Physical Methods Questions

1995

"Describe the principles and biochemical applications of two of the
following:  (i) Pulsed Field Gel Electrophoresis (PFGE); (ii) Confocal
microscopy; (iii) Circular Dichroism (CD); (iv) Stopped flow methodology."

" Either

(a) Answer two of the following:
	(i) Explain how you can obtain 3D structural information for a
	membrane protein that forms only ordered 2D crystals.
	(ii) Why are heavy metal derivatives of crystals usually needed for
	derivation of 3D structures?  How can such derivatives be obtained?  If
	such attempts end in failure, are there any alternative strategies?
	(iii) What is meant by refinement of a crystal structure?  Why is the
	refinement process important?

Or

(b) Answer two of the following:
	(i) In a protein (Mr = 15,000 Da) containing 6 leucine and 2 phenylalanine
	residues, it was found that all the proton resonances of these 9 amino
	acids could be resolved and assigned specifically.  Explain why this
	resolution can occur and how the assignment could be made.
	(ii) It is proposed that leucine-14 and valine-82 are in close spatial
	proximity within a water-soluble protein of molecular mass 12,000
	Da.  Describe NMR experiments that might be used to test this
	proposition.  Could the same strategy be used for an integral membrane
	protein with the same Mr?
	(iii) What factors determine the emission wavelength and the quantum yield
	of a fluorophore?  Describe how one of these factors could be the basis
	for assessing distances between different proteins in a prokaryotic
	ribosome."

1996

Either

"Briefly describe the advantages and disadvantages of NMR studies
of proteins in solution.  In this kind of NMR, how can:
(a) Resonances from particular groups be resolved and assigned in
a protein, Mr = 20,000 Da?
(b) Structural restraints be derived experimentally?
(c) Structural restraints be used to determine protein structure?
(d) Information about protein mobility be deduced?"

Or

"Suggest how physical methods might be used to give information
about the following:
(a)The spatial distribution of protein subunits in a prokaryotic
ribosome;
(b) The ligand geometry of an iron-sulphur protein with Mr = 40,000Da;
(c) The identity and pKa values of amino-acid residues involved in proton
pumping in a membrane protein;
(d) Growth-factor induced aggregation of membrane receptors."

1997

"Suggest how spectroscopic methods might be used to obtain information
about the following:
(a) Association and dissociation of a protein-protein complex;
(b) Intermediates in a protein folding pathway;
(c) Molecular motion of a protein domain;
(d) ATP-induced shape changes of myosin, in the solution state.

"Write and essay on either "time-resolved X-ray crystallography" or " in
vivo NMR".

"What methods, other than X-ray crystallography, are available to probe
the structure of membrane proteins?"

1998

"Suggest how appropriate physical methods might be used to give
information about the following:
(a) The dissociation of a protein homo-dimer (Kd = 0.1mM);
(b) Diffusion of a protein in a membrane;
(c) Creatine kinase activity in muscle
(d) Post-translational modification of a recombinant protein (Mr = 45
kDa)."

"The crystal structure of bacteriorhodopsin has recently been solved by
X-ray diffraction methods at 2.5Å resolution and refined to a
crystallographic R value of 22.1%.  Details of the structure are shown in
figures 1 and 2.  Use you knowledge of bacteriorhodopsin and protein
crystallography to answer the following:
(a) Describe the structure as represented in fig 1.  How does the X-ray
crystal structure compare with the structure obtained from high-resolution
EM studies with 2D crystals?
(b) Fig 2 shows a schematic representation of groups that may be involved
in the light-driven hydrogen ion transport.  Comment on the arrangement
for hydrogen ion transport and describe, where appropriate, other evidence
in support of the roles of some of the individual amino acids.
(c) Indicate briefly the problems involved in membrane protein
crystallisation.
(d) The crystals of bacteriorhodopsin used for the X-ray studies had
dimensions approximately 30 x 30 x 5 mM.  Briefly, what advances in X-ray
studies have made data collection from such small crystals possible?

1999
" 'NMR methods are now yielding about 100 protein structures per year,
while X-ray diffraction methods are producing about 400.'  Compare and
contrast the experimental difficulties encountered in the two methods, and
suggest reasons for the difference in their rates of generation of
structures."

"Suggest how physical methods other than high resolution solution NMR
might be used to provide information about three of the following:
(a) Conformational changes induced in calmodulin by peptide ligands;
(b) The solution conformation of the jun/fos dimmer and its complex with
DNA; 
(c) The kinetics of carbon monoxide binding to myoglobin;
(d) The kinetics of association of an antibody with a protein antigen.

" 'Determination of the structure of membrane proteins is no longer an
insurmountable challenge.'  Discuss, with specific examples, how recent
high-resolution EM and X-ray diffraction studies support this statement."

2000

Either
	
"Discuss, with examples, the types of information that can be
derived using protein crystallography.  Does this information complement
that obtained from other biophysical methods?"

Or

"Describe, with examples, the use of isotope labelling in NMR
studies of proteins and their ligand complexes."

"Discuss whether non-crystallographic methods are of continued relevance
to the study of the structure of integral membrane proteins."