Module Code: BIO00025H

Transcription

1 Examination Candidate Number: Desk Number: BSc and MSc Degree Examinations Department : BIOLOGY Title of Exam: Protein protein recognition Time Allowed: 2 hours Marking Scheme: Total marks available for this paper: 100 The marks available for each question are indicated on the paper Instructions: Section A : Answer all questions in the spaces provided on the examination paper Section B : Answer either question A or question B. Write your answer on the separate paper provided and attach it to the back of the question paper using the treasury tag provided Materials Supplied: CALCULATOR only: For marker use only: Office use A B Total as % DO NOT WRITE ON THIS BOOKLET BEFORE THE EXAM BEGINS DO NOT TURN OVER THIS PAGE UNTIL INSTRUCTED TO DO SO BY AN INVIGILATOR Page 1 of 10

2 Section A 1a) A class 1 MHC molecule HLA-NOV has been purified and you wish to identify bound antigenic peptides. Suggest a method for identifying antigenic peptides bound to HLA-NOV. (2 marks) Expecting a variety of bound ligands. Peptide elution/separation by some reasonable methods and mass spectrometry to infer the sequence. The answers to this component were a little disappointing with suggestions that were not really practical. Four nonapeptides were identified and their sequences are given below H 3 N- Asp Leu Asp Lys Phe Pro Val Gly Ile -CO H 3 N- Phe Leu Pro Asp Ser Phe His Thr Val -CO H 3 N- Ser Leu Phe Tyr Glu Val Pro Ser Val -CO H 3 N- Gly Leu Leu Pro Gly Val Ser Tyr Ile -CO 2-1b) Comment on these sequences in relation to the function of HLA-NOV and its expected mode of peptide binding. (4 marks) The sequences show invariance at position 2 and conservation at position 9. They are otherwise divergent [1 mark]. The latter is consistent with the function of HLA-NOV which is to display intracellular peptides and the diversity in sequence recognition is necessary/expected [1 mark]. Most of these divergent residues will be directed outwards from the protein so that they are visible to T-cell receptors on T-cells [1 mark]. At the same time, HLA-NOV must bind peptides tightly so that they are retained when displayed on the cell surface. This is achieved by tight binding of the terminal amino and carboxylate groups and the so-called anchoring side chains of residues 2 and 9 [1 mark]. Better on this component. Most spotted the conservation at 2 and 9 and as this as a binding determinant. Not always so good on explaining the sequence variety in terms of the function of class I MHC. Page 2 of 10

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4 2) The figure below shows the structure of a tetrapeptide. Give the sequence of the tetrapeptide and draw arrows to indicate the features of this peptide that are likely to be important in binding to a sequence-independent peptide binding protein such as OppA. (4 marks) Asp-Gly-Ser-Phe [2 marks]. The key groups will be those conserved in all (tetrapeptides) these being the terminal amino and carboxylate groups [1 mark] and the amide and carbonyl groups of the peptide bonds [1 mark]. Well done. Most students identified the peptide, the most common mistake being Glu instead of Asp at position 1. Most students got the important interaction features though overlooked the peptide amides. 3a) Explain how biotin labelling experiments provided evidence that there is a channel connecting the forespore and the mother cell during sporulation in Bacillus subtilis. (5 marks) SpoIIIAH a mother cell protein was expressed as a fusion protein with a biotin acceptor peptide (BAP) attached to its C-terminus [1 mark]. In two experiments, BirA, which biotinylates BAPs, was expressed in the mother cell and the forespore under the control of the compartment-specific promoter PspoIID and P spoiiq promoters respectively [1 mark] and biotinylation was detected using streptavidin in Western blots [1 mark]. The results showed that SpoIIIAH-BAP in sporulating cells was biotinylated by BirA produced in the forespore but not by that expressed in the mother cell [1 mark]. This implies that SpoIIIAH which is located in the mother cell membrane proximal to the forespore is accessible to cytoplasmic BirA in the mother cell implying the existence of a channel [1 mark]. Page 4 of 10

5 Some good answers to this question with impressive detail. 3b) Give one piece of evidence that SpoIIQ and SpoIIIAH form a complex in vivo and give one piece of evidence that they form a complex in vitro. (4 marks) GFP-tagged SpoIIQ and flag tagged SpoIIIAH [1 mark] initially localise to the sporulation septum and subsequently to the engulfing membranes, moreover the localisation of each protein depends on the other [1 mark]. The interaction of the two purified protein has been shown by surface plasmon resonance which reveals an increase in response units upon flow of SpoIIQ over a surface on which SpoIIIAH has been immobilised. [2 marks] or SEC-MALLS of a mixture of the two purified proteins reveals the elution of a species from a size exclusion chromatography column with a mass that is the sum of the two proteins. [2 marks] or X-ray crystallography following crystallisation of mixtures of the two proteins which reveals the two proteins in a one-to-one complex. [2 marks] Again manygood answers here. 4) For each of the following symmetries give two protein examples and indicate the functionally important property that is generated by this symmetry. [12 marks] One mark for each suitable protein example with correctly identified symmetry and one mark for correctly described functionally important property, generated by this symmetry in the example protein. Suitable examples would be: a) Cyclic symmetry TRAP: C11 or C12, cyclic symmetry generates extended nucleotide-binding surface around the outside of the oligomer that can bind RNA containing repetitive sequence motifs. Small terminase protein: C9, cyclic symmetry generates extended nucleotide-binding surface around the outside of the oligomer that can bind intrinsically bent DNA. Portal protein: C12 or C13, cyclic symmetry generates a channel for DNA translocation. Page 5 of 10

6 b) Dihedral symmetry TPL (tyrosine phenol-lyase): D2, dihedral symmetry generates a tetramer with 4 catalytic sites, each made up by two adjacent subunits. GroEL: D7, dihedral symmetry generates internal cage (in fact, 2 equivalent cages) where unfolded proteins can be refolded. Proteasome: D7, dihedral symmetry generates internal reaction chamber where polypeptide hydrolysis takes place. c) Cubic symmetry Capsids of most viruses: cubic icosahedral symmetry (532). The symmetry generates a capsid that is used to store, protect and transfer genetic viral genome. Anti-TRAP: cubic tetrahedral (233). Cubic assemblies, formed at lower ph (e.g. stress conditions) prevent anti-trap from binding to TRAP, facilitating efficient transcription termination by TRAP. Analysis of the lecture course and wider reading material 5) Give the most suitable biochemical or biophysical technique which could be used to address each of the following experimental scenarios: [5 marks] i) Separation of molecular assemblies by hydrodynamic radius ii) Determining the dissociation constant when large amounts of interacting proteins are available iii) Accurate estimate of the molecular weight of the complex iv) Analysis of atomic details of interacting surfaces, at highest possible resolution v) Analysis of the structure of a large assembly, with molecular weight exceeding 1MDa i) Size-exclusion chromatography ii) ITC (isothermal titration calorimetry) iii) AUC - analytical ultracentrifugation or possibly also native mass-spectrometry iv) X-ray crystallography v) Cryo-EM Deductive reasoning based on lecture course material 6a) The 80-residue KIX domain from the co-activator protein p300 interacts with an array of different partner proteins. The properties of KIX and the molecular Page 6 of 10

7 recognition elements (MREs) of its binding partners have been studied extensively using a variety of biophysical and structural approaches. Briefly describe how you could use heteronuclear solution NMR spectroscopy to measure the affinity of a protein-peptide interaction with 1:1 stoichiometry. Assume the exchange rate of the interaction ( k ex ) is fast with respect to the NMR time-scale.(4 marks) Would need isotope-labelled protein and unlabelled peptide, or other way around (1 mark). Titrate unlabelled part (e.g. peptide) into labelled part (e.g protein; 1 mark). Measure change in compound chemical shift in NMR spectrum as function of titrant concentration (1 mark). Fit data to suitable binding isotherm to calculate dissociation constant (1 mark). 6b) The table below shows the results of interaction assays performed using isothermal titration calorimetry (ITC) in which purified KIX was titrated with peptides derived from three different binding partners: c-myb, MLL and pkid. Data adapted from: Goto et al., 2002 Briefly describe how you could use ITC to investigate whether these peptides interact with KIX with positive cooperativity. (2 marks) Would need to titrate one peptide into a pre-formed complex containing KIX and another peptide (1 mark). Evidence of positive co-operativity would be a increase in binding affinity in these experiments compared to titrating the peptide into KIX alone (1 mark). 6c) You have data showing that the apo-mll peptide is unfolded in solution, but it forms an α-helical structure when in complex with KIX. Propose an NMR-based experiment to investigate whether the MLL peptide folds before binding KIX, or whether it binds to KIX and then folds. (3 marks) Page 7 of 10

8 Would need to titrate isotope labelled peptide with unlabelled KIX (1 mark). Deviation from linear changes in chemical shift as a function of KIX concentration suggests formation of a binding intermediate (1 mark). The chemical shift of this binding intermediate will indicate whether the apo-peptide folds and then binds KIX or whether it binds KIX and then folds (1 mark). 6d) How is the interaction between the kinase-inducible domain (KID) from camp-response element protein (CREB) and the KIX domain of p300 regulated by Protein Kinase A? (2 marks) KID is phosphorylated (1 mark) at Ser-133 which enhances binding to KIX 100-fold (1 mark). 6e) How might optimising the preparation of a protein sample to meet the criteria of a biophysical technique affect the conclusions of the experiment? (3 marks) The answer should address the balance between having a tractable sample that gives interpretable data with the technique and what modifications to the protein, buffer or experimental conditions are needed to make the sample tractable. Therefore, need to consider sample stability with respect to the length of the experiment conditions, monodispersity, yield, purity, ability to modify the protein sequence. It may be necessary to use of off-physiological solution (ph, salt, etc) or experimental (e.g. temperature) conditions; modification of protein sequence (truncations, tags, etc). (3 marks from above examples) Page 8 of 10

9 Essay questions Section B A) Discuss how the formation of protein-protein complexes is controlled by (i) covalent modification, and (ii) the binding of small molecule ligands, including those designed for therapeutic intervention. A good answer here would begin with an interpretation of the question and how the student proposes to address it. This is because the question has scope that would allow for the consideration of a number of lecture course examples and of course numerous wider examples the students will have encountered elsewhere as well as from their own reading. The students may discuss the regulatory system restricting SigF activity during sporulation in B. subtilis. Here adenine nucleotides (ATP or ADP) bound to SpoIIAB are needed for binding to SigF. Meanwhile phosphorylation of SpoIIAA determines whether this antagonist of SpoIIAB forms a complex with the latter. Here complex formation is prevented because the serine phosphate would abut the phosphate of the nucleotide bound to SpoIIAB, were the complex to form. A parallel might be drawn here with T-cell receptor binding to a complex of MHC bearing antigen. Here, the antigen is a peptide (small molecule). A distinction could be made to the role of similar peptides binding to OppA where the protein undergoes a conformational change that fully buries the ligand and creates a surface that is competent to bind the membrane components of the cognate transporter. For the therapeutic intervention component, the students are likely to introduce the use of nutlins and stapled peptides to block interactions between p53 and Mdm2 in cancer therapy. B) Discuss how tubulin tyrosine ligase (TTL) affects the network of proteins that associate with the plus-ends of microtubules. An interaction network involving microtubule plus-end tracking proteins (+TIPs) is presented in detail in one lecture. This essay focuses on the addition of a C-terminal tyrosine to alpha tubulin by TTL and how this modification makes a motif that can be recognised by MT-binding proteins that have CAP-Glycine domains. The essay should describe the step-wise modification of the C-terminal tail of a-tubulin to produce an acidic tail motif (e.g. EEY). The essay should also describe different +TIPs that have acidic tail motifs (e.g. p150n, CLIP170). The different domains that interact with acidic tails should also be described (e.g. CAP-Gly domains). The module focuses on protein-protein recognition and therefore a description of how CAP-Gly domains recognise acidic tail motifs should be included. Good essays will also include an explanation of why a motif lacking the C-terminal aromatic residue would not bind. Better essays will discuss the interplay between +TIPs that have the domain and Page 9 of 10

10 motifs in different +TIPs and the fact that this network is governed by the switch from intra-molecular blocked interactions to intermolecular interactions. The impact of +TIP binding to MTs should be mentioned and the fact that +TIP association has a positive impact on MT growth by reducing catastrophe and promote polymerisation. A variety of experimental data was shown in the lecture and discussed in the context of strategies that unpick the individual interactions within a larger network. Again, better essays would say how knowledge of the system has been obtained (e.g. structures, mutants, pull-down assays, ITC, etc.). Page 10 of 10