BA, BSc, and MSc Degree Examinations

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1 Examination Candidate Number: Desk Number: BA, 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 Sec on A: Short Answer / Problem / Experimental Design ques ons (50 marks) Sec on B: Essay ques on (marked out of 100, weighted 50 marks) The marks available for each ques on are indicated on the paper Instructions: Sec on A: Answer all ques ons in the spaces provided on the examina on paper Sec on B: Answer either ques on A or ques on B. Write your answer on the separate paper provided and a ach it to the back of the ques on paper using the treasury tag provided. Materials supplied: CALCULATOR For marker use only: For office use only: Module 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 9

2 SECTION A: Short Answer / Problem / Experimental Design questions Answer all questions in the spaces provided Mark total for this section: a) Explain the terms core and rim when referring to residues in protein interfaces and comment on why these definitions are helpful in characterising protein-protein interaction sites. (3 marks) b) Explain the term hydrophobic effect and explain its contribution to protein-protein interactions. 2. a) Two monomeric proteins A and B form a complex with 1 : 1 stoichiometry and a K d of 3.0 x 10 7 M. After mixing 16 μm A with B, it is found that the equilibrium concentration of the free A is 6 μm. Showing your reasoning, calculate the overall concentration of B in the mixture. (5 marks) page 2 of 9

3 b) The Figure below shows structures of SpoIIQ-SpoIIIAH and gp120-cd4-f ab complexes. Comment on the structural characteristics of the protein-protein interfaces and their functional significance. page 3 of 9

4 3. Research on protein A associated with a rare genetic disorder established the following protein-protein linkages: Pair of proteins Linkages A, B gene fusion immunoprecipitation B,C C,D gene co-occurrence negatome database isothermal titration calorimetry surface plasmon resonance cryo-em structure E,A F,A gene neighbourhood gene fusion analytical ultracentrifugation surface plasmon resonance yeast two-hybrid G,A analytical ultracentrifugation gene neighbourhood yeast two-hybrid a) Showing only possible interacting partners, draw a schematic diagram for the network formed by protein A, indicating interactions as shown below: Physical, established in one-to-one experiments Physical, established by proteome-wide analysis Functional interactions (solid line) (dash dot line) (dot line) (6 marks) page 4 of 9

5 b) Rank interacting partners of protein A, starting from the least likely interacting partner and finishing with the most likely. (3 marks) c) Name two linkages of protein A that were established by proteome-wide methods. (2 marks) d) Name two linkages of protein A that were established in vitro. (2 marks) e) Further studies revealed that protein A is a major protein interaction hub, with its N-terminal and C-terminal domains involved in one-to-many and many-to-one types of interaction, respectively. Briefly outline the structural basis for each type of interaction giving an example from lecture material. page 5 of 9

6 4. The table below presents protein binding affinities measured in a series of isothermal titration calorimetry experiments in which different fragments of microtubule plus-end tracking proteins were mixed. a, no binding detected b, the data did not allow a rigorous measurement of affinity but did suggest an affinity in the millimolar range Data taken from Weisbrich et al., (2007) Nat Struct Mol Biol, 14, page 6 of 9

7 The protein constructs used in these experiments and how they relate to full-length EB1, p150glued and CLIP170 are shown schematically below: a) Compare and contrast the information obtained from surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) experiments. b) How does mutation of the acidic tail of EB1 impact binding to p150glued. Use the data in the table and your knowledge of CAP-Gly domains to justify your answer. c) Suggest why ClipZn1 does not interact with p150n while ClipZn2 does (1 mark) page 7 of 9

8 d) What do the effects of mutation of residues 68, 69, 90 and 93 of p150n tell you about the interaction with ClipZn2. (3 marks) e) CLIP170 has been shown to form a closed, inhibited state in which ClipCG1 and ClipCG2 domains interact with the Zn12 region. Predict what might happen if p150glued is added to the closed form of CLIP170. Use the data in the table to justify your answer. (2 marks) f) The EB1c construct forms a homodimer in solution. Explain the binding affinities measured in ITC experiments involving EB1c and ClipCG1, ClipCG2 and ClipCG12. (3 marks) The space above this line should be sufficient for your answer page 8 of 9

9 SECTION B: Essay question Answer one question on the separate paper provided Remember to write your candidate number at the top of the page and indicate whether you have answered question A or B Mark total for this section: 50 EITHER A) Using appropriate examples, explain how solution NMR spectroscopy has illuminated the role of intrinsically disordered binding motifs in protein-protein recognition. OR B) Discuss how early and late compartment-specific gene expression is established in the forespore during sporulation in Bacillus. Refer to the roles of relevant protein-protein complexes, commenting on the techniques used to identify and characterize these complexes, and the supporting evidence for their existence. page 9 of 9