Exam # 1)
1) Glucagon is a 29 amino acid peptide hormone whose sequence is shown below:
NH3+-His- Ser- Gln- Gly - Thr -Phe -Thr - Ser - Asp - Tyr - Ser - Lys -Tyr - Leu - Asp –
Ser - Arg - Arg - Ala -Gln - Asp - Phe - Val - Gln - Trp - Leu - Met - Asn - Thr-COO-
Studies show that in type I diabetes, the lack of insulin is accompanied by hypersecretion of glucagon. Diabetic patients are currently treated with exogenous insulin, but some researchers suggested that treatment should include a strategy for reducing the effect of glucagon by administration of glucagon antagonists. For this purpose various glucagon analogs have been synthesized (Table below) and these were tested for their ability to bind to receptors and initiating signal transduction.
Glucagon Analog
Glucagon des Asp 9
Asp9 →Lys
Lys12 → Ala
Lys12 → Glu
Arg17 → Ala
Arg17 → Glu
Arg18 → Ala
Arg18 → Glu desHis1 des His1des Asp9 des His1-Lis9
% Binding affinity
100
45
54
17
1
38
21
13
6
63
7
70
% Activity
100
8
0
60
80
29
95
94
100
44
0
0
[*The prefix des indicates that the specific amino acid was deleted. Arrows indicate replacement of amino acid. % Binding affinity refers to the ability of the analogue to bind to membrane receptors in the liver. % activity represents the ability to stimulate the production of cyclic AMP (cAMP).] a. Why does the hypersecretion of glucagon worsen the symptoms of diabetes? Design a scheme that describes the mechanism of action of glucagon in hepatocytes. b. Calculate the isoelectric point (pI) of glucagon. c. What conclusions could you make regarding the effects of changes in amino acids at positions 12, 17 and 18? d. Of the analogss tested, which is the best glucagon antagonist? Justify your response.
2) You want to purify three proteins (A, B, C) from a mixture using a combination of gel filtration chromatography (also known as size exclusion chromatography) and ion exchange chromatography. Describe the protocol you use to separate these proteins, describing the pH of the buffer to be used and the order of elution of proteins.
The proteins in the mixture have the following characteristics:
Protein A: pI = 4.2, molar mass 40 kDa

Protein B: pI = 4.4; molar mass 70 kDa
Protein C: pI = 8.0, molar mass 42 kDa.
3) A Michaelian enzyme whose conformation is stable between pH 3 and pH 9, is only active when a particular histidine residue of the structure is protonated. On the same graph design the approximate curves of the initial reaction rate (V
0
) that this enzyme catalyzes as a function of the concentration of its substrate in the following three cases:
3a. reaction at pH 3
3b. Reaction pH 6
3c. reaction at pH 3, in the presence of non-competitive inhibitor concentration (I) such that 25% of the enzymes are in the form of enzyme-inhibitor complex (EI).
Use arbitrary values on the ordinate and abscissa but specify the units used to measure velocity and concentration.
4) Crocodiles may remain submerged for over an hour, even though they possess a myoglobin concentration about 100 less than diving mammals such as whales. The hemoglobin of crocodiles has several differences in the primary structure when compared to the human protein. The graph below was obtained from in vitro experiments using hemoglobin of crocodiles (circles) and men (squares) in two situations: in the absence (open symbols) and presence (filled symbols) of CO
2
. a. Propose a hypothesis to explain the resistance of crocodiles to lack of oxygenation.
Experiments similar to those described above were made using a hybrid hemoglobin [α chains of human + β chains of crocodiles] (circles) and a modified hybrid hemoglobin
[crocodile α chains + human β chains bearing multiple amino acid substitutions]
(squares) in two situations: in the absence (open symbols) or presence (filled symbols) of CO
2
.

b. What can be deduced by comparison of the structures α and β chains of human hemoglobin and crocodile? c. Make a hypothesis to explain the behavior of the modified hybrid hemoglobin.
5) Many animals rely on fat stores for energy during periods of hibernation, migration and other situations that involve radical changes in metabolism. One of the most important changes in fat metabolism occurs in gray bears. These animals remain in a continuous state of dormancy for periods of up to seven months.
Different from other animals, they maintain temperatures between 32 and 35 ° C, very close to the normal temperature. In this state they expend up to 6000 kcal per day, even though the bears do not eat, drink, urinate or defecate for months.
Experimental studies show that, during hibernation, the bears survive solely on stored fat. For example, the urea produced by degradation of proteins is reabsorbed in the kidneys in order that the nitrogen can be reused in the synthesis of amino acids. Explain, justifying your reasoning, how a bear during hibernation, using fat reserves can: a. obtain energy to maintain body temperature, synthesize proteins and other metabolites. b. maintain blood glucose. c. Considering that the bear loses water during hibernation (for example via the warm, moist air coming out of their lungs), explain how it is possible, with fat metabolism, to restore part of the lost water. d. Why, in these situations, do large animals use fat rather than carbohydrates (ie glycogen) to store energy?
6) The stability of body mass and overall appearance of an individual hides large daily fluctuations of metabolism. For example, food intake subjects the organism to opposing situations: the abundance and lack of nutrients. The adjustment of our metabolism to different metabolic conditions occurs by processes which are known to "metabolic

regulation". Several strategies have been developed by organisms to modulate and control the different metabolic pathways. Give at least three examples of mechanisms utilized by organisms to regulate their metabolic pathways. Explain in detail how each of these mechanisms operates increasing or decreasing the velocity of specific pathways.
7) In 2009, the Nobel Prize for medicine was awarded to Elizabeth Blackburn, Carol and Jack Grider Szostack for the discovery of the enzyme telomerase. This enzyme is a reverse transcriptase which extends the ends of linear chromosomes (telomeres) using
RNA as template. Considering the role of Okazaki fragments in the replication of the discontinuous strand, discuss the importance of telomerase to replicate the ends of chromosomes.
8) In 2010 a research group led by Craig Venter published in Science a study which reportted the creation of the first synthetic organism (bacteria of the genus
Mycoplasma), wherein the only source of genetic information was a synthetic DNA which was inserted into a recipient cell depleted of its original DNA. Discuss how the researchers could reach that feat. What kinds of genes were required for the assembly of a viable bacterial cell?

Exam # 2
1) In May this year, the journal Science published a paper by the group led by J. C.
Venter that described the creation of the first synthetic life form. This created great furor in both specialized and high circulation media. The feat was only possible due to a number of technological advances, in which some new and unpublished techniques wee combined with other well established ones. The first step was to determine the genome of the “mother” bacteria (Mycoplasma mycoideum). a. Describe in detail a technique used to sequence DNA.
After the DNA fully sequenced, the information obtained was used to chemically synthesize DNA fragments which carried the original sequence of M. mycoideum plus sequences designed by the researchers as "watermarks" that allowed their identification.
In order to combine these fragments into a single molecule, they were introduced into yeast cells which possessed the necessary machinery to perform this ligation. This was only possible because the fragments had identical sequences at their extremities. b. What process occurred in the yeast cells which allowed the connection of the fragments? Name two physiological functions of this process, highlighting its importance for the functioning of the cell and organism.
The assembled DNA was extracted from the yeast cells and inserted into Mycoplasma capricolum, creating an organism originated from a DNA that did not exist on any ancestor. Thus, this organism was considered a form of "synthetic life". c. What processes occur based on the information contained in the "synthetic" DNA that caused the recipient M. capricolum cell become and perpetuate a new organism, called
M. mycoides JCVI-1.0?
2. RNAse A is a digestive enzyme secreted by the pancreas into the lumen of the small intestine, where it catalyses the hydrolysis of RNA to its component nucleotides. The active site has two catalytic histidines active (His 12 and His 119) in the active site. The pK values of His12 and His 119 this protein are 5.4 and 6.4, respectively. a. In order to achieve maximal enzyme activity, His 12 has to be deprotonated and protonated His 119. What would be the optimum pH of the enzyme? Construct a graph of hypothetical velocity of the enzymatic reaction as a function of pH for this enzyme. b. The scheme below shows the three dimensional structure and mechanism of catalysis
RNAse. Identify secondary structures in the scheme and describe how they are stabilized.

c. In addition to the catalytic histidine, the enzyme has three lysines (Lys 7 Lys 41 and
Lys 66). How would you expect them to help stabilize the substrate (RNA) in the active site? d. Why this enzyme does not catalyze the hydrolysis of DNA?
3) A 3 year old was taken to hospital after suffering a fainting spell during a swimming lesson. The blackout had been preceded by a period of wheezing. At the hospital, exams indicated blood acidosis with high blood concentrations of lactic acid, chronic muscle weakness and a slight delay in neuromotor development. Pediatric neurologists who examined the girl suspected mitochondrial disease and, in fact, identified a mutation in the catalytic site of DNA polymerase γ, the only human polymerase which is located in mitochondria. This mutation alters the conformation of the active site such that the 3'-
OH terminus of the nascent chain is embedded in a hydrophobic pocket in the mutant enzyme.
Based on these observations, answer the following questions: a. Why was the episode of lactic acidosis accompanied by wheezing?

b. Explain why lactic acidosis occurred, discussing the metabolic pathways involved. c. Discuss the effect of the mutation in the DNA polymerase on DNA synthesis.
4) Suppose your research project has as its object of study the BQBM gene, which is normally only expressed in neurons. Although you know the coding sequence of the
BQBM gene, the function of its product is not known.
A construction in which the regulatory region of the BQBM gene was ligated to a reporter gene showed that this gene is expressed in neuronal cells, but not in fibroblasts.
However, if a specific sequence in the regulatory region is mutated, the reporter gene is expressed on both cell types. a. What would be the function of this region in the expression of mutated BQBM? What kind of element would be acting on it? b. Name three approaches that can be used to unravel the function of the BQBM gene and / or its gene product. c. Choose one of the approaches you mentioned in item (b) and describe the logic of the experiments you would perform and how the data obtained from these experiments allow progress in understanding the function BQBM. (It is not necessary to include technical details of the experiments, just how they will be designed and what kind of results would be expected and the way they would be interpreted).
5) The amino acids X, Y and Z have in their side chains a thiol group, an amino group of pKa <8 and a carboxyl group of pKa> 4, respectively. a) Identify each based on chemical structures and pKas values given in the attached table. b) Assume that X, Y and Z react with each other to form the tripeptide sequence Y-X-Z that had to be separated from the tetrapeptide Gly-Ala-Trp-Leu electrophoresis on cellulose acetate strips. Could you use the pH corresponding to the pI of this tripeptide during electrophoresis? If not, suggest another pH and justify your response.
Indicate the migration expected for each of the oligopeptides on the strip represented below. d) Suppose now that the amino acids X, Y and Z were part of a globular protein consisting of a single polypeptide chain. In what types of chemical interaction would they engage to maintain the protein tertiary structure? e) Assume that this protein was a Michaelian enzyme with amino acid Y present in its active site. If replacing Y by an alanine (Ala) would lead to a totally inactive mutant, what could you conclude about the role of Y in enzymatic function of the protein?

6) The liver is the most important organ for metabolic integration in mammals. Explain how the liver can: a. Maintain blood sugar at appropriate levels. b. Control the storage of energy reserves in the body.