Tentamen i Biomätteknik 2013-­‐10-­‐30 ENGLISH VERSION Problem 1 (10p). 358
In the experiment below, the binding of calmodulin to part of the estrogen receptor has been investigated, since it has been implied to be involved in cancer. Based on earlier experiments the study focusses on a 25-­‐amino-­‐acid peptide from the estrogen receptor; this peptide is called ERα25p. a) Which method has been employed? b) What is measured here? c) Which two fundamental principles for this method are illustrated by these results? Explain how these effects arize! d) Describe and interpret the result below! L. Carlier et al. / Biochemical and Biophysical Research Communications 419 (2012) 356–361
Fig. 1. The interaction between Ca4CaM and ERa25p monitored by Trp fluorescence and ITC. (A) Intrinsic tryptophan fluorescence spectra o
concentration (kex = 295 nm). (B) ITC data obtained upon titration of ERa25p into Ca4CaM (upper panel). The data best fit a oneCa4CaM
determination of thermodynamic parameters (lower panel).
Peptide sequence EPa25p: RAANLWPSPLMIKRSKKNSLALSLT two hydrophobic pockets at the surface
We further used NMR spectroscopy to probe the interaction beProtein sequence CaM: tween ERa25p and Ca4CaM at the residue level. Chemical shift perMet residues which play a major role in
MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGT
turbation (CSP) analysis was performed by collecting 1H–15N HSQC
proteins [6,8]. 1H–13C HSQC spectra were
IDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDE spectra on uniformly labeled 15N–Ca4CaM in the presence of
CaM as a function of added ERa25p. As
EVDEMIREADIDGDGQVNYEEFVQMMTAK increasing
amounts of ERa25p. Amide resonances of free Ca4CaM
the 9 Met methyl groups undergo drama
upon binding. They correspond to Met re
were assigned from the analysis of 3D heteronuclear experiments
N- and C-terminal hydrophobic pockets
recorded
on a uniformly labeled 15N/13C–CaM sample. Upon addi and 109, 124, 144, 145, respectively). O
tion of 0.6 molar equivalent of peptide, the majority of CaM resostrong evidence that both the N- and
nances exhibited significant chemical shift changes and
patches of Ca4CaM are involved in the
broadened or disappeared (data not shown). The resonances became sharper as the peptide concentration was increased and no
peptides.
further changes in the HSQC spectrum were detected beyond a
As stated above, CaM-binding peptid
3:1 peptide:CaM ratio. These observations indicate that the molectured in aqueous solution and adopt a
Problem 2 (10p) The diagrams below show the results of continued measurements of the interaction between calmodulin and the ER peptide. a) Which method has been used? Acronym and interpretation, please! b) Describe the result obtained in figure A (i.e. what was measured, how can we interpret these data, what do we learn about the biological system from these results?). c) How has the difference spectrum in figure B been obtained? d) Is it correct to state that the ”difference spectrum” corresponds to ”bound ER2p”, as was done in figure B? Motivate your answer carefully! 360
L. Carlier et al. / Biochemical and Biophysical Research Communications 419 (2012) 35
Fig. 3. Far-UV CD studies of the interaction between Ca4CaM and ERa25p. (A) CD
spectra recorded for Ca4CaM in the absence and presence of 2 molar equivalents of
ERa25p. (B) Difference CD spectrum representing ERa25p bound to Ca4CaM
compared with the CD spectrum of free ERa25p.
residues play a major role in the interaction. Hydrogen bonds and
electrostatic interactions also contribute to the stabilization of the
Problem 3 (8 p) The figure below shows further results from the investigation of a possible interaction between calmodulin and the ER peptide. a) Which method has been used (acronym and interpretation)? b) Which experiment has been performed? (acronym is sufficient) c) What do the peaks represent in this experiment? d) What can you say about the interaction based on the result of the experiment below? Motivate your answer carefully! L. Carlier et al. / Biochemical and Biophysical Research Communications 419 (2012) 356–361
(A) Overlay of spectra obtained for N–Ca CaM in the unbound state and in complex with ERa25p. (C) Average amide chemical shift perturbation (CSP) in Ca4CaM induced by the addition of 3 molar equivalents of ERa25p as a function of residue number. on a surface which corresponds to the C-terminal hydro
(Fig. 3B). Using the mean residue molar ellipticity of a fully helical
pocket (Fig. 4C). Therefore, unlike ERa25p, the ERa17p pep
peptide [16], the De
of the bound ERa25p indicates a partial a-
Fig. 2. Monitoring ERa25p-induced structural changes in Ca4CaM by NMR spectroscopy. (A) Overlay of 1H–15N HSQC spectra obtained for 15N–Ca4CaM in the unbo
and in complex with ERa25p. (B) Surface representation of the Ca4CaM structure (PDB ID: 3CLN). Residues that exhibited significant (CSP > 0.25 ppm) and
(CSP > 0.5 ppm) chemical shift changes are colored in pink and yellow, respectively. (C) Average amide CSP in Ca4CaM induced by the addition of 3 molar equiv
15
4
ERa25p as a function of residue number. (D) Overlay of 1H–13C HSQC spectra
showing the Ca4CaM methionine 13Ce/1He signals in the absence and presence of ERa
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
222
helical fold with a mean helicity of 35 ± 5%.
In this work, we showed by fluorescence spectroscopy that the
residues 287–294 of ERa, and in particular W292, are involved in
the interaction with Ca4CaM. However, previous studies unambiguously demonstrated the ability of the synthetic ERa17p peptide,
which lack these residues (region 295–311), to bind Ca4CaM
in vitro [5,12]. To better understand the structural importance of
residues 287–294 in CaM binding, we examined the interaction between ERa17p and Ca4CaM by NMR spectroscopy. An overlay of
the HSQC spectra collected during the titration of 15N–Ca4CaM
cruits mainly the Ca4CaM C-terminal domain, highlighti
structural importance of residues 287–294.
4. Discussion
The aim of this study was to better understand the mo
mechanisms underlying the interaction of human ERa wit
For this purpose, we used a biophysical approach to pro
interaction between CaM and two ERa-derived peptides: E
and ERa17p which was previously identified as the CaM-b
Problem 4 (8 p)
In the experiment below, different aggregating peptide fragments (NN, CC, NC, DC) derived from the protein synuclein have been studied. Synuclein is a key protein in Parkinson’s disease, and fragments of this protein form the principal component of the protein aggregates characteristic to this disease. a) Which method has been used? (acronym and interpretation) b) Describe how the experiment has been performed (sketch the experimental setup, describe measurement area and observable (i. e. what we are measuring). c) Describe based on the various curves in the figure below the procedure taken to interpret results from this type of experiment! d) What can you say about the different peptides compared to each other just by comparing the curves, without detailed knowledge or access to specialized software? From the figure legend: Curve fitting was performed with Gaussian and Lorentzian
lineshapes and with bandwidths varying between 15 and 20 cm-1. The peak position of the
components was deduced from the second derivative spectra. The sum of the fitted curves is
shown as a broken line, closely overlapping the experimental trace, shown as a continuous
line.
Problem 5 (10p) In the figure below, a complementary method has been used to study the aggregating peptides. a) Which method has been used? Acronym and interpretation, please! b) Explain the method by sketching an outline of the experimental setup; then describe how the signal is obtained and detected. c) There are two possibilities for measurement with this device. Describe the method used in the figure below. d) Propose an experiment where the alternative measurement option is used, and describe what type of results would be obtainable in this way. e) Which modes of aggregation seem apparent in the various synuclein fragments? Based on this, which fragment do you think is the most disease-­‐related? Motivate your answer! Covalent Dimers of a-Synuclein
Covalent Dimers of a-Synuclein
Figure
Representative
TEM and
AFMby
images
ofCC,
fibrils
the NN,
CC,1–4)
NC and DC
dimers
(rows
igure 7. Representative TEM
and7.AFM
images of fibrils
formed
the NN,
NC formed
and DC by
dimers
(rows
aS (fifth
row)
(left1–4) and aS (fifth row) (left
Problem 6 (12p) In the figure below, the myosin binding of the calmodulin-­‐like protein S100A4 has been studied. Myosin is a protein related to cell mobility. Study carefully the results in the figures below as well as the figure legend extract. a) Which method has been used? Acronym and interpretation please! b) Explain HOW the stochiometry of the interaction was evaluated. Use figure data and figure legend. c) Which of the measurements B, C or D do you rely on most/least with respect to affinity? Motivate your answer! d) Provide a proposal for an alternate measurement technology to determine the affinity and describe an experiment to do this; also motivate your choice of method. Fig. 2. Nonmuscle myosin II heavy chain fragments and peptides used in this
work and
Extract from ”their
Figure affinity
Legend”: to S100A4. (A) Fragments MF1 (NMIIA Ser1712B–D)
Heat
changes
were
recorded
by ITC experimentsform
at 25 °C,
at physiological
salt MF1
Glu1960) and MF2 (NMIIA
Gln1795-Lys1937)
coiled-coil
structures,
concentration.
75 μM
S100A4 (33).
monomer
titrated
with 500 μM
monomeric NMIIA and
assembles into
filaments
Thewas
mainly
negatively
(Glu1722-Asn1756)
peptides. Peptides MPN (B) and MPT (D) show nanomolar affinity to S100A4 (Kd < 8 nM),
positively charged (Ala1868-Lys1895) regions (the latter called ACD) that are
while the binding affinity of MPC (C) is in the micromolar range (Kd ≈ 1.8 μM). In all cases,
crucial for filament formation (33, 34) colored in red and blue, respectively.
the binding stoichiometry is approximately one myosin peptide per S100A4 dimer.
Within the sequence of MPT (NMIIA Arg1894-Lys1937) the residues that
are thought to form a coiled-coil in longer fragments are underlined, while
orange letters show a and d positions in the heptad repeat. Note that the N terminal basic residues of MPT overlap with ACD and MPT does not contain
the full nonhelical tailpiece. Corresponding amino acid positions of S100A4
binding site that differ in NMIIB (1901-1944) and NMIIC (1918-1961) isoforms
are indicated. An N-terminal Tyr (green) was added to the peptides MPT and
MPN to facilitate concentration measurements. (B–D) Heat changes were
recorded by ITC experiments
at 25 °C, at physiological salt concentration.
75 μM S100A4 monomer was titrated with 500 μM monomeric NMIIA peptides. Peptides MPN (B) and MPT (D) show nanomolar affinity to S100A4
(K d < 8 nM), while the binding affinity of MPC (C) is in the micromolar range
(K d ≈ 1.8 μM). In all cases, the binding stoichiometry is approximately one
myosin peptide per S100A4 dimer. Thermodynamic parameters of the interactions of S100A4 with NMII heavy chain fragments are shown in Table S1.
Kiss et al.
Phe45
cilitat
four m
ture
(Tabl
muta
finity
chiom
shows
1894–
tal str
Symm
Wher
the sa
subun
homo
acter
matri
is tha
more
A (by
(resid
Diffe
the cr
unit B
detail
N-ter
nifica
partly
surfac
bindin
the a
form
the tw
Intera
face b
dant
chain
hub b
taneo
forme
Problem 7 (12p) The study of S100A4 binding to myosin results in a hypothesis suggesting a calcium-­‐
dependent association of S100A4 to myosin, which would dissociate the fibrills and thereby affect cell mobility. a) Propose THREE methods which could be used to test whether S100A4 dissociates myosin fibrills! b) Describe pros and cons for each method in testing the hypothesis. c) Choose ONE of the methods, motivate your hcoice and propose an experimental setup to investigate the hypothesis. -­‐ What is required to perform the experiment? -­‐ How is the eperiment performed? -­‐ What results are expected? Schematic model: The Ca2+-loaded
S100A4 (yellow) binds to NMIIA at
the nonhelical tailpiece, partially
unwinds the ACD (blue) and
sterically blocks myosin-myosin
interactions causing filament
disassembly. Problem 8 (10p)
You have just successfully designed a fabulous therapeutic against cancer and as a result, you are now proceeding from small-­‐scale to large-­‐scale production. a) Choose five complementary methods by which you will analyze your product so that it complies with your deliverables. The methods you choose should have been discussed in the course Biomeasurement Technoloties. Describe for each methods in max one sentence what should be measured. b) Motivate each choice of method briefly, max three sentences per method. Congratulations!
You have now reached The End of this
examination!