Mueller Cume Classic Experiments in Biochemistry April 10 th, 2004 Name: 1. (10) 2. (10) 3. (10) 4. (10) 5. (10) EC. (5) TTAL (50)
1. Franz Knoop deduced the catabolism of fatty acid by feeding dogs fatty acid derivates with ω-phenyl groups. a. Briefly recount his results, including the products obtained from fatty acids with odd and even numbers of carbons. Feel free to use diagrams as you see fit. [5 pts] b. Briefly explain the conclusions that can be drawn from the Knoop experiments. Do not over-conclude by describing β-oxidation as we now understand it. [5 pts] 2
2. Stöckenius and Racker devised and conducted an elegant experiment to test Peter Mitchell s chemiosmotic hypothesis (that a proton gradient drives ATP synthesis by ATP synthase). They created vesicles containing ATP synthase and bacteriorhodopsin in particular orientations, and they then added ADP and P i to a solution containing those vesicles. Remember that bacteriorhodopsin is a transmembrane, light-driven proton pump. a. What product s formation was monitored by Stöckenius and Racker? [2 pts] b. What external stimulus was critical to Stöckenius and Racker s experiment? [2 pts] c. Diagram the Stöckenius and Racker experiment using the cartoons given below to denote ATP synthase and bacteriorhodopsin. Pay close attention to the orientation of each protein in the vesicle. Include the proton gradient and the location of small molecule reactant(s) and product(s). [6 pts] ATP synthase bacteriorhodopsin 3
3. Beadle and Tatum irradiated Neurospora crassa (a mold) with X-rays and then characterized mutants to formulate the one gene, one protein hypothesis. They focused on mutations in the arginine biosynthetic pathway, which was known at the time of their experiment to proceed by way of ornithine and citrulline, as shown in the simplified scheme: precursor ornithine citrulline arginine Beadle and Tatum isolated arginine auxotrophs by growing irradiated Neurospora on rich medium to get clonal cultures and then testing the ability of each of those to grow on minimal medium. Those that could not grow on minimal medium were then tested for the ability to grow on minimal medium supplemented with arginine. The cultures that grew were arginine auxotrophs. a. Describe the next experimental steps that Beadle and Tatum took to establish that each of the Neurospora that were auxotrophic for arginine had a defect in only one of the enzymes required for arginine biosynthesis. Don t describe the results until part b! [4 pts] b. Describe the results of Beadle and Tatum in terms of your answer to part a and the simplified diagram of arginine biosynthesis given above. Correlate particular results to an enzyme carrying out a particular step in the pathway. You may find a table a conveinent way to present the results, but you need to provide enough text to explain the table. To clarify your description, you may add abbreviations (for example, A, B, C or I, II, III) to the simplified pathway given above to designate the individual enzymes just make sure that the labeling is clear and consistent with your description. [6 pts] 4
4. Avery, McCarty, and MacLeod did the critical experiment to show that DNA is the genetic material, although universal acceptance of that then-radical notion did not come until the later experiments of Hershey and Chase. Avery, McCarty, and MacLeod followed up on the observation by Griffiths that smooth (type S) Streptococcus pneumoniae are virulent (mice injected with them die) but rough (type R) streptococci are not (mice injected with them live). Mice injected with heat-killed type-s streptococci are fine, too, but when type-r streptococci are mixed for a time with heat-killed type-s streptococci before injection, mice die, and type-s streptococci are recovered from their corpses. The heat-killing breaks open the bacteria, so a transforming principle is in the cell extracts and that transforming principle is the genetic material. Griffith s experiment can be simply diagrammed: type-s heat cell extracts + type-s type-r Diagram the set of three experiments that Avery, McCarty, and MacLeod conducted to demonstrate that DNA rather than RNA or protein is the genetic material. Use the same general format as shown for Griffiths experiment. [10 pts] 5
5. For each, write the name of the principal investigator who was responsible for producinng each of the following key results. [2 pts each] a. The primary structure of proteins can suffice to dictate the tertiary structure. b. For DNA base composition, A = T and G = C. c. The first crystal structure of a protein (sperm whale myoglobin). d. Yeast extracts can still carry out glycolysis, showing that live organisms are not required to effect the chemical transformations. hint: He filtered the broken yeast through a funnel to remove cell debris. e. Recombination proceeds by branched junctions between two DNA molecules. 6
EC. In one of the first uses of NMR in biochemistry, Ronald Breslow measured the 1 H NMR spectrum of thiamin pyrophosphate (TPP, below) at several times after it was diluted into D 2. Briefly describe what he observed, and explain how that observation provided critical evidence for the mode of action of TPP. [5 pts] P P S H N TPP N NH 2 N CH 3 7