Questions for Section 1 1. Describe a the structure of three
different units of supersecondary structure, one containing all
helices, one all strands and one with both types of secondary
structure. In one sentence, explain why most supersecondary
structure units are not associated with a particular protein
function. 2. Describe the structure and function of three proteins
that bind DNA using different structural motifs. For each protein,
describe the interactions between the protein and the DNA, and
explain whether or not they are sequence specific. 3. Describe, in
detail, the structures of two protein domain families that contain
just beta secondary structure that have different architectures.
Give a full set of CATH numbers for each. What is the equivalent
SCOP classification?
Questions for Section 2 1. a) What is meant by competence, when
referring to bacterial cells? [2 marks] b) How is this condition
related to transformation? [2 marks] c)Name two common approaches
to conferring artificial competence to laboratory cells of E.coli,
and note the key steps up to and including transformation. [3 marks
per approach, 6 marks] 2. What is a plasmid? [2 marks]. In order to
do a recombinant expression experiment in E.coli, the promoter and
ribosome binding sequence are key factors: describe details of
these elements, such as nucleotide spacing and interactions [4
marks]. What other DNA sequence elements are required on an
expression plasmid? Outline the roles of each named element [4
marks]. 3. The following is a small portion of the sequence of a
vector, in the vicinity of the cloning site. The vector start codon
and choice of stop codons are shown in bold type. Some unique
restriction sites in this region are listed below the sequence.
TAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTAAGAAGGAGGTATTGGCCATGGAACTGGTCTCACCCGCAGTTCGAGAAAGCTAGCGCTGTGCACCATCACCATCACCATTGAAGCTTATAAGTTAAGTATGATGAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAUnique
restriction sites in the ORF cloning region: AfeI, ApaLI, HindIII,
MscI, NcoI, NheI, PsiI. a) Using the open reading frame for the
uracil-DNA glycosylase in the following GenBank summary page
http://www.ncbi.nlm.nih.gov/nuccore/NC_008541.1?report=genbank&from=838653&to=839357&strand=true
answer the following: i) Design primers for PCR of the gene, to
subsequently permit restriction-ligation cloning at the NcoI site
for the 5'-end of the gene, and at the HindIII site for the 3'-end
of the gene. ii) Design an alternative 5'-end primer for PCR of the
gene, to create a LIC-compatible end, assuming a MscI cut to the
vector. iii) Design an alternative 3'-end primer for PCR of the
gene, to create a LIC-compatible end, assuming a PsiI cut to the
vector. b) After cutting the vector at the LIC restriction sites,
describe in sufficient detail, the steps required to complete the
LIC process.
Questions for Section 41. A protein crystal has cell dimensions
of a = 48.4, b = 48.4 , c = 138.4, = 90.0, = 90.0, = 120.0. What
possible crystal systems could it belong to? 2. In protein
crystallography the ratio of the data collected to the number of
parameters to be refined is often low. Why is this problematic and
name 3 ways in which this can be overcome? 3. What is anomalous
scattering in protein crystallography? [ 3 marks] How is it used in
phase determination? [4 marks] Which elements typically display
anomalous scattering in protein crystals and under what conditions
are they found in the crystals? [3 marks].
Questions for Section 51. Discuss the merits of expression hosts
other than E.coli for producing proteins for structural
biology.
Question for Section 61. The Fourier Transform is a mathematical
function that is used extensively in structure determination in
both electron microscopy and protein crystallography. a) Describe
qualitatively (without the use of equations), or illustrate, what
the Fourier Transform of the electron density in a protein crystal
looks like [2 marks]. b) Why is a periodic pattern not seen in the
Fourier Transform of a raw EM particle [2 marks]? The following
diagrams represent c) the Contrast Transfer Function in EM and d)
the scattering factor f for an atom in protein crystallography. For
each graph describe what these functions show (2 marks) and their
effect on the data obtained [2 marks].
e) Describe or illustrate how the Contrast Transfer Function
would look for a electron microscope with perfect optics [1 mark].
f) Describe or illustrate how temperature (B)-factors affect the
graph shown in d) [1 mark]. 2. a) Describe sample preparation for
negative stain EM and cryo EM. [5 marks] b) What are the
differences between the data that are obtained by each approach? [5
marks]
Questions for Section 71. (a) Describe the principles of
operation of an electrospray ionisation (ESI) mass spectrometer [8
marks]. b) Describe the information that can be obtained about
protein-protein complexes using ESI mass spectrometry. [2 Marks].
2. (a) Outline the principles of secondary structure determination
using Circular Dichroism spectroscopy? (b) Use the web and
literature to find out about the advantages of synchrotron
radiation CD in determining secondary structure. Briefly describe
these advantages and how they arise. Give references for your
source(s) of information. 3. Describe the mechanisms by which
proteins are separated in 2D-gel electrophoresis using the
O'Farrell technique. Describe ONE limitation of this technique in
separating complex mixtures of proteins?
Questions for Section 81. It is thought that a small peptide has
the sequence Ile-Asn-Gly-Phe. Write the chemical formula of the
tetrapeptide when dissolved in D2O solution at neutral pH,
distinguishing between protons (H) and deutrons (D) and indicate
which atoms will give distinct signals in a 1H NMR experiment under
these conditions. 2. The typical fingerprint solution NMR spectrum
of 3 proteins are shown in Figure 1 (A,B,C). These correlation
spectra are known as HSQCs and can represent the fingerprint of the
topology of the protein. Each dot or cross-peak in the HSQC
spectrum corresponds to a backbone amide nitrogen and hydrogen
pair. In these spectra, each (non-proline) residue in the protein
gives rise to one cross-peak which comes at the intersection
between the amide protein frequency (X-axis, labelled hydrogen) and
the nitrogen frequency (Y-axis, labelled nitrogen). The frequency
axes are by convention both use the parts per million or ppm
scale.
A) Green Fluorescence Protein (GFP, 238 residues, 27 KDa, 10
prolines), B) immunoglobulin (Ig) domain (106 residues, 11 KDa) 9
prolines)C) alpha-synuclein (140 residues, 14.5 KDa, 4 prolines)2.1
What does HSQC stand for? (1 mark)2.2 What quantities (in mg) would
be needed in a 0.5 ml NMR tube to achieve the concentrations stated
in the inset of the spectra in Figure 1? (1.5 marks)2.3 Answer the
following 3 questions about the spectra in Figure 1a) How many
cross-peaks would be expected in each spectrum? 1.5 marksb) What is
the spectral dispersion i.e. the x- (hydrogen dimension) and y-axis
(nitrogen dimension) ranges that are covered by peaks 1.5 marksc)
The relative thickness (linewidths) of the dots in A) and B)? Does
this relate to the molecular mass of the protein? 1.5 marks2.4
Along the x-axis (the 1H dimension), the resonances in spectrum C)
are crowded in the centre of the spectrum. Of what feature of this
proteins conformation (alpha-synuclein; see Dedmon MM, et al, J Am
Chem Soc. 2005;127:476477 if necessary;
http://www.ncbi.nlm.nih.gov/pubmed/15643843) is this likely to be
an indication? 1 mark2.5 In light of your answer to ii) what would
the dispersion of the spectra shown in A) and B) indicate about
these proteins? 1 mark2.6 How, then, is this spectrum of
alpha-synuclein likely to change on addition of lipids (useful ref:
Perrin et al, J. Biol. Chem., 2000 275, 34393-8;
http://www.ncbi.nlm.nih.gov/pubmed/10952980). Assume that we would
not see the peaks of these added molecules? 1 mark3. NMR
spectroscopy can be used to describe the structure and dynamic
aspects of a protein during folding. If the Ig domain (same protein
whose NMR spectrum was shown in Figure 1B) is subjected to
increasing amounts of urea denaturant, the NMR fingerprint spectra
at 3 points during this urea titration are shown in Figure 2.
3.1 What changes are observed in the three spectra as the
concentration of urea increases: as regards (a) the spectral range
along the hydrogen (x-axis) dimension? (b) the number of
cross-peaks? (2 marks each)3.2 What does the spectrum of Ig in 8M
urea conditions reveal about its likely topology under these
conditions? (2 marks)3.3 What is the likely explanation for what is
occurring to the Ig domain at 4.5M urea (2 marks)3.4 Using a simple
schematic representing the Ig domain as a string, depict what is
happening to the proteins structure in increasing concentrations of
urea. (2 marks) 4. NMR can also be used to provide detailed
information on protein-protein interactions at a residue specific
level. Calmodulin (CaM), is a 16.7kDa EF-hand regulatory
calcium-binding protein composed of two domains (see Figure 3A) and
mediates a range of cellular processes. CaM undergoes a
conformational change upon binding to calcium ions, which enables
it to bind to specific proteins for a specific regulatory response.
The resonances observed in the fingerprint of CaM have been
assigned (Figure 3B) to their specific residue in the CaM sequence,
which allows us to know which cross-peak in this 148 residue
protein corresponds to an amino acid within the protein. In this
experiment, a 25 amino acid peptide substrate was bound to
calmodulin in different ratios of calmodulin:peptide: 1:0, 1:0.5,
1:0.8, 1:1.2 and HSQC spectra were recorded for each sample, as
shown in Fig 3C (black= 1:0, red = 1:0.5, green 1:0.8 and cyan
1:1.2). We can measure the changes in the positions of the
cross-peaks during the titration and plot this, know as the
chemical shift change for each of the amino acids within the
calmodulin sequence as shown in panel D.
4.1. Using these data and the figures, describe what structural
changes occur in CaM when the peptide binds. (5 marks total)
Questions for Section 91. For the PDB structure 2YMV find the
original publication describing the structure determination and
give the reference. Describe the methods used for the protein
purification (NOT THE EXPRESSION) and the principles of the methods
2. Below are listed a number of factors that affect the efficiency
of protein separation during a column chromatography run. Discuss
these factors and include any equations that relate to each one. a)
Retention time and resolution [2.5 Marks] b) Band broadening [2.5
Marks] c) Theoretical Plates [2.5 Marks] d) Capacity Factor and
Peak Symmetry [2.5 Marks] 3. What are the three distinct stages in
the process of crystal formation? Illustrate using a generalised
phase diagram and a schematic for the energetics of crystal
formation
Questions for Section 101. What are the following stereochemical
parameters and how are they used in protein structure determination
[2 marks each] a) Bond length b) Phi and psi angles (Hint:
Ramachandran plot) c) Side chain conformer/torsion angles d)
Hydrogen-bond e) Distance restraint (NMR) There are several answers
we will accept to the following questions, which depend on how you
do the search in which database and what you and the database take
resolution to mean. 2. a) Using web based search facilities (in
practice the RCSB or PDBe web sites), determine and write down the
PDB codes of the protein structures that currently have the highest
and lowest quoted values for their resolution. b) Download the full
text of the main journal reference for the highest resolution
structure and tell us what the full reference is. Say in no more
than a few sentences what can be seen at this resolution. (We do
not expect you to fully understand the paper, but to be able to
give some idea of what it is talking about in broad terms). c) Tell
us the primary reference for the lowest resolution structure.
Describe the technique used to determine the structure in a few
sentences. What information can be gained from a structure of such
low precision?
Questions for Section 111. Describe four protein modules that
specifically bind to the phosphorylated state of serine residues.
2. What is a "hot spot" in protein-protein interactions? 3.
Describe four methods that can be used for measuring
protein/protein interactions in protein complexes. How quantitative
is each method? For which of obligate core, non-obligate core or
transient complexes does each give useful results?