important to continue studying the interactions among Mediator proteins
and TFs in order to reveal how Mediator works and how its subunits can
function to integrate signals from different regulatory pathways.
Figure 5. Schematic illustration of the transcription pre-initiation complex in
eukaryotes where Mediator integrates signals from promoter-bound transcriptional
regulators (repressors and activators). Mediator kinase module is not shown. Specific
subunits present in higher Eukaryotes are colored grey. The location of Mediator
subunit 25 (Med25: enlarged grey subunit) is undefined. Adapted from Ansari and
Morse117
9
1.4 Molecular recognition - Coupled folding and binding
The enormous size of Mediator, its subunit complexity and its dynamic
nature makes it a very challenging subject for applying biophysical studies.
The complete Mediator has been studied by cryo-electron microscopy118,119
but a high-resolution structure is still not available. Only 13 subunits have
been structurally characterized at atomic resolution by X-ray crystallography
or solution NMR120,121. This means that there is still an immense lack of
knowledge about the structure of Mediator and hence about key aspects of
the mechanisms that regulate gene expression. Most knowledge about
Mediator has so far been achieved by studies of interactions between specific
Mediator subunits and individual TFs. The specific interactions occur
between TADs of the TFs and the ACtivator Interaction Domains (ACIDs) in
Mediator subunits. When studied in vitro, these interactions are generally
found to be weak (in the micro-molar range), most likely since they are
meant to be transient in the cells55,122,123. However, several weak interactions
might cooperate to trigger structural shifts in other Mediator subunits in
order to propagate the regulatory signals, similar to a Domino effect.
Several studies have shown that a specific TAD can target several ACIDs in
Mediator. For example Gcn4 targets Med2, Med3, Med15 and Med16 124-128.
On the other hand, different TFs interact with the same Mediator protein,
such is the case for VP16129, IE62130, HNF4α131, SOX9132, ATF6α133 and
ERM134 that all interact with the human Med25. The question is how this is
possible and what the mechanisms for binding and recognition are. There is
not one straight answer for those questions, since the process of protein
binding is the result of several attributes at the protein-protein interfaces e.g. amino acid composition, hydrophobicity, electrostatic interactions,
hydrogen bonding and energetics of binding55,56,135. It is known that TADs
are highly diverse when comparing their amino acid sequences. However,
some studies have revealed potential motifs of short amino acid sequences
that are present in several TFs, e.g. p53, which mediate transient interaction
with different targets123,136-140. Another aspect is that TADs are typically
unfolded in their free state but can adopt structured conformations upon
binding to their targets139,141-144. These structural transitions are dynamic and
might explain the ability of TADs to adopt multiple conformations such that
different TADs can share a common target surface on Mediator (fuzzy
complex)124. Conversely, this conformational flexibility can also provide an
explanation for how one specific TAD can target multiple Mediator subunits.
Moreover, several TADs have acidic properties which makes it possible to
interact with target proteins by complementary electrostatic interactions 57.
Electrostatic interactions can promote a rapid initial contact and might
dominate during the initial binding phase to increase the rate of complex
10
formation. Formation of the final complex might depend on hydrophobic
interactions in a subsequent entropy driven phase. Finally, the binding
energetics involve a balance between entropy and enthalpy changes,
compensations and commitments of favorable or unfavorable effects that
have to be kept in balance to lend stability to the complex54,59-61,145-148.
The binding of a single TAD, for example the VP16 TAD which is composed
of 40 residues, to its target might trigger small and large conformational
changes, both in the target and in the TAD itself. This will bring functional
flexibility and it is a suggested mechanism by which regulatory signals might
be propagated112,114,115. The study of conformational changes that occur upon
interaction between Med25 and the TFs Dreb2a and VP16 is described in
papers II and III.
1.4.1 Med25
As already mentioned, Mediator is highly conserved from yeast to humans
149. However, some Mediator subunits, i.e. Med25, are specific to higher
eukaryotes such as humans and plants88,150. As discussed in section 1.2.2, the
divergence in Mediator subunit content might be connected to the evolution
of eukaryotes that require a higher level of complexity in transcription
regulation. In this section, Med25 from A. thaliana and human are
introduced.
A. thaliana Med25 (aMed25) is a 90 kDa protein (836 amino acids) and its
ACID is located between residues 551 – 680. It was originally known as the
PHYTOCHROME AND FLOWERING TIME 1 (PFT1), a nuclear protein
involved in regulation of flowering time downstream of the PhyB
photoreceptor151. PFT1 was identified as a Mediator subunit when the plant
Mediator was isolated from A. thaliana150. The identification of PFT1 as a
Mediator protein suggested that PhyB signaling targets Med25 indirectly via
one or several unidentified transcriptional factors. Later it was found that
aMed25 integrates signals from different stress- and developmental
pathways. For example, aMed25 was reported to be involved in jasmonate
(JA) signaling which is a critical plant defense response152. Both, positive and
negative effects have been identified for aMed25 upon interaction with
specific TFs. The interaction with MYC2, which regulates JA-mediated gene
expression, resulted in a positive effect on expression of target genes; while
interaction with the TF ABI5, had a negative regulatory effect on the
expression of abscisic acid (ABA)-genes153. ABA is a plant hormone and
similar to JA, it is involved in developmental and physiological processes in
plants154. Moreover, aMed25 interacts with other TFs involved in stress
11
response pathways including the Myb-like protein, the zinc finger
homeodomain 1 (ZFHD1), and Dreb2a155.
Figure 6. NMR structure of the human Med25-ACID (PDB 2XNF)156.
Human MED25 (hMed25) is also a 90 kDa protein (747 residues) and its
ACID comprises residues 394 – 543129. It was first described as the human
activator-recruited cofactor 92 (ARC92) identified in interaction studies with
VP16129. hMed25 has been more extensively studied compared to aMed25. It
has been shown to interact with several TFs involved in different cellular
processes, including retinoid activation by RARα, chondrogenesis by SOX9,
insulin secretion in pancreatic cells by HNF4α, cellular growth and
differentiation by PEA3 subfamily members, and the endoplasmic reticulum
stress response by ATF6α131-134,157. In addition, hMed25 interacts with
Varicella-zoster virus protein IE62, which activate transcription of viral
genes129,130,158,159. The structure of the hMed25-ACID (residues 394-543) has
been solved by NMR and it comprises seven β-strands forming a β-barrel
flanked by three helices156,160-162 (Figure 6). The interaction between hMed25
and VP16 has been studied in detail and the VP16 binding site on hMed25ACID has been defined129,156,158,162. Moreover, mutations in hMed25 have
been related to Charcot-Marie-Tooth neuropathy, an inherited neurological
disease which results in muscle degeneration in the limbs163.
1.4.2 The plant-specific transcription factor Dreb2a
Dreb2a belongs to the large AP2/ERBP (Ethylene Responsive Element
Binding Protein) TF family which is unique to plants164-166. These TFs
interact with cis-acting dehydration-responsive promoter elements and
regulate genes involved in drought-, salt-, and cold stress response
pathways167,168. Dreb2a functions in response to drought stress and high
salinity. The protein is around 38 kDa, its TAD is located in the C-terminal
12
within residues 254-335 and it also contains a negative regulatory domain
(NRD) in the middle region within residues 135 – 165169. The NRD might
explain the regulatory effects of Dreb2a which have been found to be both
positive and negative155,169. aMed25 was reported to interact with Dreb2a 168335 in vitro and yeast-2-hybrid assays showed that the minimal domain
required for interaction was located within residues 169-254 in Dreb2a155,168.
Phenotypes of a series of mutants, i.e. med25 mutants were drought resistant
and dreb2a mutants showed early flowering time, suggested that the
interaction of aMed25/Dreb2a had a negative regulatory effect through the
NRD of Dreb2a155.
1.4.3 The human herpes simplex virus 1 VP16 protein
VP16 is a 54 kDa protein (490 amino acids) and it belongs to the
Herpesviridae tegument protein family; proteins that form the capsid layer
which encloses the viral genome and function as transcriptional activators
during viral infection of viral immediate early genes170. Transcriptional
activation by VP16 has been reported to operate through interaction with
different general TFs, including TFIIA, TFIIB, TFIIF, TFIIH, TBP, hTAFII31
and hTAFII32129,139,171-176. Thus, the VP16-TAD has been extensively studied
and in addition to its interaction with the human Med25-ACID, it has also
been reported to interact with another Mediator subunit, Med17177. The
VP16-TAD can be divided in two subdomains, the N-terminal (residues 412452) and the C-terminal (452-490), which function independently and
complementary to each other175,178,179. The VP16-TAD is unstructured in free
state as many other TADs, however it adopts an α-helical conformation upon
binding to its target proteins139,141,144, a characteristic also observed in other
TADs e.g. p53 141-143. Within the two subdomains, the formation of α-helical
segments has been located around residues 429-450 and 465-488141. A nine
amino acid sequence (DFDLDMLGD) that play a key role for VP16-TAD
transcription activation has been located to the first segment (429-450)137,178.
In addition, the DFDLDMLGD motif has also been identified in a range of
TFs and is proposed to be a conserved domain which can be recognized by
the same regulatory protein. Such is the case for VP16, p53, HSF1, NF-kB
and NFAT1 which all contain the DFDLDMLGD motif within their TADs and
share the ability to interact with TAF9 (TAFII31)136,139,140.
13
14
2. Aims
The focus of my thesis included two major subjects. The first subject was to
investigate how folding and stability of a heptameric protein was affected by
biologically relevant environments such as macromolecular crowding. The
second subject was to study the interaction of Mediator subunit 25 with
specific TFs, and the structural shifts occurring upon binding.
Understanding these processes in more detail will improve our knowledge of
protein folding in vivo and will contribute to learn more about the molecular
recognition between proteins that regulate gene expression, which is one of
the most central cellular processes that occur in crowded environments.
Effects of macromolecular crowding on a heptameric protein:
The first specific aim was to assess the effects of macromolecular crowding,
or excluded volume effects, on unfolding and dissociation of the heptameric
human protein, cpn10. This protein represents an excellent model system for
studies of oligomeric proteins since both intra- and intermolecular
interactions are involved in its folding process. Chemical and thermal
denaturation were used to induce unfolding/dissociation in a crowded
condition. In addition, the overall stability and monomer-heptamer
equilibrium were also assessed in such conditions. To create a crowded
environment in the in vitro system, I used the inert synthetic polymer Ficoll
70 at specific concentrations. This crowding agent is usually used for
macromolecular crowding studies since it does not interact with proteins and
does not interfere with different spectroscopic measurements.
Interaction studies between transcriptional regulatory proteins:
The second specific aim was to characterize the structure, stability and
interaction between Med25 and the TF Dreb2a from A. thaliana in the
presence and absence of an oligonucleotide that contains a consensus
Dreb2a binding site. The main purpose was to study in more detail the
potential structural shifts that occur upon interaction between Dreb2a, the
Dreb2a consensus sequence and Med25 in different combinations. Many TFs
are unstructured in dilute buffers, but might gain a more ordered structure
upon binding to their targets. In addition, structural shifts might underlie
the mechanisms of action by which the Mediator complex functions as a coregulator of transcription. An additional task was to investigate the
interactions of the unrelated TFs Dreb2a and VP16 with Med25 proteins
from human and A. thaliana. The Med25 homologs share low sequence
similarity, yet their folds might offer similar surfaces for ligand recognition.
This study helps to understand the molecular recognition between
15
transcriptional regulatory proteins and thereby the conserved function and
structure of the ancient Mediator complex.
A combination of biophysical- and biochemical methods (fluorescence,
circular dichroism, surface plasmon resonance, calorimetry, NMR, size
exclusion chromatography, cross-linking, GST pull-downs) were used in the
two sub projects.
16
3. Methods
3.1 Protein stability, folding and binding
Protein folding and binding can be studied by applying the laws of
thermodynamics180. Equation (1) shows the thermodynamic principle of
Gibbs free energy which can be used to describe the favorable state of protein
folding and binding reactions. The folding stability of a protein can be
obtained by measuring the difference in free energy between the folded and
unfolded states.
∆G = ∆H - T∆S
(1)
Where ∆G is the change of free energy, ∆H is the change of enthalpy, T is the
absolute temperature and ∆S is the change in entropy. Since every system
strives for minimum energy, proteins will fold or bind into a conformation
with the most favorable ∆H and T∆S. Then, the net protein stability is
marginal and can be easily disturbed by changing its environment, e.g. by
changing the pH, the salt concentration, the temperature or by adding
chemical denaturants. The relative contributions from the enthalpy can be
gained by chemical bonding within the protein structure (hydrogen bonding
and van der Waals interactions) and the entropy corresponds to the
conformational freedom and also to hydrophobic effects.
∆G of any given reaction can also be related to the equilibrium constant Keq
in equation (2), where R is the general gas constant.
∆G = -RT lnKeq
(2)
For protein folding studies, a two-state model can be assumed where the
protein is in equilibrium between the folded (F) and unfolded (U) states. The
rate constants are defined as the ratio of the population of the unfolded and
folded states in terms of the protein fraction present (f) in those states (3).
To obtain information about Keq, the system has to be disturbed, which can
be achieved by inducing unfolding, as mentioned above, with heat or with
chemical denaturants such as urea or GuHCl.
Keq = kU/kF = [fU]/[fF]
(3)
In paper I, the thermal induced unfolding was used to extract Tm values
which are the melting temperature at the denaturation midpoint. Then, the
thermal data was fitted to a modified expression of the van’t Hoff equation
using equations (4) and (5), where ∆Hm is the enthalpy of denaturation at
17
Tm, y(T) is the CD or the fluorescence signal at specific T’s, yF and yU are the
measured signals, and mF and mU are the slopes181,182.
Kobs(T) = exp[(∆Hm/R)((1/Tm)-(1/T))]
y(T) = [yF + mFT + Kobs(yU + mUT)]/(1 + Kobs)
(4)
(5)
For chemical induced unfolding, ∆GU can be extracted using the Linear
Extrapolation Method (LEM) which describes the linear relationship
between ∆GU and the denaturant concentration [D]183. Then, the denaturant
dependence of ∆GU can be described as in equation (6) where ∆GH2O can be
calculated by extrapolating back to zero denaturant concentration. This
yields the slope or m-value which is the dependence of free energy on
denaturant concentration.
∆GU = ∆GH2O - m [D]
(6)
In paper I, ∆GU,diss was calculated by using an apparent two-state model
coupled to dissociation (diss). Then, the Keq for a heptamer was defined as in
equation (7).
KU,diss = [Umonomer]7 / [Fheptamer]
(7)
For protein-ligand binding, an equilibrium expression can also be used that
describes a two-state process (8) where L is the ligand binding to protein P
and forming the complex (PL)54.
P+L
PL
(8)
The chemical equilibrium is the ratio of the rate of association Kon (P+L PL)
and the rate of dissociation Koff (PL P+L). The constants for association
(KA) and dissociation (KD) can be described as in equation (9) and (10).
KA = [PL] / [P] [L]
KD = [P] [L] / [PL]
(9)
(10)
The KD value is generally used to characterize the strength of a binding
reaction. The KD corresponds to the concentration of the ligand at which half
of the protein molecules are bound or at which the protein is half saturated.
Consequently, the binding free energy can be calculated using K D and eq. (2).
In a saturated reaction, a binding curve can be plotted using equation (11),
where Bmax is the maximum specific binding value and Y is the degree of
saturation.
Y = Bmax [L] / KD + [L]
(11)
18
3.2 CD spectroscopy
Circular dichroism (CD) is a technique conventionally used to investigate the
structure and stability of proteins. Some advantages of CD measurements
include their simplicity and short recording times. A typical measurement to
assess the folded state of a protein can be done in some minutes using low
protein concentrations and small volumes (e.g. 5 µM pure protein, 200 µL
for a 1 mm cuvette). The instrument measures the difference in absorbance
between left and right circularly polarized light. Proteins are optically active
because of their chiral nature and they will respond differently to the left and
right circularly polarized light. Therefore, a particular protein will have a
particular CD spectrum that provides information about its secondary (farUV below 250 nm) and its tertiary structure (near-UV above 250 nm)184.
The secondary structural information obtained from the far-UV CD
spectrum of a protein corresponds to the backbone conformation and the
contribution from secondary structure components such as α-helical, β-sheet
and random coil. Some known spectral features can be used to characterize
the overall contribution from these secondary structure elements. For
instance, the content of α-helixes gives a positive signal maximum around
190-195 nm and negative signals at 208 and 222 nm. The β-sheet content
gives a positive signal around 195-200 nm and a negative signal at 215-220
nm. Random coil gives a positive signal around 220 nm and a negative signal
around 200 nm185. The amount of secondary structure elements can be
estimated by using the CD spectrum of the protein and available
deconvolution softwares such as CDNN186.
Protein stability can be assessed by monitoring CD signal changes at a
specific wavelength as a function of temperature or chemical denaturants.
Then, thermodynamic data such as the midpoint of the unfolding transition
(Tm), the van’t Hoff enthalpy (∆HU) of unfolding and the free energy (∆GU)
can be calculated. Generally the Gibbs-Helmholtz equation is used to fit the
data for a two-state transition. In paper I only Tm values were extracted from
the thermal denaturation fit. In paper II CD was used mostly to monitor
secondary structure content.
3.3 Fluorescence spectroscopy
Fluorescence spectroscopy is a very sensitive method to assess protein
folding and kinetics at very low protein concentrations (e.g. 1 µM pure
protein, 100 µL, 3 mm cuvette). It measures the intensity of light emitted by
a molecule as it returns to its ground state after excitation. Fluorescence
signal from proteins are contributed from aromatic residues such as
19
tryptophans and tyrosines. These chromophores can be excited between 260
to 295 nm wavelengths and after the system goes back to equilibrium, the
emission of the light have a different intensity or have shifted position to
another wavelength. The emission changes can be monitored from the folded
to the unfolded state since in a folded state the protein aromatic residues are
normally located in a hydrophobic environment. Then, upon unfolding the
environment will change and those residues will give lower intensities as the
signals are quenched by the solvent187,188.
Tryptophan fluorescence was used in paper II to assess the integrity of
proteins at an excitation of 290 nm with an emission maximum around 350
nm. In paper I, tyrosine fluorescence was used (cpn10 lacks tryptophan) at
an excitation of 280 nm with an emission maximum around 304 nm.
3.4 Nuclear Magnetic Resonance
Solution-state nuclear magnetic resonance (NMR) spectroscopy is a
powerful technique to study protein structures, dynamics and interactions at
atomic resolution. It provides residue-specific information and can capture
events down to the picosecond time scale. Some disadvantages are the need
of concentrated samples and the limitation when it comes to the protein size.
It is generally difficult to obtain proteins at high concentrations and some
proteins that are unstable start to precipitate above certain concentrations.
Large proteins are difficult to study by NMR either due to the number of
resonances that might complicate the spectra or just because the size of the
protein affects the tumbling rate and thereby the signal line-width and
amplitude. To be able to study proteins using NMR, the protein sample has
to be isotopically labeled and present at a concentration of at least 50 µM
and volumes around 300 µL. However, the required protein concentration
depends on the type of experiment, the spectrometer magnetic field strength
and the running time, which can help to increase the signal-to-noise ratio.
NMR measurements are based on quantum mechanical properties of the
elemental isotopes nuclei (1H, 13C, 15N, 19F and 31P) which have a
characteristic spin (I = 1/2), and a nuclear magnetic moment. When an
external magnetic field is applied, the spin will have two energy states (+1/2
and -1/2), one aligning with the external field and the other one with
opposite direction. The electromagnetic radiation is therefore used to
stimulate transitions and coherences between the energy states of the spins
in a sample where specific nucleus are being studied. The difference in
energy between the two spin states is generally small and proportional to the
external magnetic field strength. Generally, the energy difference is given in
frequency units (MHz) and the chemical shifts are referred in units of parts
20
per million (ppm). In a typical one-pulse experiment the magnetization is
flipped to 90 degrees to be able to record the resonance signals. Then the
NMR signal will be proportional to the amount of magnetization in a xyplane which oscillates with a frequency that induces current. The time
dependent current is recorded as a so called free induction decay (FID) and
Fourier transformed to a 1D spectrum that represent the intensity as
function of frequency189.
A typical protein NMR experiment is to acquire 2D heteronuclear single
quantum coherence (HSQC) spectrum, and as the name suggests more than
one nucleus is involved in the measurement. A 1H,-15N HSQC gives rise to
one cross-peak from each amino group in the backbone corresponding to
one residue in the protein. Since the nucleus in the amino acids within the
protein structure experience a distinct chemical environment, then each of
them will have a distinct chemical shift in the spectrum.
3.4.1 Chemical shift perturbation
Chemical shift perturbation (CSP) experiments can be used to study
interaction between proteins and other biomolecules. In a typical CSP
experiment, a series of 1H,-15N HSQC spectra monitors the chemical shifts
from a 15N-labelled protein upon titration with an unlabeled protein
interacting partner (ligand). From CSP data several features about the
interaction can be obtained, such as the rate and binding affinity, and in
addition the binding sites can be mapped. The binding sites can be localized
by identification of the residues that change their chemical shift position in
the spectrum upon addition of ligand. Protein-ligand complexes are very
dynamic systems and the rate at which the components of the complex
exchange between the free and bound states affects the spectra and gives rise
to specific features that can be analyzed. If the exchange rate is slow in the
chemical shift time scale, two sets of signals will be observed for the free and
bound state. If the exchange rate is fast, only one signal will be observed as
the average of the two states. Finally if the exchange rate is intermediate,
only one signal is observed but with distinctly line-broadened peaks. To trace
the peaks from free to bound state, increasing ligand concentrations can be
added to reach saturation and to facilitate the analysis of the residues
involved in interaction190-192. This method was used in paper III to map the
binding-site of Dreb2a in Med25 proteins.
21
3.5 Surface plasmon resonance
Surface plasmon resonance (SPR) is a technique that enables the detection of
interactions between proteins or other biomolecules in real-time. The
instrument measures the changes of refraction index between two interfaces
after polarized light irradiation. A typical experiment requires low protein
concentrations and small volumes (<5 µM). The ligand is immobilized on a
sensor surface or a chip, which is a conducting film composed by a thin layer
of gold. The analyte which will be the potential interacting partner is in free
solution and is injected over the chip in a continuous flow. The interaction is
recorded due to the increase of molecule density upon association, which
results in a different reflection of light at a certain angle. The SPR angle of
reflectivity at a certain resonance condition will change while molecules
associate until saturation is reached. Then, dissociation will make the angle
to return back to baseline once all analyte is completely removed from the
sensing surface. The change of the SPR angle will be proportional to the
mass of the bound molecules and the result of interaction can be displayed as
a sensorgram against time where the y axis is the binding response in
resonance units (RU). From the binding curve the rate of association and
dissociation, and the affinity can be obtained193. This method was used in
paper II and III to probe binding between Med25 proteins and the Dreb2a
and VP16 TFs, and to obtain the KDs of their binding.
3.6 Isothermal titration calorimetry
Binding processes can be fully thermodynamically characterized by using
isothermal titration calorimetry (ITC). This technique is sensitive and
detects the heat generated or absorbed when proteins or other molecules
interact. During a typical experiment one protein sample is placed in a
sample cell (~20 µM) and the protein ligand (present in at least a 10-fold
excess in concentration) to be titrated is placed in a syringe compartment.
Then the ligand is injected into the cell repeatedly at certain injection
volumes until the system reaches saturation. The peaks obtained from the
raw data and the heat flow as a function of time can be integrated and then
fitted to an appropriate binding model (e.g. one site-binding model) to yield
the binding enthalpy (ΔH) and the binding affinity (KA) from which the
dissociation constant KD can be derived. Also, the free energy of binding can
be calculated using eq. (2) and the entropy of binding by using eq. (1).
The thermodynamic data gives more detailed information about the physical
basis of molecular interactions. The favorable or unfavorable changes in
entropy and enthalpy can describe the formation of a complex. A negative
enthalpy change for instance, reports favorable interaction due to hydrogen
22
and van der Waals bonds at the proteins interface. Hydrophobic effects will
also favor association in a process that can be viewed as a transfer of the
ligand from solvent to the protein environment which increases the
entropy194. ITC was used in paper III to characterize the interaction between
Dreb2a and Med25 proteins.
3.7 Cross-linking
Chemical cross-linking is a method that can be used to probe transient and
stable interactions between proteins. It involves the formation of covalent
bonds between proteins by using reagents that contain reactive groups which
will react with the functional groups of proteins. The principal idea to probe
interaction is that if two proteins are physically interacting with each other,
then they can also be covalently cross-linked to each other by the reagent.
There is a range of crosslinking reagents available. One which is often used is
glutaraldehyde which can react with any two amino groups that are close to
each other in space. Because of its promiscuous nature, glutaraldehyde has
low specificity but can be used at mild conditions as a convenient initial tool
to investigate protein interactions. The reagent is also used to characterize
oligomerization states195-197. This method was used in paper I in combination
with denaturation of cpn10. The heptamer crosslinks efficiently with
glutaraldehyde and can be visualized using SDS-PAGE. Upon addition of
chemical denaturants at concentrations that favor unfolding, the monomers
will not crosslink and will appear as monomers in SDS-PAGE. By performing
these experiments an apparent two-state unfolding reaction for cpn10 was
probed.
3.8 GST pull-downs
This is a relatively easy and straightforward method to probe potential
protein-protein interactions using cell lysates or pure proteins at relatively
low concentrations. One of the interacting proteins has to be GST-tagged
(the bait) and can be purified in one step to further be mixed with putative
binding partners (the prey). Prey proteins can be either purified proteins or
come directly from cell lysates. The assay is based on the immobilization of
the bait protein on glutathione sepharose beads. The GST protein prebounded to beads is then incubated with bait proteins. The non-bound
material is washed off with an adequate buffer and the proteins that interact
with the bait will remain on the beads. The interacting proteins can be eluted
and resolved in a SDS-PAGE, and analyzed by Coomassie-, silver staining or
Western blotting using appropriate antibodies198. GST pull-downs
experiments were used in paper II and III.
23
4. Results
4.1 Summary of papers
4.1.1 Macromolecular crowding stabilizes cpn10 monomers
Paper I: Macromolecular crowding extended to a heptameric system: The
co-chaperonin protein 10
In vivo, proteins fold and associate in highly crowded environments.
Previous experiments have shown that macromolecular crowding stabilizes
monomeric proteins towards heat perturbation and also can modulate their
structure40-42,199. Cpn10 is a ring-shaped oligomer consisting of seven
identical β-barrel monomers and it is an important human protein that
functions to help other proteins to fold. The stability of the heptamer is
dominated by subunit-subunit interactions and the individual monomers
have low stability on their own73-75. To assess the effects of macromolecular
crowding on the cpn10 protein, where denaturation involves unfolding and
dissociation, the thermodynamic and kinetic properties of the cpn10 were
investigated both in diluted buffer solution and in an in vitro crowded
system. Ficoll 70, a sucrose-based polymer, was used to create a crowded
environment in vitro at concentrations up to 300 mg/ml.
Increased stability in presence of Ficoll 70:
The results of a set of spectroscopic experiments showed that cpn10 is
stabilized by both, thermal and chemical perturbation in presence of 300
mg/ml Ficoll 70. The thermal stability of cpn10 increased by 4°C in the
presence of Ficoll 70 (Figure 7A). The stabilizing effects toward chemical
perturbations were reflected in the transition midpoint position, which
increased by 0.5 M GuHCl in Ficoll 70 (Figure 7B). Cross-linking
experiments supported the denaturant midpoints shifts in the presence of
Ficoll 70 and the apparent two-state transition from folded heptamer to
unfolded monomers.
Free energy of unfolding/dissociation increased in presence of Ficoll 70:
The coupled unfolding/dissociation free energy for the heptamer increased
by about 36 kJ/mol (average free energy in buffer 239 kJ/mol and in Ficoll
275 kJ/mol) (Figure 7C and 8 [a]). The spectroscopic data was analyzed by a
new approach using the complete spectra (fluorescence) at each condition
and global analysis of unfolding curves at every wavelength in the span of
294 to 396 nm. The data was fitted to an apparent two-state reaction coupled
to dissociation going from a folded heptamer to unfolded monomers
24
(F7↔7U). In order to probe the unfolding mechanisms in chemical
denaturants, NMR diffusion experiments were performed and the results
indicated that the diffusion coefficient of unfolded cpn10 was approximately
the same in both denaturants indicating a monomeric unfolded state.
Further, the unfolding kinetics of the heptamer was investigated and it was
found that the unfolding rate constants were slower in crowded conditions,
which might in part explain the higher equilibrium stability found for cpn10
in the presence of Ficoll.
Figure 7. Increased stability of cpn10 in presence of Ficoll 70. A) Thermal midpoint
as function of cpn10 concentration. Inset: thermal unfolding curves for cpn10
monitored by CD 230 nm changes. B) Transition midpoints from the GuHCl induced
unfolding as function of cpn10 concentration. C) Free energy for coupled
unfolding/dissociation from GuHCl induced unfolding. D) Heptamer-monomer
transition as a function of cpn10 concentration monitored by tyrosine fluorescence
changes. F7 indicates folded heptamer, F folded monomer, U unfolded monomer.
Cpn10 in absence (blue circles) and presence of 300 mg/ml Ficoll 70 (red squares).
25
Heptamer dissociation is affected in the presence of Ficoll 70:
The macromolecular crowding impact on the heptamer-monomer
dissociation constant was assessed by measuring fluorescence changes. The
full spectra were also analyzed as above. The heptamer-monomer transition
midpoint was lower in Ficoll 70 (3.1 µM) compared to in buffer (8.1 µM)
indicating tighter binding in crowded conditions (Figure 7D). The free
energy values for heptamer-monomer dissociation were calculated to 191
and 177 kJ/mol in Ficoll and buffer, respectively (Figure 8 [b]).
Figure 8. Thermodynamic cycle of the cpn10 heptamer unfolding and dissociation.
Free energy values are shown for the reactions both in buffer and in the presence of
300 mg/ml Ficoll 70. [a] Data from coupled unfolding/dissociation going from a
folded heptamer to unfolded monomers. [b] Heptamer-monomer dissociation
assessed by fluorescence changes. [c] Monomer stability calculated from [a] and [b].
The stability increased by 8% on the protein-protein interfaces and by 33 % on the
individual monomers in presence of Ficoll 70.
Finally, using the free energy values in [a] and [b], energy values for [c] were
calculated. Based on these results, a thermodynamic cycle was constructed
(Figure 8) which reveals that macromolecular crowding increases stability by
8% on the protein-protein interfaces and by 33 % on the individual
monomers. This means that macromolecular crowding exerts larger effects
on the individual monomers.
26
4.1.2 Conformational changes upon interaction between Med25
and Dreb2a
Paper II: Interactions between DNA, transcriptional regulator Dreb2a and
the Med25 mediator subunit from Arabidopsis thaliana involve
conformational changes.
Mediator is a multi-protein complex that transmits regulatory signals from
DNA-bound TFs to the Pol II transcription machinery109,111,117. How exactly
Mediator accomplish this function is not entirely understood. Several studies
indicate that interaction between TFs and Mediator subunits triggers
conformational changes which might be the way that regulatory signals are
propagated through Mediator to the Pol II transcription machinery 112,114,115.
In this study, the interaction between the activator interaction domain of A.
thaliana Med25 (aMed25-ACID) and Dreb2a were investigated in absence
and presence of an oligonucleotide containing the canonical Dreb2a DNAbinding site. The main purpose was to explore the potential structural
changes that might occur upon complex formation.
The C-terminal of Dreb2a is unstructured in the free state:
The secondary structures of each protein individually and the mixture of
proteins were monitored by CD spectroscopy. The ACID of Med25
(Med25551-680) showed ordered structures with negative peaks at 205 and
230 nm while the C-terminal of Dreb2a (Dreb2a168-335) was unstructured,
when they were analyzed in their free state (Figure 9A). However, full-length
Dreb2a (Dreb2af.l.) fused to a GB1-tag showed some ordered conformations
with negative peaks around 205 and 224 nm, thus indicating the presence of
some α-helical content (Figure 9B).
Gain of structure upon complex formation:
The interaction between Dreb2a168-335 and aMed25-ACID was studied by
SPR. In these experiments we determined the KD for the binding between the
proteins to 1 µM. We were unable to detect any interaction-induced
structural changes in these proteins in the CD measurements (Figure 9A).
Our SPR experiments also showed interaction of aMed25-ACID and
Dreb2af.l.. However, in this case the kinetics was complex and displayed a
fast association and a slow dissociation rate. The KD was approximated to 0.1
µM. Regarding this interaction, the mixture of Dreb2af.l. and aMed25-ACID
at a one-to-one ratio resulted in an increased negative CD signal which
indicated that structural shifts occurred upon interaction. The stoichiometry
of binding between aMed25-ACID and Dreb2af.l. was determined to 1:1 using
size exclusion chromatography.
27
Human Med25 interacts with Dreb2a:
Interestingly, by using pull-down, cross-linking and SPR experiments we
showed that the Med25-ACID homolog from human (hMed25-ACID) also
interacted with the plant-specific Dreb2a TF. The amino acid sequences of
ACID from human and A. thaliana share low homology. However, secondary
structure prediction revealed that they have a similar secondary element
content. This suggests that the structural features that are important for
activator recognition might be conserved between Med25-ACID from human
and A. thaliana.
Figure 9. A) Far-UV CD spectra of aMed25-ACID showed ordered structures
(green), Dreb2a168-335 was unstructured (green dashed), the mixture of both proteins
at 1:1 molar ratio (purple) and the sum of the individual protein signals (grey dotted)
overlapped. No structural shifts were observed upon interaction. B) Far-UV CD
spectra of Dreb2af.l. in presence of specific DNA (grey), in presence of mutated DNA
(dashed), and in absence of DNA (green) at 1:1 molar ratio, showing that Dreb2af.l. is
prone to fold upon interaction with its DNA-binding site (DNA signal subtracted).
Interaction between Dreb2a and aMed25 in the presence of the canonical
Dreb2a DNA-binding sequence indicates that Dreb2a is prone to fold:
After the observation that the complex formation between aMed25-ACID
and Dreb2af.l. resulted in structural shifts, experiments with specific and
mutated DNA oligonucleotides were performed to see if it would lead to
structural changes. First, the binding of Dreb2af.l. and its canonical Dreb2a
DNA-binding site was confirmed by using SPR. Next, CD measurements
showed that Dreb2af.l. gained secondary structure in presence of its specific
DNA (Figure 9B) and the complex was more stable over time. These results
indicated that Dreb2a is prone to fold upon interaction with both its specific
DNA and aMed25-ACID.
28
Finally, the interaction of aMed25-ACID and DNA-bound Dreb2af.l. did not
result in any additional conformational changes and SPR experiments
showed lower affinity between Med25-ACID and Dreb2af.l. when the
canonical Dreb2a DNA-binding site was present. These results suggest that
the binding of Med25-ACID and Dreb2af.l. is independent of the binding
between Dreb2af.l. and DNA which might explain its previously reported
negative regulatory effect 155.
4.1.3 Similar binding affinities but different binding energetics
Paper III: Interaction studies of the Human and Arabidopsis thaliana
Med25-ACID proteins with the Herpes Simplex virus VP16- and plantspecific Dreb2a transcription factors
Mediator is present in all eukaryotes and has an evolutionary conserved
structure and function. The Med25 subunit is specific for higher eukaryotes
such as humans and plants. The A. thaliana Med25-ACID (aMed25-ACID)
interacts with Dreb2a which is involved in different plant stress responses.
Human Med25-ACID (hMed25-ACID) and its interaction to the VP16
transcriptional activator protein has been extensively studied by other
groups. Despite that hMed25-ACID and aMed25-ACID share low sequence
similarity, hMed25-ACID interacts with the plant-specific Dreb2a. The aim
of this study was to characterize the interaction of the unrelated TFs Dreb2a
and VP16 with the Med25-ACID proteins from human and A. thaliana.
Figure 10. Sequence alignment of A. thaliana and human Med25-ACID. Identical
amino acids are indicated with asterisks. Secondary structure content of hMEd25ACID was obtained from PDB 2XNF (7 β-strands and 3 α-helices) and secondary
structure content for aMEd25-ACID was predicted using Jpred 3 server (7 β-strands
and 2 α-helices). Pink color indicates α helical content (H) and blue color indicates βstrands.
29
VP16 interacts with A. thaliana Med25:
We used GST pull-down- and SPR experiments to show that the VP16-TAD
interacts with the aMed25-ACID with similar affinity (micro molar range) as
the previously reported interaction between hMed25-ACID and VP16-TAD
(Figure 12, I and II). GST pull-down experiments also showed that the
Dreb2a168-335 fragment inhibits the binding between aMed25-ACID and VP16
when Dreb2a168-335 is pre-incubated with aMed25-ACID. This indicates that
the VP16 binding site on aMed25-ACID overlaps with the binding site for
Dreb2a168-335 (Figure 12, V).
Figure 11. NMR studies of interaction between Dreb2a168-335 and Med25-ACID
proteins. A) NMR structure of human Med25-ACID (2XNF)156, residues which
showed significant chemical shifts upon addition of Dreb2a168-335 are enlarged (gold)
and labeled with the residue name and number. Asterisks indicate residues that have
been previously described to be involved in the interaction surface with VP16TAD156,162. B) Comparison of the fraction of residues which were affected to different
degrees by interaction of Dreb2a168-335 with Med25-ACID proteins. Color coding as
in (A), unaffected residues (blue), broadened beyond detection (light yellow),
chemical shift changes (CS) above 0.1 (gold) and CS under 0.1 (white).
Dreb2a and VP16 have overlapping binding site in hMed25-ACID:
To map the interaction sites for Dreb2a168-335 in Med25-ACID proteins, NMR
chemical shift perturbation experiments were performed. The results
showed that the Dreb2a168-335 interaction surface in human Med25-ACID
overlapped with the earlier reported VP16-TAD binding site (Figure 11A and
Figure 12, I and III). The structure for aMed25-ACID has not yet been
solved, and therefore its Dreb2a binding site could not be mapped. Still,
NMR titrations showed that both aMed25-ACID and hMed25-ACID behaved
in a comparable manner upon interaction with Dreb2a 168-335 (Figure 11B).
NMR chemical shift perturbations also indicated that similar conformational
changes occurred in both interactions (Figure 11B, 12 III). These results and
30
the secondary structure prediction for a Med25-ACID (Figure 10), which
indicated a similar content of secondary elements as hMed25-ACID,
suggested that ACID from human and A. thaliana Med25 have structural
similarity and display a similar interaction surface recognized by Dreb2a.
Similar binding affinities but different binding mechanisms:
ITC was used to investigate in more detail the mechanism of binding
between Dreb2a168-335 and the two Med25-ACID proteins. The results from
complex formation between hMed25-ACID/Dreb2a168-335 and aMed25ACID/Dreb2a168-335 showed similar binding affinities but different binding
energetics. The complex formation between aMed25-ACID and Dreb2a168-335
displayed larger unfavorable negative entropy and larger favorable enthalpy
changes compared to complex formation between hMed25-ACID and
Dreb2a168-335. Dreb2a168-335 is unstructured in its free state and the large
conformational entropy penalty is presumably associated with formation of
structured elements or conformational changes upon binding of Dreb2a168-335
to aMed25-ACID (Figure 12, IV). The different binding energetics for the
hMed25-ACID/ Dreb2a168-335 complex formation indicated less structural
shifts since the changes in enthalpy and entropy were not as large in
magnitude as for the aMed25-ACID/Dreb2a168-335.
We conclude that the unrelated TFs Dreb2a and VP16 interact with
overlapping regions in the ACIDs of A. thaliana and human Med25 (Figure
12) even though their ACIDs show a low sequence homology. These results
suggest that subunits of the conserved Mediator complex, despite being
evolutionary diverged, still retains a conserved structure and function.
31
Figure 12. Summary of the results illustrating similar folds for the hMed25-ACID
and aMed25-ACID proteins upon interaction with VP16-TAD and Dreb2a168-335. In
our model, the binding sites on the two ACIDs are overlapping but the
conformational changes upon binding are somewhat different for aMed25ACID/Dreb2a168-335 compared to hMed25-ACID/Dreb2a168-335. I) Illustration of the
previously studied interaction between hMed25-ACID and VP16-TAD shown by other
groups using NMR. II) Illustration of results from this study where we used SPR and
pull-down experiments to study the interaction between aMed25-ACID and VP16TAD. III) Illustration of our NMR results for the interaction between Dreb2a168-335
and Med25-ACID from human and A. thaliana. IV) The same interactions as in III
but here studied using ITC. The deduced results from our ITC data indicated larger
structural shifts for the aMed25-ACID/Dreb2a168-335 complex formation highlighted
in green. V) The area within the orange dashed lines represents our results from GST
pull-down experiments which showed that pre-incubation of Dreb2a168-335 with
aMed25-ACID inhibits the interaction between aMed25-ACID and VP16-TAD, thus
suggesting overlapping binding sites on aMed25-ACID.
32
5. Discussion
5.1 Protein folding studies in cell-like environment
In the first project we studied the model protein cpn10, which is part of an
ATP-dependent chaperonin complex-system that assists in the folding of
other proteins. Cpn10 is a stable heptamer which has been previously
studied in vitro75-79. The specific aim was to test the effects of
macromolecular crowding, or excluded volume effects, on its stability and
unfolding mechanisms. According to macromolecular crowding theory,
excluded volume will favor compact states and self-associations that lead to
compact states. A folded globular protein have a compact state and marginal
thermodynamic stability, while an unfolded protein have a more extended
state with large configuration entropy. Then, a way to favor the folded state
is by restricting the entropy of the unfolded state, which can be modulated by
increasing the volume occupancy, e.g. adding macromolecular co-solutes to
the system being studied29,30. Previous in vitro studies have shown that the
stability of cpn10 heptamer is governed by the monomer-monomer
interfaces and that the individual monomers have low stability by their
own73-75. The results from our study showed that the stability effect on the
cpn10 heptamer originated in the monomers that have lower stability than
the interfaces, and they appear to be stabilized more than the interfaces by
macromolecular crowding. Even though the effect is small in absolute values
the magnitude of the effect could still be significant inside the cell. Moreover,
only the excluded volume effects were taken in to account and other effects
such as high viscosity, weak or non-specific interactions were not taken into
consideration.
A complete picture for the effects of the crowded cell environment on protein
folding would be the ultimate goal to reach. Nevertheless, the limits of
current biophysical techniques restrict us from monitoring the folding of a
single protein molecule in real time inside the cell. During the last years, new
methodologies have been developed to make the study of proteins possible at
in vivo conditions. Optical spectroscopy and NMR have been adapted to
study proteins inside of living cells200-202. Several studies have directly
investigated the stability and folding of proteins by using in-cell NMR
techniques. The results from these experiments differed. While some
proteins gained structure in a cellular environment, others failed to fold203205. Major challenges are however encountered by performing these kinds of
in-cell experiments, for instance unspecific weak protein-protein
interactions and the high intracellular viscosity of the system206-208. For incell NMR, these challenges are difficult to overcome, not to mention the
33
restriction when it comes to the protein size. Also, new computational
approaches have recently been applied to study protein folding and the
combination of in silico simulation methods and experimental work are
helping us to understand protein folding for small proteins in conditions
similar to those found in the cell209-211. Still, much remains to be learned to
understand how proteins behave in their natural complex cell environment.
5.2 Interaction between transcriptional regulatory proteins
In the second project the specific aim was to characterize the interaction
between the Mediator subunit Med25 and Dreb2a from A. thaliana, but also
to compare this interaction with the one between human Med25 and the
VP16 TF. In this study only the interaction domains from each protein, ACID
(from aMed25 and hMed25) and the TADs (from Dreb2a and VP16), were
used except for Dreb2a which was also used as a full-length protein. In
contrast to the first study, these proteins were relatively unstable, especially
at high concentrations. The Med25-ACID proteins showed more stability
compared to Dreb2a and VP16, which both lacked structure as free proteins.
It was therefore challenging to find appropriate conditions for the
interaction experiments. Macromolecular crowding was used in an attempt
to increase the stability of the proteins. However, we found that Dreb2a and
VP16 were more unstable in the presence of the crowding agent Ficoll 70
which resulted in precipitation of the proteins (data not shown). The
marginal stability of the Dreb2a and VP16 TADs might be due to the fact that
they are isolated from their natural environment inside cells. These domains
are part of larger proteins or complexes which are normally residing in the
crowded environment of the nucleus. These proteins are also very dynamic
in their original environment and their stability and function depends on
interactions with their partners.
5.2.1 Small contacts might end up in great connections
The main purpose of the second project was to account for potential
structural changes upon interaction of aMed25-ACID with Dreb2a, since
conformational changes have been proposed to explain how Mediator
conveys transcription regulatory signals from promoter-bound TFs to the Pol
II transcription machinery. The detected conformational changes upon
interaction were very small when using the full-length Dreb2a and could not
be detected by CD when using Dreb2a168-335. However, NMR and ITC results
showed that interaction between aMed25-ACID and Dreb2a168-335 triggered
conformational shifts. Those structural shifts are probably contributions
from the most unstructured interacting protein, Dreb2a 168-335. The gain of
structure of TADs upon interaction with their targets has been reported
34
before. For example VP16-TAD has been shown to undergo helical
conformational transitions upon interaction with hTAFII31, TFIIB, TFIIH
and Tfb1139,141,142,144. Likewise, p53, c-Myc and Gcn4 have been shown to gain
structure upon interaction with Tfb1, TBP and Gal11 (Med15),
respectively124,142,143. The last mentioned, Gcn4, that was reported to interact
with the yeast mediator subunit Med15, was described to form a ‘fuzzy’
complex in which the binding interface could not be defined by a single
conformation state. Forming a fuzzy complex could allow the functional
flexibility needed to activate transcription and could explain how TADs have
the ability to interact with multiple targets. In this perspective, our results
showing that the unrelated TFs VP16 and Dreb2a interact with both human
and A. thaliana Med25-ACID are perhaps not that unexpected. Still, the
larger conformational changes of Dreb2a upon interacting with the aMed25ACID compared to when it binds to hMed25-ACID, might indicate that the
interaction between A. thaliana proteins are more specific. In this context,
proteins with different amino acid sequences might interact in similar ways;
still every interaction has a purpose. In the case of the mediator proteins that
we have studied, it is to propagate transcriptional regulatory signals by
interacting with specific partners.
On the other hand, the structural shifts that occur in aMed25-ACID upon
interaction with Dreb2a168-335 were hardly detected by CD. However, NMR
results showed that both human and A. thaliana Med25-ACID proteins
might adopt similar folds upon interaction with the TFs which means that
the ACIDs exhibit similar modes for recognition of different TFs. The fact
that the Med25-ACID from human and A. thaliana interacted with the
unrelated TFs, VP16 and Dreb2a, despite their low sequence homology,
indicates that even though the amino acid sequences of the Mediator
subunits have diverged, the structure and interaction properties of its
subunits remain conserved.
Thus, the project’s aim - to explore conformational shifts in Med25-ACID
upon interaction with TFs - was only partly achieved, mostly because only
the well folded ACID domains were used. The optimal situation had been to
use full-length Med25 which has around 50 % predicted disordered regions
similar to most mediator subunits. Those disordered regions are most likely
to acquire transient conformations upon interaction, which is the prevailing
hypothesis for how Mediator complex propagate transcriptional signals. Still,
working with the full-length protein which is around 90 kDa might restrict
the use of certain biophysical methods as commented earlier.
35
The investigation of specific protein-protein interactions gives insights into
the nature and mechanisms of protein recognition and function. To
characterize an interaction, the binding sites are required to be identified
which is very challenging to do experimentally. Prediction of protein-protein
interactions based on the primary amino acid sequence is currently partially
possible, but still known structures of the participating components are
required to amplify the efficiency of the prediction studies 212,213.
The contribution of future studies will expand our knowledge about how
protein machines work in the cell and hopefully soon we will be able to
predict their structures and functions.
36
6. Conclusions
Proteins are small machines that do the hard work in cells which offer a
hostile environment. Their function relies on proper folding and on their
ability to recognize and to interact with a range of partners in order to
transmit regulatory signals. This thesis reviews results from two projects
intended to explore two different aspects: protein folding and proteinprotein interactions. First, we studied the biophysical properties in a cell-like
environment of cpn10, a stable oligomer which has previously been well
studied in vitro. Second, we describe the interactions in vitro between
unrelated TFs, Dreb2a and VP16, and human and A. thaliana Med25
proteins.
The results from the heptameric cpn10 subproject showed that
macromolecular crowding or excluded volume had a stabilizing effect on
cpn10 upon thermal- and chemical induced unfolding. Such effects were
larger for the monomer stability than for the monomer-monomer interface
stability. The cpn10 stability effect that we detected in cell-like conditions
endorses the already known fact that the intracellular environment affects
the biophysical properties of proteins. Consequently, it is important to
continue comparing the results between in vitro and in-cell like conditions in
order to quantify the magnitude of such effects. Taking these effects in to
account will increase the understanding of protein folding and thereby
misfolding in the cell. Better knowledge of protein misfolding mechanisms is
critical since misfolded proteins are associated to neurodegenerative
diseases such as Alzheimer’s and Parkinson's.
Results from our studies of interaction between the A. thaliana Med25 and
Dreb2a proteins indicated that conformational changes are induced upon
binding. The gain of structure originated from the unstructured TF Dreb2a
and direct contributions from Med25 were not detected. These results
endorse the already reported potential of TFs to recognize different partners
and to adopt transient conformations upon binding. Still, the binding
between A. thaliana proteins gave larger conformational changes which
might indicate a more specific interaction. The unrelated VP16 and Dreb2a
TFs both interacted with overlapping regions in the ACIDs from both human
and A. thaliana Med25. This interaction occurred despite that ACID from
human and A. thaliana share low sequence homology, and showed that even
though Mediator sequences have diverged, the function of its subunits
remain conserved.
37
7. Acknowledgements
I would like first to acknowledge all the co-authors and collaborators behind
the papers, without your help this thesis would never have been written.
Först och främst vill jag tacka alla forskare som har varit inblandade i båda
projekten och som har bidragit till de publicerade artiklarna och
manuskriptet. Utan er hade denna avhandling inte funnits.
Tack till min handledare Stefan Björklund för att ha hållit dörrarna öppna
och tagit emot mig i sin grupp. Stefan, min tid på ditt labb har varit oerhört
lärorik och utvecklande på alla tänkbara sätt. Jag tackar dig för att du gav
mig utrymme för att tänka själv, för att jag fick vägledning för att få mina
spretande tankar samlade åt rätt håll och för att ha gett mig inspiration och
motivation igenom Mediator-projektet. Jag känner mig mycket stolt över att
ha fått vara en del av din forskning.
Pernilla Wittung-Stafshede, jag har mycket att tacka dig för. Tack vare
dig finns jag här idag och är på väg att disputera. Jag fick ditt förtroende och
det slutade med att jag vågade flytta norrut till en främmande vinterstad. Jag
lärde mig mycket under den tid då jag var i din grupp och är tacksam för det.
Jag tar med mig flera fina minnen från våra gruppaktiviteter. Jag önskar dig
fortsatt framgång, du är en stark forskningskvinna som jag beundrar stort.
Elisabeth Sauer-Eriksson, också tack vare dig står jag här idag för att
disputera. Ditt stöd och dina kloka råd fick mig att få mod att fortsätta på
min forskningsutbildning. Det finns inga ord som kan uttrycka min
tacksamhet.
Jürgen Schleucher, I’m very thankful for all your help with the NMR
experiments and also for your valuable input on my work. Thank you for the
discussions about science and also for the laughs.
Thank you Michael Kovermann, Anders Öhman and Katja Petzold
for all help with NMR and invaluable comments that contributed to my
work.
Gerhard Gröbner, tack för all input och allt engagemang som jag fick från
dig på våra korridorsmöten. Tack också för att du skapar en trevlig
arbetsmiljö men din charmiga tyska personlighet.
38
Lars Backman, tack för ditt äkta intresse för forskningsprojekten, inte
bara riktat till mig, utan allmänt till alla oss doktorander. Tack för att du
alltid ställer kluriga frågor på doktorandseminarier vilka får oss att tänka till.
Jag uppskattar mycket all konstruktiv kritik du gett oss.
Christin Grundström, tack för att du alltid ställde upp och hjäpte mig
med diversa metoder och tekniker. Du tog dig alltid tid till att svara på mina
frågor. Jag är också mycket tacksam för ditt engagemang i försöket att få
kristaller.
Patrik Andersson och Henrik Antti, tack för ert engagemang i mitt
projekt under den gånga tiden och för all uppmuntran.
In addition to those who have been involved in my work, I’m grateful to all
scientists at Umeå University, I’ve been privileged enough to get to know you
and work with some of you. Thank you also to all my colleagues at the
Department of Chemistry and Medical Biochemistry and Biophysics, for
contributing to a nice working environment and for all the laughs.
I have to add that this thesis would never have been possible to finish
without the support of many friends and here comes special thanks for them.
De tre musketörerna, modiga och ärofyllda vänner som kämpade och
kämpar vidare på var sitt sätt emot livets strider. Maria, tack för all hjälp jag
fick för att hitta rätt i labbet när jag började i gruppen. Tack för tålamodet
och den tid du gav mig för att på ett pedagogiskt sätt instruera mig i Äkta
användandet. Tack för att du har den ordningsamma och lugna
personligheten som får mig att tänka klart. Du är den trovärdiga och
hjälpsamma vännen som alltid ställer upp och jag kommer alltid att bära din
vänskap i mitt hjärta. Alex, min ficoll-vän och compadre som delade kontor
med mig ett par år. Jag var vilse i labbet och du hjälpte mig att komma igång
med mitt projekt, utan dig hade jag inte lärt mig att på så kort tid använda
alla dessa instrument. Du är inte bara en stor, stark och smart man, utan du
är också en ödmjuk och klok person. Jag tackar dig oerhört mycket för att ha
funnits till hands när jag som mest behövde det. Moritz, den vise, du är min
favorit tysk hårdrockare, tack för att du spelade musik på labbet som fick en
att vakna till. Jag uppskattar verkligen all input jag fick av dig under min tid i
gruppen. Tack för att du alltid tog dig tid och upplyste mig om alla
förundrande fakta kring olika metoder. Tack också för allt stöd och för att du
är en ärlig vän som inte drar dig för att säga det du tycker.
Istvan, you get your own sentence. Thank you for helping me sometimes at
the lab and for not having lunch in the office during those beautiful times.
39
Jeanette Blomberg, forskaren bakom alla utmaningar för proteinrening.
Du får en stor eloge för att ha lyckats få till metoden för att rena fram
besvärliga proteiner. Tack för ditt bidrag till projektet, för all hjälp och
handledning under min tid på labbet. Du var inte bara min höger arm i
våras, du var den som lyssnade och tröstade när frustrationen tog över. Tack
för din vänskap.
Tobias Sparrman, vad vore kemiska institutionen utan din expertis och
vad vore fikarummet utan dina sociala kompetenser. Först ska jag tacka dig
för bidraget till cpn10-projektet. Dessutom vill jag tacka för din hjälpsamhet,
du ställer alltid upp när man som mest behöver det. Du är otroligt
pedagogisk och lyckades till och med få en guldfisks hjärna som min att
förstå lite grann kvantmekanik.
Matteus, lugn och sansad själ som lyssnar på en och viskar vishet. Tack för
din hjälp med labbassitans och för alla trevliga luncher och morotskakor du
bakade till torsdagsfikat. Tack också för att du försökte lära mig åka skidor,
det kommer jag aldrig att glömma.
Marcus och Lan, tack för er vänskap och för alla dessa trevliga stunder vi
fick spendera tillsammans. Marcus, du får ett speciellt tack för att du fick
mig trivas på jobbet redan från första dagen. Din sympatiska personlighet
fick mig att känna mig välkommen.
Michael Hall, tack för stödet och all hjälp i form av labassistens, metoder
och allmänt om forskning. Jag är också mycket tacksam för hjälp med
engelsk granskning. Lotta, tack för att du är så hjälpsam och för alla trevliga
stunder och kattsamtal.
Tack Johan först och främst för att du alltid fanns där för att vädra tankar
och idéer. Stort tack för att jag får vara med på UBH och får chansen att
tillsammans med er skapa de spännande projekten.
Jörgen Ådén, tack för att du tog dig tid och hjälpte mig med diverse frågor
kring NMR och andra metoder. Tack också för att du bidrog till en trevlig
och rolig atmosfär på korridorsmöten.
Mina underbara animekompisar, Martin och Tomas, tack för att ni
syresatte min hjärna med fantastiska anime-maraton. Våra animekvällar fick
mig att detoxifiera alla ansamlade frustrationer från labbet och fick mig att
åter ladda motivationens batterier för att börja med nya krafter på labbet.
40
Therese och Lina, tack för alla trevliga luncher och samtalen vi hade när
jag inte pratade om anime med Martin. Ni är härliga att umgås med och jag
fick mycket rofyllda stunder tack vare er.
Bengtsson, min gnällkompis. Att få träffa en västkustare med härlig dialekt
här i norr fick mig att känna mig på något sätt i Göteborgstrakterna. Tack för
din vänskap och den tiden vi fick umgås tillsammans. Tack också för allt stöd
och all uppmuntran du gav mig för att inte ge upp.
Tack alla kolleger som deltog på torsdagsfikat och bjöd på utsökta
delikatesser. Jag hoppas att Ulrikas fikatradition lever.
I would like also to thank all current and former members in my group for all
help and for creating a nice work environment in the lab. Thank you Céline,
Nils, Rajesh, Khalil and thanks again Jeanette for making thinks work in
the lab.
Kristoffer Brännström, forskaren bakom det interagerande projektet.
Tack för all hjälp med Mediator-proteinerna som var svåra att få ett samspel
med. Du är så hjälpsam och tålmodig och bidrog mycket till projektet. Tack
för assistensen med SPR och ITC experimenten. Tack också för alla trevliga
luncher på IKSU och de goda hemmagjorda delikatesser du bjöd på.
Göran Bylund, tack för att du så glatt hjälpte en vilse doktorand att hitta
rätt på instrumenten och apparaterna på MedChem. Vad jag än behövde så
fanns du alltid tillhands. Du hjälpte mig dessutom med tunga
centrifugrotorn. Tack också för den goda julklappen jag fick, du anar inte
den glädje du förde den dagen i mitt liv.
Dominika and Irina, thank you for being so cheerful and for all the fun
times, coffee- and lunch breaks. Special thanks for introducing me to
Lokalen.
Björn, Ulf, and Rais, thank you for being so kind with me. I really enjoyed
our discussions about science and also our amusing chats.
Melissa, Maite, Mar och Maria, todas empiezan con Ms. Gracias por
brindarme su amistad, por el apoyo y todos los consejos recibidos. Gracias
por escuchar y por mantener mi español vivo.
Clas, hur ska jag tacka dig för all hjälp jag fått av dig, mitt begränsade
ordförråd förhindrar mig att hitta de fina orden du förtjänar att få. Gracias.
41
Sociala gruppen på kemiska institution, tack för att jag fick vara del i
gruppen och för att ni är så sociala, glada och trevliga människor. Speciellt
tack till Rosita som fick mig att gå med och för att du är en ros som utstrålar
glädje och positiv energi med ditt soliga leende. Maria, Lars-Göran och
Erik, ni ska också få tack för alla trevliga möten och aktiviteter vi haft.
Janice, thank you for bringing sunshine to our coffee room, I’m grateful to
you for being thoughtful with me at any time.
Tack till klubben som tillät en liten bolivian att försöka berika sin kunskap
om single malts, jag kommer att sakna er.
The social group at MedChem, thank you for arranging great activities and
also for good laughs on Fridays.
Tack alla vänner i Göteborg som alltid öppnar dörrarna till en liten bolivian.
Ellen, du har alltid funnits där för mig och har lyssnat och lyssnat om och
om igen på alla mina frustrationer och problem. Tack för att du erbjuder mig
lugn och ro och målar upp ett annat perspektiv för att se på livets problem.
Tack också för att du och Christoffer alltid tar hand om mig när jag är på
besök i Göteborg. Hannah, min rofyllda och snälla vännen banana. Du har
också hjälpt mig att klara av de svåra stunderna i övergången från Borås till
Umeå och allt som ingick i den flytten. Jag är enormt tacksam för att ha
träffat dig och för att du finns som vän i mitt liv. Tack också Daniel och
underbare Eric, som får mig att känna mig som en del av er familj. Agnes,
stilla och rogivande röst, tack för att du ger mig sinneslugn.
Juan Miguel, jag har så mycket att tacka dig för och de följande orden
kommer inte täcka allt som jag vill få sagt. Tack får två underbara år i Umeå,
tack för att du var sansad och klok och fick mig ner på jorden när jag var och
navigerade bland moln. Det är också tack vare dig jag fick avsluta något som
jag var på väg att ge upp.
Thank you Aki-san for your friendship. Your words reached me and could
cheer me up in those hopeless moments of life.
Peter, den ovillkorliga vännen som slumpvis dök upp en vacker dag. Du går
oviss och förstår inte hur mycket du har hjälpt mig för att utkämpa denna
svåra och slutliga fas på min doktorandtid. Tack för att du lyssnat och velat
vara en del av den mest stressande tiden hittills i mitt liv.
Tack Umeå och alla underbara människor jag har fått äran att lära känna
här. Var och en finns i mitt hjärta, ni har varit som en familj för mig och har
gjort min vistelse i Umeå till en oförglömlig tid.
42
Hugo, hoy me encuentro aqui gracias a tí. Cada paso dado en Suecia fue
soportado por tu sabiduria. Gracias por la ayuda en matemáticas y fisica, por
enseñarme sueco, por alentarme y empujarme para seguir mis metas. Tu me
enseñaste muchas cosas valiosas, sé que nada es gratis en la vida y que sólo
tus esfuerzos limitan tus logros. Gracias.
Gracias a mi fiel amiga Daniela. Que hubiera sido de mi sin tu amistad. Me
has escuchado y visto llorar desde que tenía 17 años. Pese a la distancia, me
has brindado una sincera e incondicional amistad durante tanto tiempo. Tu
apoyo moral me ha ayudado a sobrellevar tantas crisis. Gracias amiga, te
quedo eternamente agradecida y mi aprecio por ti queda grabado en este
libro para siempre.
Finalmente, gracias querida familia por el apoyo incondicional, por
ofrecerme ayuda y cariño a pesar del océano que nos separa. Quiero que
sepan que ustedes han sido la motivación para continuar y terminar este
doctorado. Mami y Mary, gracias por haber inculcado en mi responsabilidad
y buenos hábitos, espero que esten orgullosas de mi, éste logro es sólo el
reflejo de sus enseñanzas.
43
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