BIMS 503 Exam I. Sign Pledge Here: Questions from Robert Nakamoto (40 pts. Total)

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1 BIMS 503 Exam I September 24, 2007 _ / Sign Pledge Here: Questions from Robert Nakamoto (40 pts. Total) Questions 1-6 refer to this situation: You are able to partially purify an enzyme activity from the membrane fraction of the bacterium, Thermotoga maritima. The enzyme activity hydrolyzes phosphate from deoxyribonucleotides. Silver stained SDS-PAGE shows three distinct bands that have co-purified but you do not know which protein band(s) correspond(s) to your enzyme. 1. (10 pts) Give a strategy for identifying the complete primary structure of the unknown protein. Provide the names or descriptions of the biochemical manipulations and the analytical tools you would use to identify the protein. Specific names of the tools are not necessary but state their purpose and what information they provide. Give the expected results from each step and potential problems. The strategy should include more than one approach to demonstrate convincingly that a particular protein band is responsible for the deoxyribonucleotide phospho-hydrolase activity. You may assume that the complete Thermotoga maritima genomic sequence is known. 1

2 2. (5 points). Thermotoga maritima, as the name implies, is a thermophilic bacterium which normally lives at temperatures above 80 C. What structural features of this protein would you expect to allow it to remain folded and active at such high temperatures? Please explain your answers. 2

3 3. (3 pts) Now that you have the primary structure, you find there are no obvious stretches of hydrophobic amino acids that would indicate a transmembrane segment. Furthermore, hydropathy analyses also predict a low likelihood of an integral membrane protein. Suggest possible structural motifs that could explain association with the membrane. 4. (3 pts) In taking an absorbance spectrum of the purified protein, you find that there is an unexpectedly high absorbance in the range of wavelengths 270 to 295 nm. What amino acid(s) would you expect this protein to have in exceptional abundance? 5. (3 pts) Despite thermal stability, you find that the enzymatic activity is very sensitive to molecular oxygen or oxidative conditions. What amino acids may be involved in this oxidative inactivation? 6. (3 pts) What methods might you use to estimate the types of secondary structure that make up this protein? 3

4 7. (7 pts) In Anfinsen s classical protein folding experiment, what do you expect to be the results in Experiment A versus Experiment B? Please explain your answers. A B 8. (6 pts) In Anfinsen s experiment discussed on the previous question, the ribonuclease will fold properly even if kept in a reducing environment. What are the main driving forces that lead to the proper folding? 4

5 September 24, 2007 Name: / Questions from Sepideh Khorasanizadeh (60 pts. Total) Question 1 (10 pts): a) What is Stokes shift? b) What is fluorescence quantum yield? c) Tyrosine and tryptophan have similar values of quantum yield, why is the fluorescence sensitivity of tryptophan four times that of tyrosine? 5

6 Question 2 (10 pts): a) The Circular Dichroism bands of proteins occur in two distinct regions of the UV spectrum, name these regions? b) Draw hypothetical CD spectra for two types of protein secondary structure? c) Write the appropriate units on the x and y axes of the above CD spectra? d) How would you expect these spectra to change upon addition of 6 M guanidinium hydrochloride? 6

7 Question 3 (10 pts): a) Define NMR chemical shift. b) Draw a hypothetical 1D ^1 H NMR spectrum of a protein? c) Mark the expected locations for backbone amides and side chain methyl groups? d) Which coupling interactions are measured in 2D experiments ^1 H-^1 H NOESY and ^1 H-^1 H COSY? 7

8 Question 4 (10 pts): a) List three nuclei that are NMR active and useful for protein structure analysis? b) How may the HSQC experiment be used for analysis of protein structure and function? Question 5 (10 pts): a) What is the van der Waals radius? b) What is the length of the van der Waals radius associated with a methyl group? c) What is a hydrogen bond? d) Draw an example of a hydrogen bond occurring in protein structure and indicate the distance of this bond? 8

9 Question 6 (10 pts): a) How is NMR spectroscopy used to detect the dynamics of hydrogen bonds in a protein? b) Give two examples for ^15 N relaxation NMR measurements, and indicate what information is revealed from each experiment? 9