MRes in Molecular Biophysics
Course Handbook 2014-2015
1
CONTENTS
Page Number
Short History of Biophysics at King’s College
Why study Biophysics
The course and method of assessment
Handing in Work
Introduction to the Information services and the library
Synopsis of important dates
Study of Classic papers in Biophysics and Presentation skills
Advanced Biophysical Techniques I (ABT I)
ABT I timetable
ABT I synopses
ABT I and ABT II Recommended texts
Course Practicals
Advanced Biophysical Techniques II (ABT II)
ABT II timetable
ABT II synopses
Molecular Biology Techniques and Biochemical Techniques Course
Map of Waterloo Campus
MRes Biophysics Projects
January Examination paper
Preparation and Submission of the Thesis
Guidelines for the Final Presentation
3
4
5-7
6
6
[8-18]
10-12
13-15
15-16
17-18
[19-24]
19-20
21-24
25-27
27
[26-43]
44-50
51-53
54
Cover picture (From Crystal to Structure) illustration prepared by Dr. Ivan Laponogov
The figure illustrates stages in the structure a type II topoisomerase-DNA-quinolone complex
using X-ray crystallography. In the Figure (Top left) is shown a crystal of complex, cryocooled
in a loop and mounted at the Diamond synchrotron ready for data collection, the direct X-ray
beam will strike the centre of the cross-hairs .
(Top right) one frame of the resulting X-ray diffraction pattern from a 0.1 degree oscillation of
the crystal.
(Bottom left) a section of the crystal lattice showing the molecular packing following solution
using MR (Molecular Replacement).
(Bottom right) the final structure solved structure. Solved using Molecular Replacement using
the Diamond synchrotron native X-ray data and refined using the PHENIX refinement package
written at Stanford University by Paul Adams et al.
Type II Topoisomerases as targets for rational drug design in order to improve current anti-bacterial
and anti-cancer therapies.
Ivan Laponogov, Xiao-Su Pan, Dennis A. Veselkov, Isabelle M-T.Crevel, Trishant Umrekar, L. Mark
Fisher and Mark R. Sanderson. IUCr Meeting, Montreal 2014 and EuroQSAR2014, St. Petersberg.
2
Short History of Molecular Biophysics at King’s College.
Molecular Biophysics started at King’s College in 1948. After the successful X-ray
crystallographic experimental work on the DNA structure by Rosalind Franklin, Maurice
Wilkins and Ray Gosling the MRC Biophysics Unit relocated from the Physics building in
the Strand (where Prof. J.T. Randall was director of the MRC Unit after being appointed
Wheatstone Professor of Physics in 1946) to Drury Lane. A long term lease was secured on
the premises at 26-29 Drury Lane, a former seed -warehouse from the Company of Mercers,
the property was then modified for laboratories and opened in November 1963.
Dr. David Dover coordinated the running of the very popular BSc. course for many
years through the 1970s and early 1980s until his retirement. This course produced many of
the world leaders in the field (testimonials of some of these may be found on our website
http://www.kcl.ac.uk/biohealth/research/divisions/randall/postgrad/alumni.aspx
King’s College Biophysics Dept. at Drury Lane was renamed the Randall Institute in 1989 in
honour of the first Professor Sir John T. Randall, who came to fame during the world war
together with Dr. Jessie Boot through the development of the cavity magnetron, which is the
essential component in Radar and produces the necessary pulses of microwave radiation.
Prof. Maurice Wilkins FRS was appointed the second head of the Biophysics Dept.
(1970-1981), upon Prof Sir John Randall’s retirement, followed by Professor Bob Simmons
FRS (1983-2001) and Professor Malcolm Irving FRS (2001-present) the current Director.
The Randall Institute relocated to the 3rd floor of New Hunt’s House on the Guy’s
Campus between 2000-2001 and has been renamed the Randall Division of Cell and
Molecular Biophysics.
Bibliography
The Double Helix, J.D. Watson, New American Library, 1969.
King’s College London 1828-1978, G. Huelin, King’s College publication 1978.
What Mad Pursuit, F. Crick, Penguin Books, 1990.
DNA Genesis of a Discovery, S. Chomet ed. , Newman-Hemisphere Press, 1995.
Encyclopedia of Genetics, Discovery of DNA, M.R. Sanderson, ed. S. Brenner and J.H.
Miller, Academic Press 2001.
The Dark Lady of DNA, B. Maddox, Harper-Collins, 2002.
The 3rd Man of the Double-Helix, M.H.F. Wilkins, OUP 2003.
DNA: The Secret of Life, J.D. Watson, Random House, 2004
Present at the Flood: How Structural Molecular Biology Came About, R.E. Dickerson,
Sinauer, 2005.
Francis Crick: Discoverer of the Genetic Code, M. Ridley, Harper-Collins, 2006.
Francis Crick: A Biography, R.C. Olby, Cold Spring Harbor Laboratory Press, 2009.
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Why study an MRes in Molecular Biophysics
In the post-genomic era now that the DNA sequence of the human genome and an
ever increasing number of genomes of eukaryotic, prokarotic and archael organisms are
know there is great interest, both in knowing the 3-Dimensional structure of the proteins
and RNA molecules encoded by the DNA and how they functionally interact within the cell.
Although High-throughput structural programmes using the powerful robotic based
structural techniques of X-ray crystallography and NMR are increasingly solving the
structures of easily expressed and crystallizable proteins (‘the low hanging fruit’), some of
the most interesting proteins are still proving to be elusive to express/crystallize and
difficult to solve by using direct robotic methods and will have to be in many cases solved
in complex with their cellular partners using imaginative approaches of expression,
crystallization and structure solution.
The next exciting area will be the furthering of our understanding of the temporal
and spatial expression of proteins and nucleic acids within the cell and knowing the
structures of the complexes formed, ultimately at high resolution. Examples of complexes
which have been very recently solved are those of the human spliceosomal U1 snRNP
complex at 5.5 Ǻ (Pomeranz-Krummel and co-workers, LMB, Cambridge), the structures of
the intact 70S ribosome in complex with tRNA, messager RNA and release factors (H.
Noller and co-workers, UC Santa Cruz), and the first structures of protein complexes
involved in chromosome segregation (Andrea Musacchio and co-workers, IFOM-IEO,
Milan). Membrane structure and that of embedded channels and receptors is also at the
forefront of research with the high resolution structure of a G-protein coupled receptor
GPCR being solved this year (Gebhard Schertler and co-workers, LMB Cambridge and now
at the Paul Scherrer Institute, Switzerland).
This course covers the techniques of X-ray crystallography, NMR (solution and solidstate), EM and other spectroscopy and microscopy techniques which when used in
combination, reveal at ever higher resolution the detailed working of eukaryotic and
prokaryotic cells. The pharmaceutical industry are increasingly using structural methods to
design better drugs on a highly rational basis, four examples of which are the directed
targeting of drugs to the prokaryotic ribosome (T.A. Steitz’ group, Yale and Melinta
pharmaceuticals) and in our own laboratories structure based drug design to molecules in
the immune system, muscle proteins, oxygenases, age-related proteins, the topoisomerases
of pathogenic organisms and new targets in HIV.
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The course
The taught part of the MRes course consists of 4 modules.
(1) Advanced Biophysical Techniques I [ABT I] (on the Guy’s Campus, Course
coordinator Dr. Roberto Steiner), which runs on a Monday, Henriette-Raphael Building
Room 4.16.
(2) Advanced Biophysical Techniques II [ABT II] (on the Guy’s Campus, Course
Coordinator Dr. Mark Sanderson) and runs on a Tuesday, room 3.16, seminar next to the lift
Randall floor NHH. The X-ray crystallographic and NMR lectures in these two modules
dove-tail so that ABT I covers introductory material and ABT II more advanced.
(3) Biochemical and Molecular Biological Techniques
(Course coordinators Dr. Nic Bury, Dr. Wolfgang Maret and Shirley Coomber), which runs
on Thursday and Friday at the Franklin-Wil building at Waterloo, laboratory 4.163 with a
9.30 start for Dr. Coomber’s part and a 10 am for Drs. Bury and Maret’s.
N.B. You have to meet the Course Coordinators in Waterloo Bridge Wing (WBW),
Waterloo Campus, lecture room G552 at 10 am on Thursday 25th September. There will
be a ½ hour introduction to the Course.
(4) Research project
A research project is selected from one of the projects listed at the end of this handbook
and commences in January and will be written up in July and marked in August.
Method of Assessment
The course is marked to a total of 280 credits made up of
(1)
7BBBM110 Principles of Advanced Biophysical Techniques
15 credits
(2)
7BBBM106 Advanced Bioscience Research Laboratory Techniques
30 credits
(3)
7BBBM111 Molecular Biophysics Research Project
135 credits
(1) The Principles of Advanced Biophysical Techniques module will be assessed by an
examination in January (15 credits).
(2) Advanced Bioscience Laboratory Techniques requires submitting the required in course
assessments which will be marked (30 credits).
(3) Research Project (135 credits).
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THE HANDING IN WORK, DEADLINES AND THE RETURN OF MARKED WORK
The Biophysics Practical write-ups have to be handed in within a week of doing the
practical and will be marked and returned by the faculty member one week afterwards.
Late work must to be accompanied by an Mitigating Circumstances form.
The practical write-ups for Advanced Bioscience Research Laboratory Techniques have to
be handed in as instructed by the module leaders for that course.
The deadline for handing in the project write-up is a hard deadline and write-ups
will not be accepted after this date without an Mitigating Circumstances form.
Introduction to the Information services and System (ISS)
Ms. Clare Crowley will give an introduction to the Library facilities offered by King’s
College as well as how to access electronic journals and other facilities such as databases and
endnote usage. It is essential that you attend his talk and on-line demonstration.
This will be held in the
New Hunt's House Training Room on Wednesday 1st of October 10 am-12 am
Please arrive promptly as this is an interactive session with you learning
how to navigate the system from your own allocated computer in this room.
Study of classic papers in Biophysics and Presentation skills
This part of the course will start on Wednesday the 24th of Sept at 10.00 and run through the
first semester. A list of papers will be given and you have to prepare one of these as a 25
minute power-point presentation on a subsequent Wednesday. There will be 2 presentations
per week. All the papers which are presented as power-points have to be read by all
students, not just those presenting.
First meeting Wednesday 24th of Sept 2014 Henriette Raphael classroom 2.24 at 10am
Second meeting when you decide which paper you are going to present – Wednesday
Oct 15th Hodgkin Classroom 11 at 10 am.
Presentation dates will be on Wednesday at 10 am on dates
12th of November 10am Classroom 3.17 Henriette Raphael (HR)
19th of November 10am Classroom 2.24 HR
26th of November 10am Hodgkin Classroom 4
3rd of December 10amHodgkin Classroom 5
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Assessment pattern - For Research project
Method
Unseen written
examinations
Assessed
coursework
(please specify ie
essay, project,
seminar work, lab
work)
Practical
examinations
Mandatory
to pass/
qualifying
mark
% of
final
grade of
module
15,000
50%
75%
~20min
50%
10%
25min
50%
15%
Number/
amount
Duratio
n/
length
Research
Report
Pass
Mark
(40 for
level 4, 5
and 6; 50
for level
7)
SI set up
if
different
Clinical
examinations
Oral examinations
Dissertation
Other (please
specify)
Other (where
attendance/
completion is a
requirement in
order to pass but
does not
contribute a mark
to the final grade)
Presentation
Programme Directors
Dr. Mark R. Sanderson,
Dr. Roberto Steiner.
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Synopsis of important dates on the Course
At the start of January 2014 -- There is an examination lasting 3 hours and requiring the
answering of 3 questions one from each section. The exact date is released later in semester 1
By the examination office. This examination will be held at the EXCEL centre.
Monday the 3rd of August 2015 – this is the Hand in date for your completed research
project thesis – Theses are handed in to the Randall Division Office on this date.
From 3rd of Aug till the end of August – you have to arrange with your supervisor and an
examiner appointed by your supervisor to have a Viva voce examination on your thesis
once they have had time to read it thoroughly. Please do not plan to go on holiday this
month without first fitting in this Viva date and having it (we have had problems with this
in the past)
At or around the 6th of Sept 2013– you will have to give a 25 minute presentation in front of
an audience on your research work for your thesis. The external examiner is also present.
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1st Module Advanced Biophysical Techniques I
This module is attended also by MRes students in Chemistry with Biomedicine and some 3rd
year Biomedical and medical students.
Location (site) of module: Henriette Raphael building HR 4.16, Guy’s Campus.
.
Objectives (knowledge and skills to be acquired) the objectives of the course are as
follows:






To investigate the structure of a range of biophysical techniques.
To understand the range of techniques which are used to obtain the structural and cellstructural information (X-ray diffraction, Electron Microscopy).
To understand the fundamentals of NMR and its use in protein structure determination.
To gain experience in growing crystals of a protein and recording their diffraction
patterns.
To gain expertise in analyzing NMR data.
To understand the working principle of research microscopes and fluorescence based
microscopy methods.
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Advanced Biophysical Techniques I
(Monday Morning, 1st semester)
Lectures from 10.00-12.30 with ½ hour break except on days when we have also course
work when the afternoon will be used.
Henriette Raphael Building HR 4.16
22nd Sept
1) 10.00 Introduction to the Course
X-ray crystallography 1
2) 11.30 X-ray crystallography 2
Dr. Roberto Steiner
Dr. Mark Sanderson
Dr. Mark Sanderson
29th Sept
3) 10.00 X-ray crystallography 3
4) 11.30 X-ray crystallography 4
Dr. Roberto Steiner
Dr. Roberto Steiner
6th Oct
5) 10.00 X-ray crystallography5
Dr. Roberto Steiner
6) 11.30 X-ray crystallography 6
Dr. Roberto Steiner
2.00pm Pract 1) Start of the X-ray crystallography practicals- divided into groups.
Dr. Roberto Steiner
13th Oct
7) 10.00 Nuclear Magnetic Resonance 1
8) 11.30 Nuclear Magnetic Resonance 2
Pract 1) 2.00pm X-ray crystallography practical
Prof. Jim McDonnell
Prof. Jim McDonnell
Dr. Roberto Steiner
20th Oct
9) 10.00 Nuclear Magnetic Resonance 3
10) 11.30Nuclear Magnetic Resonance 4
Pract 1) 2.00pm X-ray crystallography practical
Prof. Jim McDonnell
Prof. Jim McDonnell
Dr. Roberto Steiner
27th Oct
11) 10.00 Nuclear Magnetic Resonance 5
12) 11.30 Nuclear Magnetic Resonance 6
Pract 2) 2.00pm NMR practical (all groups)
Prof. Jim McDonnell
Prof. Jim McDonnell
Prof. Jim McDonnell
5th Nov No Lectures - Reading week for Undergraduates only, not for MRes students.
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10th Nov
13) 10.00 Electron Microscopy 1
14) 11.30 Electron Microscopy 2
Prof. Helen Saibil
Prof. Helen Saibil
17th Nov
15) 10.00 Introduction to Fluorescence
16) 11.30 Flourescence Labelling
Dr. Simon Ameer-Beg
Dr. Simon Ameer-Beg
24th Nov
17) 10.00 Fret/FILM and Fluorescence data processing
18) 11.30 Basics of Fluorescnce Microscopy
Pract 3) Data Handling
Dr. Simon Ameer-Beg
Dr. Susan Cox
Dr. Simon Ameer-Beg
1st Dec
19) 10.00 Light Detectors and Acquisition Parameters
20) 11.30 Multiphoton microscopy
Pract 4) 2.00 pm Microscopy practical
Dr. Susan Cox
Dr. Simon Ameer-Beg
Dr. Susan Cox
8th Dec
21)10.00 Advanced Microscopes
22) 11.30 High Resolution Microscopy
Pract 4) 2.00pm Microscopy practical
Dr. Susan Cox
Dr. Susan Cox
Dr. Susan Cox
12th Dec - 1st Semester ends
Outline of the module content.
The aim of the module is: (1) to provide students with an understanding of the fundamental
Principles of a range of advanced biophysical techniques; (2) to give an understanding of
how to use these techniques to solve the structure of macromolecules and macromolecular
complexes at high resolution together with studying the dynamics of cellular processes. The
techniques covered include: X-ray crystallography, cryo-Electron Microscopy, Nuclear
Magnetic Resonance, fluorescence spectroscopy and advanced microscopy.
Workload, approximate no. of hours spent by the student in:
Lectures
22
Practicals
4
11
Methods of assessment.
The module work for Advanced Biophysical Techniques I consists
of a
1) a Crystallography Practical 2) A demonstration of an NMR
spectrometer and a Data handling exercise discussing their
approach 3) A practical on adjusting and operating widefield and
confocal microscopes 4) A Data handling exercise on fluorescence.
Each practical is written up and handed in. The write-ups are
assessed and the marks count to course total for MRes.
Course Organisers
Dr. Roberto Steiner and Dr. Mark Sanderson,
The Randall Division of Cell and Molecular Biophysics,
New Hunt’s House,
Guy’s Campus
London Bridge
London SE1 1UL
Tel. 0207-848 8216 and 0207-848 6403
Contributors to the course
Dr. Simon Ameer-Beg,
Dr. Andrew Beavil,
Dr. Yu-Wai Chen,
Dr. Sasi Conte,
Dr. Susan Cox,
Dr. Sergi Garcia-Manyes,
Prof. Jim McDonnell,
Dr. Franca Fraternali,
Prof. Mathias Gautel,
Prof. Hannah Gould,
Dr. Mark Sanderson,
Dr. Roberto Steiner,
Prof. Brian Sutton, all of the Randall Division of Cell and Molecular Biophysics, King’s
College London.
Dr. David Barlow,
Dr. Alex Drake,
Dr. Lindsay McDermott,
Dr. James Mason,, all of the Division of Pharmaceutical Sciences, King’s College London.
Prof. Helen Saibil FRS, Dept. of Crystallography, Birkbeck College, Malet St., London.
Dr. Brendan Orner,
Dr. Edina Rosta,
Dr. Riki Eggert, all of the Division of Chemistry, King’s College, Hodgkin Building, Guy’s
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Lecture Synopsis Advanced Biophysical Techniques I
There is no one text book which covers all the material in the course. Suggested texts are
given below.
Start of Lecture 1) Introduction to the course (Dr. Roberto Steiner)
Lecture 1) X-ray crystallography 1 (Dr. Mark Sanderson)
Generation of X-ray radiation Data collection using In-house ‘rotating anode’ and Synchrotron
sources. Macromolecular crystallization.
Lecture 2) X-ray crystallography 2 (Dr. Mark Sanderson)
Discussion of symmetry, point-groups and space groups. Techniques for obtaining the image of a
macromolecule. Diffraction of X-rays by the crystal lattice. Analogy with the diffraction of visible
light. The Laue Equations. Braggs Law. Fourier syntheses. Electron density as a Fourier series.
Lecture 3) X-ray crystallography 3 (Dr. Roberto Steiner)
The phase problem. Classical methods for obtaining phases. Heavy atom derivatisation.
Isomorphous replacement techniques, Patterson method of locating heavy atoms.
Lecture 4) X-ray crystallography 4 (Dr. Roberto Steiner)
Incorporating the derivative directly into the protein. Selenomethionine incorporation and the use
anomalous dispersion for obtaining phase information.
Lecture 5) X-ray crystallography 5 (Dr. Roberto Steiner)
Molecular replacement technique.
Lecture 6) X-ray crystallography 6 (Dr. Roberto Steiner)
Macromolecular refinement. Electron density maps. Structure validation.
Lecture 7) Nuclear Magnetic Resonance 1 (Prof. Jim McDonnell)
General spectroscopy and electromagnetic radiation
Nuclear spin phenomenon and basic spin physics; Free induction decay and the Fourier transform
Lecture 8) Nuclear Magnetic Resonance 2 (Prof. Jim McDonnell)
Basic NMR concepts and a simple NMR experiment
Chemical shift; Relaxation; Other basic NMR concepts; A simple NMR experiment; NMR spectra of
simple chemical compounds; The NMR spectrometer
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Lecture 9) Nuclear Magnetic Resonance 3 (Prof. Jim McDonnell)
Information available from NMR experiments
NMR experiments (COSY, NOESY, exchange phenomena, HSQC, two-dimensional NMR);
Coupling constants and the Karplus relationship; Information from an NMR experiment and
different types of NMR experiments
Lecture 10) Nuclear Magnetic Resonance 4 (Prof. Jim McDonnell)
Solution state NMR in structural biology
Peptide and protein NMR spectroscopy; The assignment problem
Lecture 11) Nuclear Magnetic Resonance 5 (Prof. Jim McDonnell)
High-resolution structure determination
Structural constraints and structure calculations
Lecture 12) Nuclear Magnetic Resonance 6 (Prof. Jim McDonnell)
State-of-the-art of NMR in structural biology
Triple-resonance and multi-dimensional NMR spectroscopy; TROSY experiments; Residual dipolar
couplings; Hydrogen bond measurements
Lecture 13) Electron Microscopy 1 (Prof. Helen Saibil)
Introduction to Cryo Electron Microscopy and image reconstruction.
Lecture 14) Electron Microscopy 2 (Prof. Helen Saibil)
Cryo Electron Microscopy and application to GroEL structure solution.
Lecture 15) Introduction to Fluorescence (Dr. Simon Ameer-Beg)
Principles of fluorescence spectroscopy in solution. Photophysical properties of molecules,
properties of fluorescence, fluorescence in biology, measurement techniques.
Lecture 16) Basics of Fluorescence Microscopy (Dr. Susan Cox)
Introduction to Microscopy: Widefield, Confocal, Aberrations, Transfer Functions, the Abbe limit,
the Missing Cone Problem.
Lecture 17) Fluorescence Labelling Techniques (Dr. Simon Ameer-Beg)
Fluorescence Labelling Techniques: Fluorescent proteins, intercalating Dyes, antibody
labelling, QDots, Autofluorescence.
Lecture 18) FRET/FLIM and Fluorescence Data Handling (Dr. Simon Ameer-Beg)
FRET/FLIM and measuring distances. Theoretical basis of resonant energy transfer, practical
implementation in biology, using FRET to measure intermolecule distances on the nanometer
scale.
Lecture 19) Light Detectors and Acquisition parameters (Dr. Susan Cox)
Detection: PMTs, APDs, CCDs. Optimum sampling for different applications, deconvolution.
14
Lecture 20) Multiphoton microscopy (Dr. Simon Ameer-Beg)
Multiphoton Microscopy, SHG, THG, adaptive optics and wavefront correction.
Lecture 21) Advanced Microscopes (Dr. Susan Cox)
Advanced Microscopes: Apotome, programmable array microscope, Spinning Disk
Lecture 22) High resolution microscopy (Dr. Susan Cox)
Recent developments: STED, linear and non-linear strutured illumination, and localisation methods
(PALM and STORM).
Recommended texts
General Molecular Biophysics texts
Methods in Molecular Biophysics, Serdyuk, Zaccai and Zaccai, CUP.
Biophysical Techniques, I.D. Cmpbell, OUP.
Principles of Physical Biochemistry, K.E. van Holde, C. Johnson and P.S. Ho, Prentice
Hall.
Physical Biology of the Cell, R. Phillips, J. Kondev, J. Theriot, Garland Press.
X-ray diffraction
Outline of Crystallography for Biologists, D. Blow, OUP.
Crystallography made crystal clear. G. Rhodes, Academic Press.
Conventional and High-throughput macromolecular crystallography,
Eds M.R. Sanderson and J.V.Skelly, OUP 2007
Principles of Physical Biochemistry, K.E. van Holde, C. Johnson and P.S. Ho, Prentice
Hall.
Biomolecular Crystallography: Principles, Practice, and Application to Structural
Biology, Bernard Rupp, Garland Press, 2009.
Crystals, X-rays and Proteins; Comprehensive Protein Crystallography
D. Sherwood and J.Cooper, O.U.P. due out in November 2010.
15
NMR
NMR Spectroscopy: Basic Principles, Concepts, and Applications in Chemistry, H.
Gunther, Wiley.
NMR a physicochemical view, R.K. Harris, Longman.
Magnetic Resonance of Biomolecules, P.F. Knowles, D. Marsh and H.W.E. Rattle, John
Wiley.
NMR of proteins and nucleic acids, K. Wuthrich, Academic Press.
Protein NMR Spectroscopy. Principles and Practice. W. Fairbrother et al., 2nd edition,
Academic Press.
Understanding NMR Spectroscopy J. Keeler, Wiley.
Also James Keeler on Youtube – Lectures on NMR for ANZMAG, Univ of Queensland.
Excellent and goes beyond level of this course, follows closely his book.
Introduction to solid-state NMR spectroscopy, M.J. Duer, Wiley-Blackwell.
Microscopy Optical Spectroscopy
Handbook of Biological Confocal Microscopy, J.B. Pawley (editor), 3rd edition, Springer
Verlag
Confocal Microscopy for Biologists, Alan R. Hibbs, Kluwer Academic/Plenum
Publishers, 2004
Microscopy, Optical Spectroscopy and Macroscopic Techniques, A.F. Drake : Methods
in Molecular Biology, 1994, Vol 22 : (Edited by C. Jones, B. Mulloy and A. H. Thomas).
Spectroscopic Methods and Analyses: NMR, Mass Spectrometry, and Metalloprotein
Techniques, Methods in Molecular Biology, 1993, Vol 17 : (Edited by C. Jones, Barbara
Mulloy, Adrian H. Thomas
Membrane Biophysics
Membrane Structural Biology, M. Luckey, Cambridge University Press
Useful Web sites
On Fourier transforms
http://www.ysbl.york.ac.uk/~cowtan/fourier/fourier.html
On Macromolecular Crystallography
Randy Read’s
http://www-structmed.cimr.cam.ac.uk/course.html
Microscopy Basics with Interactive Diagrams
http://micro.magnet.fsu.edu/primer/
http://www.microscopyu.com
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COURSE PRACTICALS
1) Growing protein crystals and recording their diffraction patterns
Introduction
In this practical you will be using two pieces of equipment currently employed in
High-throughput X-ray crystallography, namely a X-ray crystallisation robot, known
by its trade-name, Mosquito, and a scanner to acquire the X-ray diffraction
patterns from a crystal within the crystallisation plate, known as a Oxford Diffraction
PX scanner.
a)Protein crystallisation
Summary
In this practical you will grow lysozyme crystals using a crystallisation robot and then view
their X-ray diffraction patterns using an Oxford diffraction plate scanner. You have been
provided with highly purified solutions of the proteins to be crystallised and precipitant
solutions required for their crystallisation.
Materials
Protein solution
Precipitant solutions
20 mg/ml lysozyme
10% sodium chloride, 20 mM sodium acetate,
pH, 4.7 and other precipitants to be screened.
Procedure
To be handed out at the practical
2) NMR Practical
Site visit to NMR spectrometer within the Spectroscopy Centre at Guy’s Campus. Basic
explanation of instrument components and demonstration of experiment acquisition on a
protein sample (including shimming, tuning, 1D proton NMR, 2D 1H-15N HSQC).
17
Data Handling Excercise
Interpretation and prediction of 2D/3D COSY and NOESY NMR spectra of aminoacids and
proteins
3) Microscopy Practical
In this practical you will carry out sample preparation, and then carry out imaging on a
confocal microscope, widefield microscope, and programmable array microscope. Basic
acquisition parameters will be optimised. Basic image processing will be carried out on the
acquired images.
4) Fluorescence Data handling exercise
Calculation of Förster radius from absorption and emission spectra of known fluorophores.
Interpretation of fluorescence lifetime images and associated data.
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2nd Module Advanced Biophysical Techniques II
Advanced Biophysical Techniques II for MRes students
This module will run on a Tuesday in the first semester (A)
Lectures to be held in Room 3.16 Randall Floor, 3rd Floor NHH unless otherwise indicated
23rd Sept
10.00-11.00
11.30-12.30
Prof. Brian Sutton
Introduction to Protein Structure
Protein Folding
(1.)
(2.)
30th Sept
10.00-11.00
11.30-12.30
Prof. Brian Sutton
Protein Mis-folding and Thermodynamics
Protein Dynamics
(3.)
(4.)
7th Oct
10.00-11.00
Dr. Andrew Beavil
Methods of measuring protein-protein affinities
(5.)
(Biacore and other methods)
Physical methods for determining size,shape and
(6.)
molar mass of macromolecules (size exclusion chromatography,
Ultracentrifugation, dynamic light scattering)
11.30-12.30
14th Oct
10.00-11.00
11.30-12.30
16.30-18.00
Dr. Arianna Fornili
Bioinformatics
Bioinformatics
Hands on Computer workshop in Dr. Fornili’s Offices,
Randall Div.
21st Oct
10.00-11.00
15.30-16.30
Dr. Sergi Garcia-Manyes
Optical Trapping
Optical Trapping
(9.)
(10.)
14.00-15.00
Prof. Mathias Gautel
Atomic Force Microscopy
(11.)
28th Oct
10.30-11.30
12.00-13.00
14.30-15.30
Dr. Alex Drake (please note later start at 10.30)
Optical Spectroscopy
Circular Dichroism I
Circular Dichroism II (G12 NHH for this lecture only)
(12.)
(13.)
(14.)
(7.)
(8.)
19
4th Nov
10.00-11.00
11.30-12.30
14.00-15.00
15.30-16.30
11th
Dr. Mark Sanderson
X-ray crystallography (7) Robotic crystallisation techniques
X-ray crystallography (11) Phasing (advanced)
Dr. James Mason
Biophysics of Membranes
Biophysics of Membranes
(15.)
(16.)
(17.)
(18.)
Nov
Roberto Steiner
X-ray crystallographic
Phase improvement techniques
X-ray crystallography Model Building
X-ray crystallographic Refinement
(19.)
(20.)
(21.)
10.00-11.00
11.30-12.30
Dr. Sasi Conte
Nuclear magnetic resonance (7) Relaxation
Nuclear magnetic resonance (8) Advance 2Dspectroscopy
(22.)
(23.)
14.00-17.00
Mark Sanderson
X-ray crystallographic workshop with laptops MRes Office
10.00-11.00
11.30-12.30
14.00-15.00
18th Nov
25th Nov
10.00-11.00
11.30-12.30
Dr. Sasi Conte
Nuclear magnetic resonance (9) Sequential assignment
Nuclear magnetic resonance (10) Structure determination
(24.)
(25.)
14.00-15.00
15.30-16.30
Dr. James Mason
Biophysics of Membranes Solid-state NMR I
Biophysics of Membranes Solid-state NMR II
(26.)
(27.)
2nd
Dec
10.00-11.00
11.30-12.30
14.00-16.30
Dr. Sasi Conte
Nuclear magnetic resonance (11) Homo and hetero decoupling (28.)
Nuclear magnetic resonance (12) Chemical shift mapping
(29.)
NMR tutorial or workshop
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Lecture Synopsis Advanced Biophysical Techniques II
Professor Brian Sutton’s Lectures
Lecture 1
Introduction to Protein Structure
The basic concepts of secondary, super-secondary, tertiary and quaternary
structure of proteins, and the forces that stabilise these structures.
Lecture 2
Protein Folding
How do proteins fold? The process of protein folding and discussion of
recent experimental and molecular dynamics approaches to determine folding
pathways.
Lecture 3
Protein Mis-folding and Thermodynamics
The structural basis for protein mis-folding, the thermodynamics of protein
folding, and a quantitative analysis of the contribution of the various noncovalent forces that determine the folded structure.
Lecture 4
Protein Dynamics
Is mobility essential for protein function? A description of the various types of
dynamics within a protein structure, their different time-scales and the
methods available for their experimental study.
Dr. Andrew Beavil’s Lecture/Tutorials
Lecture/
tutorial
5 and
6
A simple introduction to the principles and applications of
biophysical techniques for the study of the size, shape and interactions of
proteins. AUC, static and dynamic light scattering and assays of
protein-protein interactions such as TR-FRET and SPR (BIAcore) will be
covered.
Dr. Arianna Fornili ’s lectures and tutorial
Lecture 7
and 8
Protein Structure Prediction
Protein Structure Prediction. Conformational preferences of aminoacids.
Secondary structure prediction. Tertiary structure prediction: Comparative
Modelling, Ab-initio Modelling. Refinement: Energy Minimization and Force
Fields.
Tutorial:
Modelling of a protein and Evaluation of the model
Dr. Sergi Garcia-Manyes’ lecture
Lecture 9 Principles of Optical and Magnetic traps and their application to interesting
Lecture 10 Biological problems.
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Prof. Mathias Gautel’s lecture
Lecture 11
An introduction to atomic force microscopy and its general physical
principles, the design of the instruments and the qualities of the
cantilever probes. The application for nanoscale imaging, cellular
imaging, and nanoforce spectroscopy for assaying protein conformation
will be discussed using pioneering examples.
Dr. Alex Drake’s lectures
Lecture 12
Optical Spectroscopy
The electromagnetic spectrum & energy levels, Beer’s law, the transition
electric dipole moment & the extinction coefficient, the determination of
concentration, chromophores.
Lecture 13
Circular Dichroism I
Linear & Circular Polarization. The definition of optical activity (optical
rotation and circular dichroism). The CD spectrometer.
Lecture 14
Circular Dichroism II
The molecular origin of CD (transition electric and magnetic dipole moments)
and its application to monitoring protein & nucleic acid conformation and
interactions.
Dr. Mark Sanderson’s lectures
Lecture 15
Robotic crystallization techniques
Current state of the art High throughput (HT) crystallization techniques will
be discussed. The mode of action, advantages and disadvantages of the
current line of commercially available robots will be discussed. Robots which
use vapour diffusion crystallisation- the ‘Mosquito’ from TTP labtech Ltd., the
‘Cartesian/ Honey bee’ from Digilab Ltd., Phoenix, Rigaku Ltd. Robot which
uses batch crystallization under oil- Douglas Instruments Ltd. Crystallization
using chips as in the Fluidigm microfuidics Topaz system.
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Lectures 16 X-ray phasing Phasing (Advanced)
Discussion of the software which is used in practice to solve protein structures
by Multiple Anomalous Dispersion. Determination of the Selenium sites either
by Patterson solution or by using Direct methods. The different
software packages used to solve for the position of the Selenium sites and
subsequent phasing using multiple wave-length data will be discussed- Solve
written by Tom Terwilliger, ShelxD written by George Sheldrick. Use of CCP4
for site determination and phasing multiple wavelength anomalous data.
Dr. James Mason’s lectures
Lecture 17
Probing structure in Biological membranes
Structure and composition of biological membranes – phospholipids – sterols –
differences between eukaryotic and prokaryotic membranes – Gram positive
and Gram negative bacteria – phase transitions, what they are and how to
measure them – importance of membrane proteins – available structures structural motifs – problems with obtaining membrane protein structures –
strengths, weaknesses and adaptations of main structural techniques – x-ray –
electron diffraction – NMR?
Lecture 18
Bacteriorhodopsin – a case study
Function – photocycle – optical spectroscopy – 2D crystals – neutron
diffraction – electron diffraction structure – retinal conformation (2H ss-NMR)
– retinal structure (13C ss-NMR) - early models and comparison with
rhodopsin – hydropathy plots - crystallisation strategies – structures –
photostate intermediates – loop structure and dynamics (13C/15N ss-NMR) –
shunt states and DNP
Lecture 28
Solid state NMR I
Solid-state NMR in biology?- anisotropy – anisotropic interactions – using
anisotropy – removing anisotropic interactions – obtaining structure from
solid-state NMR – CP MAS techniques – oriented sample techniques – MAOSS
Lecture 29
Solid state NMR II
Understanding function and dynamics using solid-state NMR – membrane
packing – peptide binding and (dis)ordering – molecular voltmeters –
membrane depth probes – membrane protein backbone dynamics – inter
molecular distances – drug binding
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Dr. Roberto Steiner’s lectures
Lecture 19
Non-crystallographic symmetry & density modification. Solvent masks; Noncrystallographic symmetry (NCS); Solvent flattening; Histogram matching; NCS
averaging.
Lecture 20
Map interpretation and different types of maps; Secondary-structure elements;
Skeletonization, Side-chain assignement; Atomization and automated tracing.
Lecture 21
Restrained refinement and restraints; Least-squares and maximum likelihood
targets; TLS parametrization; Validation.
Dr. Sasi Conte’s lectures
Lecture 22
Detailed analysis of magnetisation and pulse lengths; relaxation: measurements of
T1 by inversion recovery and T2 by spin-echo; NOE: mechanisms and effects for
different correlation times.
Lecture 23
2D spectroscopy: general mechanisms. COSY, TOCSY AND NOESY. Example and
applications for amino acids and peptides.
Lecture 24
General uses of NMR. Amino acids chemical shifts. Principles of resonance
assignment (sequential and side chains) and NOE determination. 3D spectroscopy.
Isotope labelling of proteins.
Lecture 25
Structural determination of proteins - details of individual steps. Principles of
structure calculation using NMR restraints – evaluation of the results.
Lecture 28
The laboratory and the rotating frame. Coupling constants and their behaviour in
the rotating frame. Spin echoes and homonuclear decoupling. Spin echoes and
heteronuclear decoupling.
Lecture 29
More Biomolecular applications: mapping interactions with NMR (chemical shift
analysis). Relaxation and structure. Conformational equilibria.
24
3rd Module
Biochemical Techniques and Molecular Biology Techniques 7BBBM106
Timetabled on Thursday and Fridays starting Thursday 25th September. Please go to
Waterloo Bridge Wing Room G552 at 10am for the introduction to the course. Please be
prompt [MAP on page 27 and a map is also on the web]
Protein Biochemistry practical
Dr. Nic Bury and
Dr. Wolfgang Maret
Starts 25th September and runs until 24th October (no break for a reading-week like
undergraduates)
Please refer to Dr. Nic Bury and Wolfgang Maret’s handbook for the practicals.
Molecular Biology Techniques course
Dr. Shirley Coomber
Please refer to he handbook for the practicals.
Starts 30th of October and ends 28th of November
The aims of these modules are:
 To develop the scientific and practical skills of the participating students.
 To develop the students’ abilities to follow written instructions and carry out
advanced experiments in biochemistry, molecular genetics and molecular biology
and cell biology areas, and to describe, interpret and effectively present results and
written laboratory reports.
 To train students to trouble-shoot, analyse results when experiments fail, and design
investigative steps to determine sources of problems, and possible solutions to them.
 To train students to plan discussion for results and apply basic stats to data and
express error levels.
 To train students in advanced biomedical techniques, required by typical biosciences
researchers, and turn participants into highly competent laboratory workers.
25
By the end of the course students should be able to:
 Understand and follow written experimental instructions, identify key issues &
prepare summaries for multi-step experimental procedures.
 Carry out advanced biochemistry and molecular biology experiments, analyse,
present, and interpret results.
 Safely use instruments found in typical bioscience laboratories.
 Apply confidence criteria to their results including reproducibility and accuracy
estimates.
 Prepare experimental reports of laboratory experiments.
 Prepare discussion material and be prepared to relate results to published material.
 Describe the outcome of work done and identify the key elements in a typical
procedure.
 Collect data and apply statistical analysis methods.
 Identify safety issues in a laboratory environment.
Assessment
For Protein Biochemistry you will be asked to write short 1-page reports on the experiments
preformed and these will account for 20% of the overall mark. The further 80% is assigned
to a longer, 3000 word ( 10%), written report in the format of a scientific paper. The
Molecular Biology assessment is split into 3 reports written during the course. Further
details on these assessments will be provided by the academics running the courses.
26
Map of the Waterloo Campus showing the Franklin-Wilkins Building and the Waterloo
Bridge Wing.
27
MRes Molecular Biophysics Projects and supervisors
(Dr. Simon Ameer-Beg)
High Speed Multifocal Multiphoton Fluorescence Lifetime Imaging
Multiphoton microscopy is a three dimensional imaging technique which offers a number of
key advantages over confocal microscopy for imaging at depth in live biological samples. In
particular, the use of near-infrared excitation enables penetration to depths of ~1 mm with
limited phototoxicity and photobleaching. Multiphoton microscopy is easily adapted to
provide fluorescence lifetime data which enables intensity independent measurement of
protein-protein interaction and/or environmental parameters. The project will involve using a
state of the art high speed FLIM system to measure protein-protein interactions at the
cellular interface in live cells. The student will have the opportunity to develop new imaging
techniques to localise complexes in the ErbB family of proteins as they are activated by
various ligands. In addition, the student will correlate these functional data with single
molecule tracking to help interpret and support the ensemble imaging.
(Dr. David Barlow)
Development of a method for amino acid sequence-based prediction of residues
comprising a protein’s hydrophobic core.
Although genomics projects have provided us with the amino acid sequences for
tens of millions of different proteins, the 3D structures are known for fewer than 70,000
of these. There are significant efforts being devoted, therefore, to devising computerbased methods for predicting protein 3D structures using just their amino acid sequences.
Most of the methods developed to date have focused on the prediction of secondary
structures (that is, which residues form alpha-helices, beta-sheets, etc.). There have also
been attempts made to predict which residues in a protein are likely to be buried and
which are likely to be exposed (to solvent). There have no methods to date that have
been devised for predicting the residues that comprise a protein’s central hydrophobic
core.
In this project, the aim will be to development a method for amino acid sequencebased prediction of residues comprising a protein’s hydrophobic core.
Various in-house and commercial molecular modeling programs will be used to
analyse a set of high resolution protein crystal structures (a) to identify those residues
that are buried deep within the interiors of the molecules, (b) to study their local
environments and sequences, and (c) to determine patterns that might be used to predict
the involvement of these residues in the proteins’ hydrophobic cores.
Other Student Requirements: The project will involve extensive use of a PC – running
various in-house and commercial molecular modeling programs – but will NOT involve
computer programming. Some straightforward statistical analyses will also be
performed using MS Excel.
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(Dr. Andrew Beavil and Prof. Brian Sutton)
Engineering the IgE receptor CD23 to examine the role of Calcium
Brief background to project: The Immunoglobulin E receptor CD23/FcεRII is a pleiotropic
cell surface receptor found on B cells where it has roles including the regulation of IgE
synthesis1 and enhanced antigen presentation, which leads to epitope spreading. CD23
has an unusual structure for antibody receptor, because it is one of only a few that are not
members of the immunoglobulin gene super-family; it is instead a C-type Lectin. Other
members of the C-type Lectin superfamily typically bind two calcium ions and use one of
them to bind carbohydrates. Human CD23 (but not mouse or rat) seems to have lost the
ability to bind the calcium ion involved in carbohydrate interactions and has no clear need
for carbohydrate in binding to IgE. It seems from our crystal structures2-5 of CD23 (±
calcium and IgE) that the knock-on effect of the modified calcium ion-binding site may be an
enhancement of IgE binding.
This is an idea that we want to test directly by restoring the missing calcium binding site in
human CD23 and testing the effect on IgE binding. This may help us to understand some
of the many inter-species differences in IgE biology and help us develop CD23/IgE blocking
molecules for the treatment of allergy.
Project aims/objectives: This project will undertake a programme to engineer the Calcium
and IgE binding sites of CD23 using knowledge of the protein interaction interfaces recently
gained from our crystal structures of CD23 and its complex with IgE. We will generate a
panel of unique CD23 mutants with calcium binding restored to the canonical calcium
binding site 2 as well as some mutants that are expected to influence a polypeptide loop
within the CD23 binding interface for IgE. We will examine the effect of these mutations on
the binding stoichiometry and affinity of CD23 for Calcium and IgE. If time is available, we
will aim to solve the structures of the mutant proteins.
Techniques/methods involved: The project will involve extensive molecular biology and
protein production followed by characterisation of the mutant proteins in in vitro BIAcore
kinetic protein binding assays and Calcium binding by isothermal titration calorimetry (ITC).
Crystallisation of the mutant proteins in complex with IgE-Fc and structure determination by
X-ray crystallography using protocols that are well established within the group are a
realistic option if time allows as are MD simulations of the mutant proteins.
Relevant literature:
1) Cooper AM et al. Soluble CD23 controls IgE synthesis and homeostasis in human B
cells. J Immunol. 2012 188(7):3199-207. PMID: 22393152
2) Borthakur S, et al. Mapping of the CD23 binding site on immunoglobulin E (IgE) and
allosteric control of the IgE-FcεRI interaction. J Biol Chem. 2012 287(37):31457-61. PMID:
22815482
3) Dhaliwal B et al. Crystal structure of IgE bound to its B-cell receptor CD23 reveals a
mechanism of reciprocal allosteric inhibition with high affinity receptor FcεRI. Proc Natl Acad
Sci USA. 2012 109(31):12686-91. PMID:22802656
4) Dhaliwal B, Pang MO, Yuan D, Yahya N, Fabiane SM, McDonnell JM, Gould HJ, Beavil
AJ, Sutton BJ. Conformational plasticity at the IgE-binding site of the B-cell receptor CD23.
Mol Immunol. 2013 Dec;56(4):693-7. PMID: 23933509
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(Dr. Sasi Conte)
Structure-function investigations of the La-related proteins (LARPs)
Protein-RNA interactions control the fate of mRNAs in the cell and therefore regulate protein
synthesis at the translational level. It might be possible to say that almost no area of biology
or biomedicine will remain untouched by the science emanating from understanding the
regulation of gene expression at the level of mRNA translation. Nonetheless, the number of
protein-RNA complexes deposited in the protein data bank (PDB) remains scarce, making it
impossible to decipher a recognition code for RNA-protein interaction. Accordingly,
computational prediction of RNA binding sites for proteins remains unachievable.
An interesting example RNA binding proteins involved in regulation of gene expression has
emerged recently with the identification of the superfamily of La-related proteins (LARPs).
LARPs have only begun to be characterised, but it is already clear that they are involved in
a wide variety of essential cellular processes including muscle differentiation, development,
cell motility and cytoskeletal remodelling. LARPs also play prominent roles in many
diseases: mutations can lead to autoimmunity, muscle and fibroproliferative disorders,
cancer and viral infections.
The common feature of LARPs is the RNA binding unit called the ‘La module’, formed by
pairing a La motif and an RNA recognition motif, which was first characterised in our work
on the La protein, the archetypal LARP. Despite the high sequence conservation in the RNA
binding domain, surprisingly LARPs recognise very different RNA targets in order to perform
their different function, but the molecular basis of such RNA recognition remains elusive.
The aim of the project to investigate LARP structures and mode of interactions to RNA
targets in order to: (i) drive forward the studies of their cellular function; (ii) clarify their role
in diseases; (iii) contribute to the understanding of the essence of protein-RNA binding
specificity. This work will be conducted in parallel with groups investigating the cellular
functions of LARPs.
For more information on the Conte laboratory, see:
http://www.kcl.ac.uk/lms/research/divisions/randall/research/sections/structural/conte/index.
aspx
Key techniques / transferable skills
Protein expression and purification, molecular biology, biophysical techniques (ITC, NMR,
CD, MST, X-ray, UV), biochemistry.
30
(Dr. Sarah Barry)
Investigation of protein – protein interactions in the enzymatic assembly lines of natural
product biosynthesis
Bacteria produce a diverse range of small molecules known as natural poducts. Many of
these compounds are clinically useful as for example antibiotics (erythromycin) and anticancer agents (bleomycin) (Fig 1).
Due to their therapeutic importance we are interested in investigating the metabolic
pathways in bacteria responsible for producing these molecules, with the ultimate goal of
genetically engineering the pathways to produce novel bioactive compounds.
One of the problems associated with the genetic engineering of such systems is that the
enzymes involved in the pathways interact with each other creating a linear assembly line
system in which building blocks are added sequentially to create the desired product. In
some cases an individual enzyme must interact with up to five other proteins in a defined
order and at the correct time. If we wish to manipulate such systems we must first
understand the nature of these protein-protein interactions and how they avoid non-specific
interactions.
In this project the student will use PCR to amplify bacterial genes involved in the
biosynthesis of a natural product. The genes will be cloned and overexpressed in E. coli
and the resulting proteins will be purified using affinity chromatography and characterised.
The proteins will then be investigated to determine if and how they interact with each other
using biophysical techniques such as isothermal calorimetry; CD spectroscopy and protein
NMR.
31
(Dr. Susan Cox)
Correlative super-resolution fluorescence microscopy and atomic force microscopy of
cardiac myocytes
The structure and function of the heart centres around force; therefore, it is no surprise that
applied force can influence the structure and development of cardiac myocytes. It is
therefore critical to understand the mechanical properties of the sarcomere, the cytoskeletal
structure that allows the heart to beat. In this project different proteins in the sarcomere will
be imaged using super-resolution microscopy, achieved either through image processing or
by altering the optical configuration of a widefield microscope. Simultaneously, these cells,
which will have been stripped of their outer membrane using Triton, will be probed to
determine how the mechanical properties of the sarcomere vary across the structure.
(Dr. Carmen Domene)
Computational investigation of anaesthetic binding sites on cell membranes
Successful anaesthesia relies on achieving the appropriate time course and quality of
sedation during surgery. Despite the importance of this clinical procedure, the physiological
mechanism of anaesthesia is not well understood, although it is known that anaesthetics
partition into cell membranes and affect current flow through neurological ion channels. At
present, there is no systematic structural information about the effect of different
anaesthetics in membranes. Hundreds of substances with chemically unrelated structures
have been found to possess anaesthetic activity, which suggests that a nonspecific binding
mechanism to either the membrane or the proteins is responsible, or possibly that several
mechanisms can give rise to the same biological effect. Computational modelling and
molecular dynamics simulations are ideal tools to apply to this problem as they can reveal
both the location and the effect of anaesthetics in membranes, and complement the
available structural, spectroscopic and electrochemical data. Here, we will investigate the
effects of representative general anaesthetics on the structure of lipid bilayers of varying
composition with and without membrane proteins to determine how the chemical structure
of anaesthetics and their location relate to how they modulate membrane structure and
function.
32
(Dr. Ulrike Eggert)
Evaluation of small molecules and RNAi treatments on Rho signalling using FRET
Many mechanisms underlying cytokinesis, the final step in cell division, remain poorly
understood. The overall goal of our laboratory is to use chemical biology approaches to
address unanswered mechanistic questions by studying cytokinesis at the process, pathway
and protein levels. It has been challenging to study cytokinesis by traditional methods
because it is a rapid and dynamic process that occupies only a small portion of the cell
cycle. New approaches are needed to overcome these barriers to deeper understanding,
one of which is to develop new tools. We recently discovered several small molecule
inhibitors that target a key regulator of cytokinesis, the Rho pathway (Castoreno et al.,
Nature Chemical Biology, 2010) and used a genome-wide RNAi screen to identify new
proteins connected to Rho signalling.
This MRes project will focus on developing a FRET-based imaging assay to study if our
newly discovered small molecules and Rho pathway proteins directly affect Rho GTPase
activation. We will use a FRET biosensor that has been developed to report on localized
Rho activity. We will transfect cells with this sensor and will analyse Rho activity at the
cleavage furrow during cytokinesis and at other cellular structures that are controlled by Rho
signalling.
(Rivka Isaacson)
Biophysical Studies of SGTA
Small, glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA) is a human
cochaperone with roles in cancer, polycystic ovary syndrome and infection by viruses such
as HIV and SARS. Our lab has recently solved the structure of the SGTA N-terminal domain
(see figure and Simon et al. PNAS 2013) in the context of its role in tail-anchored
membrane protein insertion. This project will
investigate full-length SGTA and its interactions
on a structural level using a combination of
NMR, X-ray crystallography, isothermal titration
calorimetry and microscale thermophoresis. The
student will also use atomic force microscopy, in
collaboration with the lab of Sergi Garcia
Manyes, to gain revolutionary insights into the
mechanism of action of SGTA and its modes of binding to other proteins. If time permits,
the student will also investigate the role of SGTA in prostate cancer by characterising its
binding to the androgen receptor.
33
(Dr. Sergi Garcia-Manyes)
Conformational dynamics in the folding trajectory of a single protein under force
The MRes Project aims at studying the conformational dynamics of single protein molecules
under the effect of a calibrated constant pulling force. We will track the individual folding
pathways experienced by a single protein during its journey to the native, folded state from
highly extended states. Using a force-quench approach, we will dissect the individual folding
trajectories in order to understand the physical mechanisms that govern each state involved
in the folding trajectory of a variety of proteins. Contrary to previous belief, our force-clamp
experiments demonstrate that the acquisition of the protein’s native conformation occurs
after dynamic maturation of an ensemble of collapsed states. Remarkably, the existence of
such newly discovered ensemble of collapsed states that hold the key to explaining how an
extended polypeptide folds while regaining its mechanical stability is likely to have profound
implications on the onset of conformational diseases, occurring at the level of a single
molecule. The student will study the individual folding trajectories of proteins naturally
designed to fold within biological timescales (e.g. different Ig domains) and compare them
with the folding trajectories of proteins that misfold, being the ultimate cause for many
conformational diseases (such as ß42, responsible of Alzheimer’s disease, or gammaD
crystallin, triggering eye’s cataract). The uncanny ability of single molecule force clamp
techniques to capture the different conformations visited by an individual during its folding
process will allow identification of the toxic conformation that act as the molecular seed for
misfolding.
(Prof. Mathias Gautel)
Characterisation of unusual protein kinases
The project will use biophysical and enzymatic methods to study the activation of three
members of the cytoskeletal branch of calmodulin regulated kinases, expressed as
recombinant proteins in insect cells.
One of these kinases, obscurin kinase-2, does not show an obvious calmodulin binding
motif, whereas obscurin kinase-1 shows a classical binding site. Their activation mechanism
will be compared to a paradigmatic member of the family, myosin light chain kinase. The
work will involve expression and interaction analysis with potential upstream regulators and
downstream effectors using biosensor assays. Cell imaging using FRET and will
complement these studies.
34
(Prof. Hannah Gould)
Mathematical Modeling of the Class Switch Programme in Human B cells
Class switching to IgE, the key antibody in the allergic response, is a black box. There are
several parameters that determine the outcome, which is the level of IgE produced in vivo,
related to the risk of allergy. We have an in vitro model of this process. It involves the
incubation of purified human B cells with the cytokines that stimulate IgE production in vivo.
The various parameters are the birth and death of the B cells emerging from each type of
precursor B cell (expressing IgM, IgG or IgA antibodies) after every cell division (1-8) and
the particular switch, starting from IgM and going to each of the other isotypes or
sequentially through these isotypes to IgE. We have attempted mathematical modeling of
the observed experimental outcome, estimating the probability of each of the critical events,
P(birth), P(death), P(division up to 6-8 cell cycles), P(switch) by varying the values of the
different parameters. Our questions are, for example, does P(death), P(division), P(switch)
remain constant, or do they vary with the isotype or number of divisions. The parameters
that are determined to provide the best fit to our data must then be measured by
manipulating the system to specifically stimulate or inhibit them in vitro or isolating the B
cells of the other switched isotypes (IgG or IgA) at different division numbers and reincubating them in vitro to determine the value of the parameters associated with further
(sequential) switching to IgE. This project therefore has components of both cellular and
molecular immunology and computer-based mathematical modeling. Some knowledge and
experience in both fields would be advantageous. We envisage that the understanding
gained in this study could be used to develop biomarkers of allergy risk and therapeutic
strategies.
For references on IgE structure, function and regulation, see Gould and Sutton, Annual
Reviews in Immunology, 2003, and Nature Reviews in Immunology, 2008.
(Dr James Mason)
The role of peptide structure and membrane interactions in antibacterial and gene delivery
strategies
We use a multidisciplinary platform comprising biophysical methods, including CD and
fluorescence spectroscopies, solid‐state NMR and in silico molecular modelling, alongside
systems biology approaches, including NMR metabolomics and genomic methodologies, to
understand how linear cationic peptides bind to, disturb and cross biological membranes.
This function is crucial to their role as next generation antibiotics or nucleic acid delivery
systems. The project will focus on the structural effects of peptide modifications that are
designed to enhance these functions whilst increasing their specificity and reducing their
potential toxicity to host cells.
35
(Dr. Lindsay McDermott)
Fluorescence characterization of ZAG’s metal binding function
Zinc alpha two glycoprotein (ZAG) participates in the chronic weight loss and muscle
wasting exhibited by certain cancer patients. The protein binds long chain fatty acids and is
thought to participate in lipolysis by an as yet undefined mechanism. ZAG was first
identified and named due to the fact that it precipitated from blood plasma with zinc salts.
It’s three dimensional structure has been solved using x-ray crystallography and resembles
that of a class I major histocompatibility complex (MHC) heavy chain.
Our recent data shows that zinc is able to displace bound fatty acid upon addition to a
ZAG:fatty acid complex. The student interested in this project will continue to characterize
zinc binding by ZAG using modelling, fluorescence and circular dichroism. This will allow us
to better understand ZAG’s in vivo function and the interplay between metal and fatty acid.
The project involves growth and induction of E. Coli genetically engineered to over-express
ZAG protein; protein purification, SDS PAGE, dialysis, UV spectroscopy and use of
fluorescent probes.
(Prof. Jim McDonnell)
Molecular interaction analysis and inhibition of IgE network components
In collaboration with the Beavil, Gould and Sutton laboratories, we are carrying out a
detailed structure/function analysis of a set of proteins that regulate allergic and
inflammatory immune responses.
Using a variety of biophysical methods we are determining three- dimensional structures of
proteins and complexes, mapping interaction sites, and characterizing the mechanisms of
molecular recognition of defined partners in the IgE network. This information is used in the
rational design and development of small molecule inhibitors of protein-protein interactions,
using either library screening methods or more traditional structure-based inhibitor design
approaches.
Training will be available in a wide variety of biophysical techniques, with a likely emphasis
on one or more of the following:
NMR spectroscopy, mass spectrometry, surface plasmon resonance, isothermal titration
calorimetry, fluorescence and optical spectroscopies.
36
(Dr. Brendan Orner)
High Throughput Screening of Protein Cage Libraries in Living Cells
Keywords: Protein Engineering, Chemical Biology, Synthetic Biology, Nanomaterials,
Protein Cages, FlAsH, Self-Assembly, Protein-Protein Interactions
Proteins that form cage-like architectures have been of intense recent interest due
both to their complex assembly which is a result of highly symmetrical protein-protein
interactions and to their application in bioconjugate and materials chemistry. Designing or
discovering cage proteins with new properties and structures could be impactful to these
fields as well as expand our knowledge of how self-assembly occurs and what governs
quaternary structure. Rapid and direct methods to probe the assembly of protein cages and
to assess the ‘most fit’ structures of mutant cages have been lacking. In this project we aim
to apply split cysteine display technology, which uses the fluorescent fluorescein-based
reagent, FlAsH, to monitor assembly of the Bacterioferritin (Bfr) and DNA binding protein
from starved cells (Dps) cages from E.coli and to detect specific oligomerization states in
both cell lysates and purified proteins. We will design several potential FlAsH binding sites
along different axes of symmetry using both a bipartite and a new tetrapartite binding site
design. These designs will take advantage of protein-protein interactions that are formed
only upon cage formation and other oligomerization states. These designs will be applied to
screening libraries to identify proteins with enhanced cage forming potential and to directly
monitor the assembly process. This will be one of the first examples of designed discovery
of nano-scalled protein quaternary structure and will lend insight into the study of both
protein folding and protein-protein interactions. We will eventually apply these proteins with
novel properties to the generation of protein-gold nanoparticle hybrids for the downstream
development of materials with nonlinear optical properties.
37
(Dr. Dylan Owen)
Quantitative fluorescence microscopy to analyse membrane structure and dynamics in subsynaptic T cell vesicles
This project aims to investigate the structure of the cell membrane using advanced, live-cell
fluorescence microscopy techniques. In particular, we will perform high resolution imaging
of environmentally sensitive membrane dyes – those which change their fluorescent
properties in response to environmental changes e.g. the density of lipid packing or the
viscosity of the bilayer. The structure of the bilayer will be investigated in terms of its affect
on membrane protein distributions especially during the activation of T cells when they kill
infected target cells during an immune response; which has important applications in
disease and cancer. The student will learn cell culture, a variety of advanced fluorescence
microscopy methods (confocal, multiphoton, TIRF, FLIM) as well as having a go at
programming and new image processing techniques.
(Dr. Mark Pfuhl)
Exploring the dynamics of Titin kinase by NMR spectroscopy
Titin is not only the the largest protein known (~3000 kD) but also the longest: it spans an
entire half-sarcomere in striated muscle, covering a distance of ~1μM. By extending to such
an extraordinary length the protein is able to connect the Z-disk to the M-band and inbetween linking with both the thin and thick filaments, thus forming a new, 3rd filament
system. Titin fulfils many roles, amongst them being a molecular ruler that defines the size
of sarcomeres Most importantly is its elasticity in its I-band portion, required to allow it to
remain anchored at the Z-disk and M-band while muscle is contracting thus keeping all
filaments in register. This feature allows it to act as a force sensor to add a novel approach
to signalling by connecting the force production of muscle to a change in gene expression
(1). This additional signalling appears to be coordinated by the kinase domain of Titin which
was shown recently to be activated by force (2). However, at present there is only a crystal
structure which limits the information about local dynamics and flexibility, a key prerequisite
to properly understanding the mechanism of the mechanical activation. It recently became
possible to produce the kinase in bacteria, an essential condition for NMR studies. We are
now in a position to characterise the kinase domain in solution and study its dynamics to
understand how it can be activated by force.
The project will involve the optimisation of protein expression, protein purification, sample
preparation and the measurement and interpretation of NMR spectra including the analysis
of experiments to study protein dynamics and experiments to monitor interactions with
ligands such as ATP, substrates and other regulatory proteins.
References:
1. Lange et al., The kinase domain of titin controls muscle gene expression and protein
turnover., Science 308, 1599-1603, 2005
2. Puchner et al., Mechanoenzymatics of titin kinase., PNAS 105, 13385-13390, 2008
38
(Dr Argyris Politis)
Integrating mass spectrometry and electron microscopy data with molecular modeling for
generating 3D structural models of protein complexes
Understanding, describing and modulating many of the cells’ functions requires
structure characterization of its macromolecular assemblies. However, the study of many
heterogeneous assemblies by conventional methods remains a challenging task, thus
impeding structural information of many important biological machines. Hybrid structural
biology approaches, which combine information from various sources, can address this
challenge enabling insights for systems that remain elusive by a single method. We have
developed a groundbreaking approach, which uses four types of mass spectrometry (MS)based data, proteomics, native MS, ion mobility–MS and cross-linking MS to derive
structural information to guide modeling of large protein assemblies (Politis et al., Nature
Methods, 2014).
Here, in this interdisciplinary project, we aim at integrating MS based and electron
microscopy (EM) experiments with a state-of-the-art modeling strategy to generate
structural information that can be utilized as restraints for building 3D models of
heterogeneous assemblies. For this project, the student will be involved in developing the
computational tools and workflow for integrating MS-based data, acquired in house, with EM
data obtained through collaborations. Benchmark analysis will be performed on a known set
of complexes using experimental datasets to assess the ability of the approach to generate
near-native structural models.
39
(Dr. Edina Rosta)
Activation mechanism of RAF kinases
Kinases are ubiquitous group of enzymes essential in regulating key cellular processes
such as growth and proliferation. One of the most important processes in which kinases are
involved is the ERK pathway. This pathway consists of a chain of interacting proteins, RAS,
RAF, MEK, and ERK, where the latter three are kinases that transfer the signal via
phosphorylation. Importantly, this pathway is hyperactivated in >30% of all cancers, with
RAS and RAF being the main oncogenic factors. Single-residue mutations of BRAF, in
particular V600E, are linked to 66% of malignant melanomas, and also to ovarian, colon
and papillary thyroid cancers. A recently FDA-approved drug, Vemurafenib, is the first drug
targeting specifically V600 BRAF mutations. However, the high efficacy of Vemurafenib in
melanoma is quickly offset by the development of drug resistance, presenting acute clinical
challenges.
Here, we will study the key structural and functional changes upon phosphorylation that lead
to the activation of RAF kinases. Phosphorylated structures are largely unavailable
experimentally, however their essential roles are straightforward to study using molecular
modeling methods.
We aim to carry out simulations that provide novel structural insights about kinase activation
and dynamics. In particular, we will investigate the paradoxical activation mechanisms of
certain inhibitors, and perform inhibitor screening studies using our newly obtained
phosphorylated protein structures.
40
(Dr Maria Eugenia Sanz)
Towards understanding the mode of action of anticonvulsant valproic acid
Valproic acid (VA) is unique for its many therapeutic applications, including epilepsy, bipolar
disorder and migraine, with potential roles being proposed in cancer, HIV and Alzheimer’s
disease. Despite its varied effects, VA mechanisms of action in treating these diseases are
still unclear. No specific binding sites are known for VA and therefore it has been suggested
that it can act by perturbing the plasma membrane. Although some computational studies
have been carried out trying to shed light on the translocation of VA across membranes,
there is no detailed experimental data on VA or its interactions with membrane components
or water.
In this project we will investigate VA and VA-water complexes using a combination of
molecular modelling and rotational spectroscopic techniques to characterise VA preferred
conformations in isolation and their changes upon interacting with water molecules.
Structures of VA and its complexes with water will be determined from analysis of the
rotational spectrum.
(Dr. Victoria Sanz-Moreno)
Studying changes in physical properties of the matrix and their role in drug resistance during
cancer metastasis
Abnormal cell migration and invasion is a characteristic of malignant cancer cells and
is required for metastasis, the major clinical problem in cancer. Ninety percent of cancer
patients die from metastatic disease and drug resistance. To metastasize, tumor cells must
move through tissues and cross tissue boundaries, which requires cell motility, loss of cellcell contacts and remodeling of the extracellular matrix (ECM). The acto-myosin cytoskeletal
plays a crucial role in controlling migration of many types of cancer cells through generation
of contractile force (1,2,3,4,5).
On the other hand, several drugs used for cancer treatments have failed in the clinic,
even in some cases promoting metastatic dissemination and changing the surrounding
ECM. Therefore, it is important to understand how physical changes in both cancer cells
and in the surrounding matrix are responsible for therapy failure, in order to design better
strategies that integrate this information. In the lab we study how cells can migrate and
invade different matrices found in the human body, for example collagen I- present in the
dermis- or hyaluronic acid- present in the brain (1,2,3,5). Using 3D systems of different
physiologically relevant ECMs, the student will combine confocal imaging, atomic force
microscopy (AFM) technique and matrix anisotropy measurements. All these approaches
will help to assess the physical changes occurring in the matrix as a result of different drug
treatments that have failed in the clinic. Furthermore, using traction force microscopy (TFM),
the student will explore perturbations in the force that cells can apply to the matrix due to
the same drug treatments.
We want to understand if changes in the acto-myosin cytoskeletal force are
responsible for drug resistance, and if such changes have an impact on the physical
properties of the matrix. This will guide in the design of better therapeutics, that will aim to
provide the strongest physical barrier against disseminating cancer cells by keeping the
ECM in a “non-permisive” physical state and impairing tumour cell migration.
41
References
1.
2.
3.
4.
5.
Sanz-Moreno et al, Cell (2008)
Sanz-Moreno et al, Cancer Cell (2011)
Orgaz et al, Nature Communications (2014)
Orgaz et al, Pigment Cell and Melanoma Research (2013)
Ortanon et al, Stem Cells (2013)
(Prof. Brian Sutton)
Crystallographic studies of IgE-receptor interactions, and complexes with potential
inhibitors.
The interactions between IgE antibodies and their receptors are targets for therapeutic
intervention in allergic disease, including asthma. We have determined the structures of
IgE-Fc and its receptors (FceRI and CD23), and are engaged in a programme to discover
small molecule inhibitors. The work will involve crystallising one or more of the protein
components and soaking with inhibitors, or co-crystallisation, and X-ray structure
determination.
(Dr. Mark R. Sanderson)
Structural studies of proteins of viral origin
Structural studies on proteins of viral (either Herpes or HIV) origin using X-ray
crystallography and/or NMR. The project will involve studying either a thymidine kinase from
a herpes virus or will be a study of proteins involved in HIV Vif protein-protein interactions.
42
(Dr. Roberto A. Steiner)
Structural analysis of the heavy chain subunit of the kinesin-1 molecular motor
Cells possess many specialised components that must be in the right place at the right time
to fulfil their function. Mis-regulation or disruption of these transport processes can
contribute to many human diseases ranging from neurodegenerative conditions such as
Alzheimer's disease to cancer and even contribute to viral infections by HIV-1 or bacterial
infections such as Salmonella. To move components around, cells use molecular motors
(1).
Kinesin-1 is a tetrameric motor formed by two heavy chains (KHCs) and two light chains
(KLCs). We have recently elucidated the structural basis for cargo recognition by KLCs (2).
This breakthrough now gives us the exciting opportunity to study other intriguing facets of
kinesin-1 biology. At their N-termini KHCs feature the ATP-dependent motor domains
required for movement along microtubules. Structurally, very little is known about KHCs
outside their motor domain.
Sequence-based predictions suggest
presence of several coiled-coil regions
KHCs. One of these has been mapped
biochemically as responsible for the
interaction with a coiled-coil region on
KLCs. Large conformational changes
within the KHCs are engendered in
response to kinesin-1 activation (see
To understand this process a better
structural description of KHCs is
needed. The student will work on a
number of KHC expression constructs
provide an initial characterization of the
KHC-KLC interaction.
the
in
the
pic).
to
Vale RD et al. (2003) Cell. 112:467-480.
(1) Pernigo S et al. (2013) Science.
340:356-359.
43
Examination paper
King’s College London
University of London
This paper is part of an examination of the College counting towards the
award of a degree. Examinations are governed by the College Regulations
under the authority of the Academic Board
MRes Examination
Advanced Biophysical Techniques
January 2010
Time allowed: THREE hours
Answer THREE questions, ONE from each of the sections (A,B,and C).
PLEASE USE A SEPARATE ANSWER BOOK FOR EACH QUESTION
AND WRITE ITS NUMBER ON THE COVER
CALCULATORS MAY BE USED.
NOT REMOVE THIS PAPER FROM THE EXAMINATION ROOM
TURN OVER WHEN INSTRUCTED
2010 © King’s College London
44
SECTION A
1)
2)
3)
Answer all parts to this question
a)
Describe the principles of the Nuclear Overhauser
Effect (NOE) in NMR spectroscopy (30%).
b)
Describe how the NOE depends on the correlation time of the
molecule (30%).
c)
Discuss how NOEs may be used for the analysis of
protein structure (40%).
Homology models represent an important tool in bioinformatics
analyses of the functional properties of new proteins.
(a)
Describe the fundamental requirements for choosing a
particular template structure to construct a homology
model (30%).
(b)
How can the quality of a model constructed by
homology be evaluated? Describe the main plots and
analyses that could be performed and explain in
detail their meaning (70%).
Describe the principles, applications, advantages and
weaknesses of two biophysical methods (not NMR or X-ray
crystallography) for determining the size, shape and molar
mass of biological macromolecules in their native state (50% for
each).
See Next Page
45
SECTION B
1)
2)
Answer both parts to this question
a)
Draw the Patterson function of a benzene ring
considering only its six carbon atoms. Please indicate the weight
of each peak (50%).
b)
Discuss phase improvement techniques in X-ray
crystallography and the basic principles on which they are based
(50%)
Answer both parts to this question
a)
Describe the static and dynamic quenching processes
that may occur in the excited state of a fluorescent
molecule (75%).
b)
In the case of purely dynamic quenching of a
fluorescein solution with Iodide:
Given that the bimolecular quenching rate,
kq , is 2x109 M-1s-1. What concentration of quencher would be
required to reduce the fluorescence lifetime of fluorescein by 1ns
from a control of 4ns ? (25%).
See Next Page
46
3)
Answer all parts to this question
a)
What are the advantages of using an optical trap in
which there are two opposing light beams relative to the
simpler single beam trap ? (30%).
b)
Discuss the different optical trapping methodologies
required to study kinesin, myosin V and skeletal
myosin II (30%).
c)
From the point of view of a virus, detail the
challenges, energetic and other, involved in
packaging the genome into the capsid. How have
optical trapping methods helped to evaluate the
completeness of our picture of the process ? (40%).
See Next Page
47
SECTION C
1) Answer all parts to this question
a)
Circular Dichroim is a form of optical spectroscopy.
Explain what is Circular Dichroism (25%).
Examine the peptide sequence given below and with
reference to the chart below answer the
questions (b),(c),(d) and (e).
C
O
H
2
C
H
O
H
H HO
2
H
N C C NC C N
2
HO
C
H
2
N
H HO
C NC
HO
C
H
2
N
H C
H
2
H
H HO
C N C C NC
H HO
HO
C
H
2
H
NC C
O
H
2
HH
C
H
2
N
H
C
H
2
N
H
2
C
H
N
O
W
=
8
6
2
.
4
;
=
1
1
0
0
0
3
6
5
4
1
2
1
3; M
2
8
0
O
H
N
H
3
b)
How many amino acid residues are there and what is
the amino acid sequence ? (10%).
c)
How many pKa’s does this peptide have and what is
the overall charge at pH1, pH8 and pH12 ? (15%).
d)
A sample of this peptide dissolved in 10mM Na
phosphate pH7 buffer gave A280=0.5 in a 5mm
pathlength cuvette. What is this absorption at 280nm
due to and what is the concentration in mg/ml?(15%).
e)
How does a Circular Dichroism Spectrum help assess the
folding of this peptide (35%).
See Next Page
48
Th e Na tu r a l L-Am in o a c id s
Hy d ro p h o b ic
C
H
3
H
C
3
H
H
H
N
2
C
H
3
C
O
H
2
Gly c in e , (Gly ) G
7 5 │-│9 . 6 │2 . 3 4
H
H
N
2
C
H
3
H
C
3
C
H
H
H
N
2
C
O
H
2
Ala n in e (Ala ) A
8 9 │-│9 . 8 7 │2 . 3 5
H
2
C
C
H
H
H
N
2
C
O
H
2
Va lin e (Va l) V
1 1 7 │-│9 . 7 2 │2 . 2 9
C
H
C
H
3
H
C
2
C
H
3
H
H
N
2
C
O
H
2
Is o le u c in e (Ile ) I
1 3 1 │-│9 . 7 6 │2 . 3 2
C
O
H
2
Le u c in e (Le u ) L
1 3 1 │-│9 . 6 │2 . 3 6
N
H
S
H
C
H
2
C
H
C
2
C
H
2
H
C
2
H
C
2
H
CH
2
N
H
C
O
H
2
Pr o lin e , (Pr o ) P
1 1 5 │-│1 0 . 6 │1 . 9 9
H
H
N
2
C
O
H
2
Cy s te in e (Cy s ) C
1 2 1 │8 . 3 3 │1 0 . 7 8 │
1.71
H
H
N
2
C
H
3
H
C
2
C
S
H
C
2
H
H
N
2
C
O
H
2
Me th io n in e (Me t) M
1 4 9 │-│9 . 2 1 │2 . 2 8
H
H
N
2
C
O
H
2
Ph e n y la la n in e (Ph e ) F
1 6 5 │-│9 . 2 4 │2 . 5 8
C
O
H
2
Tr y p to p h a n (Tr p )
W
2 0 4 │-│9 . 3 9 │2 . 3 8
Hy d ro p h ilic c h a rg e d
N
H
H
C
H
2
C
C
O
H
2
H
C
2
H
C
2
H
C
2
H
H
N
2
C
O
H
2
As p a r tic , (As p ) D
1 3 3 │3 . 6 5 │9 . 6 │1 . 8
8
H
H
N
2
C
H
C
H
2
C
C
O
H
2
H
2
C
C
H
2
H
2
C
N
H
2
H
C
2
H
N
C
H
2
Glu ta m ic , (Glu ) E
1 4 6 │4 . 2 5 │9 . 1 3 │2 .
17
N
H
2
H
H
N
2
C
O
H
2
Ly s in e , (Ly s ) K
1 4 6 │1 0 . 2 8 │8 . 9 │2 . 2
N
H
C
C
O
H
2
H
H
N
2
H
C
2
N
H
C
O
H
2
Ar g in in e , (Ar g ) R
1 7 4 │1 3 . 2 │9 . 0 9 │2 . 1 8
H
H
N
2
C
O
H
2
His tid in e , (His ) H
1 5 5 │6 . 0 │8 . 9 7 │1 . 7
8
Hy d ro p h ilic
n e u tra l
O
H
O
H
2
C
O
H
H
C
2
H
H
N
2
H
C
2
C
O
H
2
H
H
N
2
C
H
2
C
C
O
H
C
O
H
2
H
C
2
H
H
N
2
N
H
2
C
O
H
2
H
C
2
H
H
N
2
N
H
2
H
C
2
C
O
C
O
H
2
H
H
N
2
C
O
H
2
Ty r o s in e , (Ty r ) Y
See Next Page
49
2)
3)
Answer all parts to this question
a)
What does “optical sectioning” mean in the context of
microscopy? Why is it important? (20%).
b)
Draw and label the optical transfer function (OTF) of a
widefield fluorescence microscope and indicate the
"missing cone". The drawing should comprise the axial
and one lateral direction. Explain the significance of the
OTF “support” and the “missing cone” (40%).
c)
Explain how the technique of structured illumination
microscopy (SIM, e.g. as used in the Zeiss Apotome)
achieves optical sectioning (40%).
Describe the three principal anisotropic NMR interactions that you are
likely to find in a biological solid (30%). Describe what information
could be obtained (and how) from EITHER an 15N OR 13C labelled
protein OR peptide in a phospholipid membrane (70%).
50
Submission of the Research Thesis and Presentation
of research
You will be expected to submit three copies (two bound {instuctions of layout please see below} and
one soft bound copy) and a electronic copy on CD of your dissertation to Ms. Helen Rudkin in the
Randall Division office (not the Academic Centre) on Wednesday 1st of August 2010 or to Dr. Mark
R. Sanderson.
The internal examiners will both receive a copy that they will mark, the soft copy will be sent to the
external examiner. Please note that the MRes examiners may request that the electronic version be
checked for word count or scrutinised by plagiarism software if necessary. Please make sure that
your CD is labelled with your name, and hand it in at the same time as the paper copies of your
dissertation.
The MRes in Molecular Biophysics thesis
1) Written dissertation
The total length of the dissertation, should be 10,000 – 18,000 (including figures, tables and
references within text but not bibliography) calculated as a word count using the “Tools/word count”
option of the word-processing package. The CD version of your dissertation is checked to ensure
that the word count listed on the front cover sheet truly reflects the dissertation length: examiners
will be asked not to read beyond the 18,000 word limit and submit their marks based on this upper
limit. If your dissertation is overlong you will not gain credit for a thoughtful discussion that an
examiner does not read.
A numbering system for Sections/Chapters helps. Pages should be numbered in sequence, and
each Section should start on a fresh page. Figures and Tables should be incorporated within the
text. Each Figure or Table should have a number, a title, and a legend that describes its main
features.
The dissertation should consist of:
- Front cover sheet - with details of number of words
- Title page - with name of student and supervisor
- Abstract - similar to a scientific paper (no more than one page).
- Acknowledgements
- Abbreviations
- Table of contents
- List of tables and figures
- Introduction – including a good review of the literature (appropriately referenced), showing brief
51
historical development and recent work, and the chief questions to be addressed. Make sure that
you give a balanced coverage, not just mentioning work from your host laboratory. If figures or
tables are reproduced from published material, the source must be stated in the Figure legend
and cited in the reference list.
- Aims – it must be clear to the examiners what you are attempting to do in your project. It may be
useful to give the aims as a series of bullet points with a brief outline of the approaches taken to
address each aim.
- Methods – given more fully than in a scientific paper, so that the examiners have a clear idea of
what you did. It is useful to describe, in brief, the underlying principles of methods used. If you
are involved in any in vivo experimental work on animals (even if you are not performing the
procedures yourself) you MUST state the Home Office scheduled procedures that have been
used. Similarly, dissertations that include work performed using human subjects MUST provide
details of the ethical approval that was granted for the study.
- Results – as in a paper, but you may include more examples of raw data. You should provide
numerical data wherever possible, so if you obtain results in a non-quanitifiable format (e.g.
experimental traces, autoradiographs, etc.), you should attempt to quantify them (e.g.
measurements of area under the curve, densitometric analyses, etc.). Wherever possible you
should perform appropriate statistical analyses on the data obtained – you should seek advice
from your supervisor about the most appropriate statistical tests to apply to your data if
necessary. The results text should describe the results presented, highlighting the main
observations, and commenting on the statistical analyses.
- Discussion – commenting intelligently and critically on the results obtained, and showing how
they fit in with the body of knowledge. Do not be afraid to criticise your own work, if you feel some
parts are weak, and try to offer an explanation if your results are different from those of previous
studies.
- Conclusion – key points from the work, and suggestions for further study.
- Appendix – the appendix contains information that you may not want to include in the main text
such as large tables of raw data summarised in the Results section, checks on methods, DNA
sequencing data, mathematical or theoretical considerations. You do not need to include an
appendix if you do not have any additional information that has not been included in the Results
section.
- References – full details (title, year, journal, page numbers) of each reference cited in the text, in
alphabetical order. If several different techniques are used, or different sub-projects done within
the whole, it may be better to keep the sub-topics separate (e.g. by presenting Methods and
Results of sub-project 1 together, then same for sub-project 2 etc).
[This document is a modified version of Stephen Struzenbaum and Nic Bury’s MSc
BIOMEDICAL & MOLECULAR. SCIENCES RESEARCH original instructions to make the MRes in
Molecular Biophysics thesis of the same format]
52
Guidelines on Format for the Research Project (Dissertation)
-
Use standard font (Times New Roman, Arial or similar).
The main body of text should be font size 12. Text in figures can be smaller but must be
easily legible.
Lines of the main body of text should be 1.5 or double spaced (not single spaced)
Top and bottom: 3 cm; Margins: left side: 3cm, right side 3 cm. Headers and footers can be
added within these set margins.
You will need to submit 3 copies (Two Hard and One soft, (velo binding)) plus an electronic
version (CD) of your thesis
For the hard bound copies they should be bound in standard University of London Blue,
known as Blue 550 to the Binders.
The thesis should be lettered on the spine only in the up-spine direction
Lettering on the spine should be in gold 14 pt Times
MRes Molecular Biophysics
2013
SMITH J.
Hence Upper/lower case
year
Name in Capitals
(I have an example in my office if you wish to see an example)
There is no strict requirement to use a specific binding service, but for reference only, an
example for a local service is:
The Document Centre,
92 Southwark Bridge Road,
Southwark,
London
SE1 0EX
Telephone 0845 345 9181
53
Guidelines for the presentations for the MRes in Molecular Biophysics.
Your presentation should consist of 4 main elements.
(1)
An introduction describing the background and aim of the project
(3-4 minutes)
(2)
Discussion of the project and results obtained. This should be the main part of the
presentation (18 minutes).
(3)
Conclusion. In this section you could describe where you would take the project in the
future (2-minutes)
(4)
Acknowledgement slide- Acknowledging the head of the lab., the people you worked with
and were helped by, as well as any funding agency who funded you or the Research in the
laboratory (1-2 minutes).
Since the presentation is for 25 minutes for a power-point presentation. 1 slide
per minute is a good rule of thumb, so that the material on a slide can be fully described.
Avoid too many more slides as this will result in cursory explanation of slides and slides
being too rapidly flashed on the screen.
Theory should be included only where relevant to the experiments undertaken.
There is not enough time in 25 minutes to start describing a particular biophysical
technique in great depth and then continuing into the experiments done.
There will be around 5-10 minutes for questions.
54