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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.
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]
c) The relative thickness (linewidths) of the dots in A) and B)? Does this relate to the
molecular mass of the protein? 1.5 marks
2.4 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 mark
2.5 In light of your answer to ii) what would the dispersion of the spectra shown in A) and B)
indicate about these proteins? 1 mark
2.6 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 mark
3. 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 calciumbinding 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)
1. 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