1. General themes in protein function. a) Vanishingly few proteins work in isolation; nearly all interact with other molecules to perform their biological work. Please think about HOW proteins interact with other molecules. In one or two sentences, please describe the types of forces that typically mediate these intermolecular interactions, and please provide an example to illustrate one of these forces. (6 points) These are typically weak intermolecular forces: H-bonds, Hydrophobic interactions, ion pairs b) Following up on part (a), please indicate one common protein denaturing agent that we have discussed which you would NOT expect to have a similarly disruptive effect on typical interactions of proteins with their binding partners. (For the sake of this question, assume that this protein denaturing agent could be targeted only to the interacting region and thus would have no impact on the protein itself.) (4 points) Anything that breaks covalent intrachain bonds would work here – e.g. 3M BME proteases c) Consider an enzyme which is active in a solution environment of very high pH, around 13. The function of this enzyme depends on the presence of a protonated lysine residue at one position in the structure. Please tell me why this poses a problem, and describe how the protein's structure might alleviate this problem by altering the local microenvironment of the lysine. Please be explicit in your answer, and provide an example of how the structure could minimize the problem. (8 points) This is a problem because at pH 13, free lysine would be deprotonated. Thus, the microenvironment around this lysine must inhibit its deprotonation, e.g. , by stabilizing the protonated, positive form. An example of this could involve one or more Aspartic or Glutamic acid side chains arranged so that the positive charge on lysine is stabilized by ion pairing.

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2. Hemoglobin structure and function. The other day (in casual conversation, of course) Julian and I were discussing affinity chromatography, and the scenario arose that one could purify hemoglobin from a complex biological mixture on the basis of its affinity for BPG. Specifically, in this scenario, one could construct a column from agarose beads to which were attached BPG molecules. Joe has suggested that this could be turned around to use hemoglobin-beads as a means of purifying BPG. Which sets up the following situation: Suppose that I want to purify carbon monoxide using this same principle: make a column with agarose beads to which are attached hemoglobin tetramers. In order to preserve hemoglobin function, my column is specifically linked to the hemoglobin in such a way that it does not interfere either with access to the heme group nor impede the flexibility of inter-subunit contacts. (Please ask me for clarification if you are not comfortable with this scenario thus far!) a) Please explain briefly why this column should have an affinity for carbon monoxide. (4 points) Carbon monoxide looks a lot like O2, and so binds hemoglobin. As the hemoglobin is the column, essentially, the column binds CO. b) Please explain briefly why I would especially need to ensure that I do not impede inter-subunit interactions of the bound hemoglobin if I plan to elute my carbon monoxide by shifting the pH of my wash buffer by a small amount (that is, not a large enough pH change to denature the enzyme). (If you are not sure of your answer to part (a), please ask me for assistance.) (6 points) The release of CO by decreasing pH shifting hemoglobin from R to T forms requires movement at the intersubunit interface. c) On the back of this page, please give me a detailed explanation addressing ONE of the following questions:

• which would be a more effective way to elute the carbon monoxide, increasing the pH or decreasing the pH?

• which would be a more effective way to elute the carbon monoxide, adding cyanic acid (HCN) to the medium, or adding sodium cyanide (NaCN) to the medium? (or will they be the same? will neither be any good?)

• which would be a more effective way to elute the carbon monoxide, adding sodium chloride to the medium, or adding sodium nitrate to the medium? (or will they be the same? will neither be any good?)

Your answer should include a careful exposition of why each eluting agent should or should not work, making reference to relevant aspects of hemoglobin structure and cooperativity, and relating this artificial situation to the physiological role of hemoglobin. (26 pts) -- Decrease -- HCN both complete and RT -- NaCl (Cl-) favors T form

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3. Kinetic and thermodynamic analysis of enzymes. a) Consider an enzyme which catalyzes the hydrolysis of two substrates with differing efficiency. For substrate X, the KM is 1.45 mM, and for substrate Y, the KM is 1.63 mM. The catalytic rate constant (kcat) for the two substrates differs by a factor of 100, with kcat for substrate X being higher. Please sketch out what you would expect to see if thermodynamic and kinetic parameters for these two reactions were determined and plotted on the axes below. Please clearly distinguish and label your curves! A reference curve is given on the free energy plot; please treat this as (enzyme + substrate X). Assume that the origin on both plots is at (0, 0). (10 points)

V max X Y as substrate V max Y Y KM X ≈KM Y

b) Which binds more tightly to its ligand, a protein with a Kd of 1.6 nM or a protein with a Kd of 1.6 pM? (Or can't you be sure based on this information?) (no explanation necessary; 3 points) The protein with Kd of 1.6pm is less likely to dissociate; it binds more tightly. c) If the two proteins in part (b) are enzymes, which one would you predict would yield a higher maximal catalytic rate? (Or can't you be sure based on this information?) (no explanation necessary; 3 points) Impossible to tell; Kd and Vmax are not necessarily linked.

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1. Fundamental concepts in prgtein-pqrification, architecture, and function. Eachof the following questions is wo{th I poin\, and most can be answered in a few words.

a) The term "heterozygote advantage" implies that individuals who are homozygous areat a dlsadvantage. For the HbS allele (i.e., the E6V mutation on the hemoglobin B chain).please briefly state what the potential disadvantages are for individuals who arehomozygous for HbA (the wild-type) and for those who are homozygous for HbS.

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b) Consider two tansiently interacting proteins, such as an antibody binding to a proteinantigen, or a peptide hormone binding to a cell-surface receptor. Which of the followingtreatments is likely to disrupt the interaction between the proteins without denaturingthem?r.0 M l3ME 1.0 M NaNO, hot 6.0 M HCI all of thesc l*r""f , ir;?orf1

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, t r . €Dc) Consider a protein wittr motafrn#s or:ioil *o n, of 4.5. Woutd you expect thisprotein to be more soluble in dbuffer at pH 4.7 or in a buffer at pH 6.0, or should thepH make no difference whatsoever to protein solubility?

Les> ssl,rfto .Jcse -{o p Ai . '/v\b@ d,rble, & 60.

d) Suppose that I am in the process of purifying Tyrosinase by affinity chromatographyon a column that consists of agarose beads covalently coupled to the amino acid tyrosine.I have passed a mushroom cell extract over this column, and washed it, and now I wantto elute the tyrosinase off of my column. Please suggest one way to do this (in amanner that is not likely to denature the tyrosinase).

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[ . a ls^ttle) Free glutamic acid in solution has a side-chain pKa of about 4. In a protein, due to theinteraction of glutamic acid with surrounding residues, however, its pKa is apt to bedifferent. For instance, if you found a protein in which a glutamic acid side chain was inclose proximity to an aspartic acid side chain, would you expect it to have a pKa lowerthan 4, equal to 4, or higher than 4?

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u _H 6l ' t * H+ + 6l ' . -

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Question #1, continued...

f) I mentioned in class that the disease phenotype in Sickle Cell Anemia is much moresevele when blood pH drops. Please briefly outline the molecular basis for ho*-achange in pH affects red blood cell sickling.

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g) Sequences of the protein elongation factor Tu from all known life forms show thepresence of a histidine at a particular position. Please propose why this might be so. (Iwant a specific mechanistic proposal here, not simply "it must be important',. yourproposal need not be true, but it must be biochemically credible.)

e.5. HisO -' \@ "1"r.,J f". st-+.a-.

h) Suppose that you could replace the conserved histidine in part (g) with another aminoacid by site-directed mutagenesis. Ifyou wanted to test your hypothesis from part (g),what amino acid would you substitute for the conserved histidine, and what impact-.----a-r---..-.

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i) Both carbon monoxide and carbon dioxide impair the ability of hemoglobin to bindoxygen, but only one of these is an allosteric effector. Which is it?

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.j) Hemoglobin and myoglobin have very similar tertiary structures, both bind oxygen,and they almost certainly share a common evolutionary origln. Yet hemoglotlin has asigmoidal oxygen binding curve while myoglobin does not. Why not?

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2. Hemoglobin structure and biological function. Over the past week and a half, wehave examined several agents that affect the ability of hemoglobin to bind, transport, anddeliver oxygen in the blood. The resulting impact on hemoglobin can often be visualizedas a change in the shape of the oxygen binding curve.

Several such agents are listed below. Please pick one of the substances or conditionsfrom the list and

. sketch the change you would expect to see in the oxygen binding curve forhemoglobin relative to the reference curve shown below for adult

(4 pointsThen, on the next page,

. explain the physiological impact of this perturbation -- what happenshemoglobin funct ion? (6 points)

. carefully explain the molecular mechanism underlying the biochemical ihow does the change in hemoglobin function occur? (In your answer, you

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. briefly outline the biochemical logic that explains why this is a desirable"feature" of hemoglobin function -- why does it happen? (2 points)

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a decrease in blood pH (such as occurs with heavy physical exertion) .-q di lh

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3. Applying what you've learned: aflinity techdquf ;'€frH d

characterizatiorr. r1r" on"optoi"in Ras 1a protein implicated in a lergs nffi ol

cancers) is berieved to'"1"" Jiffir.' i'ii int"'u"ion YfffXJ:::i:IJlllfr ,l{;'TilL;;it of several years' worth of investigations' a key argl

t""",ft".t-a to be critical to the interaction between Ras and Raf'

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l) The Raf and Ras-GST proteins are incubated together in the p'L-esence ot

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2) The samples ^-" 'pu" 'it*i ^icrocentrifuge' and the supernatant is removed'

3) Samples of the supernatani a"O ttre peUe; are analysed by SDS-PAGE'

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iffi#;il',# riag ;" n"l i' i-po'iunt ror tr'e tindinq of Ras to Raf? Please

srppoft your answer wlrn "

')i'i-J^il^"ition that refers to specific elements of the data

figure. (8 Points)

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The enzyme acetylchlolinesterase (AChE) is an important target for insecticides. It is also, not surprisingly, the site of several known mutations in pest insects that have developed insecticide resistance. A paper published in 2001 in the journal Insect Biochemistry and Molecular Biology (volume 31, pages 805-816) reported the systematic analysis of several of the mutations found in the Australian sheep blowfly (a major pest species) regarding their impact on enzyme function and likely roles in conferring insecticide resistance. Data from this paper are the subject of questions 1 and 2; copies of the paper are available if you wish to look through it. 1. The authors report the following kinetic data for the wild-type enzyme and one of the mutants: Form of enzyme Km +/- SD (uM) Vmax +/- SD (nM/min) Wild-type 23 +/- 1 19.1 +/- 0.3 mutant #1 430 +/- 20 18.1 +/- 0.4 Broadly speaking, the AChE enzyme contains three spatial regions that are functionally important: • the aromatic residues responsible for binding to the quaternary amine portion of the substrate • the catalytic triad (entirely analogous to the triad in the serine proteases) • an allosteric regulatory region called the Peripheral Anionic Site. Given these data, please tell me whether the behavior of this mutant is consistent with involvement in any of these three sites. In your answer, please be sure to:

• indicate what biochemical 'phenotype' you would expect for mutations to each region in terms of the kinetic parameters Km and Vmax

• justify each biochemical impact in terms of the structural and/or functional role of the region

• compare your expectation for each region with the observed activity for this mutant enzyme

• state explicitly whether you think that the effects of mutant #1 are consistent with your expectation

Diagrams and figures are welcome if they clarify your analysis! (28 points) Km +/- SD (uM) Vmax +/- SD (nM/min) KM goes WAY up No big change Aromatics binding quaternary amine: mutation here should primarily inhibit substrate binding rather than catalysis; expect KM to rise, Vmax had no change This matches the phenotype of the mutant. Catalytic triad: mutations here should primarily hurt catalytic efficiency rather than substrate binding; expect KM to have no change, Vmax decreases This does NOT match the phenotype of the mutant.

PAS: mutations here (this is a regulatory region), as with the catalytic triad, should primarily affect catalysis and hence Vmax decreases. This doe NOT match the mutant phenotype. 2. The authors of the paper introduced on the previous page had intended to use a detergent known as Triton X-100 in the extraction of the enzyme (as part of their initial lysis step -- where we used the bead-beater in our yeast cytochrome c extractions). To their surprise, however, they found that this component of their extraction buffer altered the activity of AChE, as shown in their figure 5:

a) Please analyze the impact this buffer component is having on the enzyme -- is it acting as an inhibitor (if so, what kind?)? an activator? What kinetic parameters are being primarily affected? Would you expect to find this substance bound at/near the active site or far from it (or can't you tell)? From these data, can you infer anything about the likely molecular shape of Triton X-100? (15 points) 2. a) The figure shows that in the presence of Triton X-100, Vmax is essentially unchanged, but KM appears to increase considerably (about a factor of 2). This is the hallmark of COMPETITIVE INHIBITION. These data suggest that TX-100 is competing with acetylcholine for binding to the ACLE active site. If this is the case, one might expect TX-100 to bear structure similarly to acetylcholine, or to intermediate in the hydrolysis of the ACh ester bond.

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Question #2, continued... b) The authors only provided a Lineweaver-Burk plot of these data. Please fill out their presentation with a sketch of a Michaelis-Menton plot of the same data shown above. Please be sure to include curves for both the enzyme alone and AChE plus Triton. Your sketch need not be painstakingly accurate, but primary features should be roughly to scale. (6 points) Vmax control (no TX-100) Plus Triton X-100 Michaelis-Menton plot V Kmapp increases [S] approx 2X (note that the TX-100 curve appears straight here due to the limitations of my computer graphics ability and not by intent; it should have the usual MM shape...)

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3. Bits and pieces. For each of the following questions, please answer in a few words or a brief sentence. Long explanations are neither necessary nor likely to serve you well. Diagrams that help you understand the question or provide an answer are fine. (3 pt each) a) At first glance, it seems counterintuitive that blood BPG levels would be higher in individuals adjusted to life at high altitude than in individuals living at sea level, because BPG favors the T state of hemoglobin and thus reduces hemoglobin's affinity for oxygen. Since oxygen is relatively scarce at high altitude, why is reduced oxygen binding affinity a good thing? (a few words should suffice here) Reduced binding at high altitude equates to better delivery of oxygen to the periphery. b) Interleukin-2 (IL-2) is a peptide cytokine, a molecule that serves as a soluble chemical messenger between cells involved in the mammalian immune response. IL-2 achieves biological action by binding to a cell-surface receptor protein, which can exist in three forms -- a low-affinity, medium-affinity, or high-affinity state. Which of these three forms should give the highest Kd for binding to IL-2? A high Kd = weak binding. Therefore the Low Affinity state should have the highest Kd. c) In comparing the free energy diagrams for two enzyme catalyzed reactions, you note that although the relative free energies of substrate and ES complex are similar for the two reactions, the energy of the transition state for the conversion of ES to E+P for enzyme #2 is considerably lower than for enzyme #1. Would you expect to see a substantial difference in the KM values for these two enzymes? If so, which would be higher? Km may be slightly lower for enzyme #1, but should be essentially the same for both. d) Considering the same two enzyme-catalyzed reactions as in part (c), would you expect to see a substantial difference in the Vmax values for these two enzymes? If so, which would be higher? Vmax2 > Vmax1 e) Suppose that you encounter a protein which contains an arginine residue with an unusually low pKa, around 10. Would you expect this side chain to be in a neutrally-charged, negatively-charged, or positively-charged microenvironment? A low pKa indicates that the protonated form of arginine is being de-stabilized. This implies that the microenvironment is positively-charged.

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1. Fundamental concepts in structure and function of proteins you've met. These questions require only very short answers. You may provide explanations if you wish, but they are not necessary. (2 points) a) Please circle all the statements in the following list that you feel are accurate:

All proteins contain aromatic amino acids and hence absorb light at 280 nm.

This is true for nearly all proteins; very few are devoid of aromatic AAs

All proteins contain heme groups and hence absorb light at 410 nm.

No.

All proteins are enzymes.

No.

All hydrolases contain a catalytic triad to enhance the nucleophilicity of the side-chain that

promotes hydrolysis of substrate.

No.

All enzymes use a Peripheral Anionic Site to regulate catalysis allosterically.

No.

b) Suppose that one of your classmates overstuffs himself at Thanksgiving dinner, causing a spike in his blood sugar level. This increases the levels of glycolytic intermediates in his blood, one of which is 1,3-BPG. The high concentration drives up the concentration of the structural isomer 2,3-BPG, which you know to be a modulator of hemoglobin function. Ignoring other effects for the moment, will your classmate experience improved oxygen-delivery capacity of his blood and hence, temporary euphoria as a result of his binge? Ignoring other effects, yes, he'll get higher oxygen delivery. c) Consider two enzymes that bind to the same substrate. The first binds very well, but the second binds poorly. Which ES complex will have a higher value for Kdiss (=KM)? The second one binds poorly (that is, weakly), so it will have the higher KM. d) Suppose that I test the velocity of an enzyme-catalyzed reaction at several substrate concentrations, and plot the data on a Lineweaver-Burk plot. How can I determine the value of KM from this plot? KM is found by taking the negative reciprocal of the X-intercept e) What is the impact of a non-competitive inhibitor on the apparent KM and Vmax of an enzyme? the apparent KM will be unchanged, but the apparent Vmax will be reduced.

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2. Thinking about protein function. For each of the following situations, please answer the question in one or a few words, or indicate that there is INSUFFICIENT INFORMATION for you to be sure of the answer. Although you need not do so for full credit, if you would like to ensure that I understand the reasoning behind your answer, please justify your choice in one short sentence. (3 points each) a) Consider two enzymes. One has a KM of 85 µM and the other a KM of 13 µM. Will the first enzyme require a greater, lesser, or equal concentration of substrate to reach a rate that is one-quarter that of its maximal rate? Greater. KM1 > KM2 <<NOT FOR 2009>> b) What impact, if any, would you expect to see on the fractional occupancy curve for fetal hemoglobin in a fetus whose mother has just moved from Rio De Janeiro, on the Brazilian coast, to La Paz, Bolivia at 12,000 ft elevation in the Andes? Little change. Short term altitude aclimatization operates via increased [BPG]blood, but fetal hemoglobin is relatively BPG-insensitive. c) Consider an enzyme functioning at saturating levels of substrate. What would happen to the KM for this enzyme/substrate pair if the substrate concentration were suddenly cut in half? Nothing. The KM is a property of the enzyme, not a response to [S]. d) In comparing the free energy diagrams for two enzyme-catalyzed reactions, you note that while the stability of ES complexes is similar in both cases, the transition state for the conversion of ES to E and P for enzyme #2 is much higher in energy than the equivalent transition state for enzyme #1. Which enzyme should have a higher maximal rate (or will they be the same)? Eact is much high for enzyme #2, so Vmax will be lower for enzyme #2.

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Question #2, continued... e) Imagine a version of hemoglobin that has been engineered to not only bind oxygen, but to cleave it into two oxide anions. Other aspects of the molecule are unaffected by the change to the active site. What would be the impact on the Vmax of this engineered enzyme if the pH of the solution were increased? We know that increasing the pH of blood favors the R form of hemoglobin, and so increases its average affinity for oxygen. Thinking in terms of enzyme kinetics, this could be expected to decrease KM, but should have no effect on Vmax. f) Relative to the wild-type enzyme, what impact would you expect to see on the KM and Vmax of a mutant form of trypsin in which the serine in the catalytic triad were replaced by a tyrosine? Please justify your answer briefly. This mutation would interfere with the geometry of the catalytic triad, and so I'd expect it to decrease Vmax. It might also introduce enough bulk into the active site to inhibit binding, too, in which case we'd see an increase in KM. g) Thermolysin is a metalloprotease. With the peptide substrate GLGA, it has a KM of 14 mM and a kcat of 420 sec-1; with the substrate FGLA, it has a KM of 1.0 mM and a kcat of 450 sec-1. Please indicate the effect you expect this to have on observable kinetic parameters by sketching out velocity versus [substrate] and Lineweaver-Burk plots for both reactions. (6 points)

The big difference between proteolysis of these two substrates is the decrease in KM (14-fold change; the kcat values are nearly identical). So plateaus in the M-M plot should be nearly the same, with a change in steepness; in L-B, y-intercepts should be much the same with different X-intercepts.

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3. Thermodynamic and kinetic analysis of enzymes. In 2004 a fascinating report appeared in the journal Molecular Biology and Evolution on the evolutionary and environmental tuning of kinetic properties of an enzyme (Johns and Somero (2004) Mol Biol Evol 21: 314). The authors were studying an important metabolic enzyme, lactate dehydrogenase (LDH), from closely related fishes adapted to different environments, and found that • the cold-adapted (temperate) fish live at an average temperature of ~15 oC, and the warm-

adapted (tropical) fish live at an average temperature of 29 oC • the kinetic properties of LDH from cold-adapted fish were different from those of LDH from

warm-adapted fish • specifically, when assayed at the same temperature, both KM and kcat are higher for LDH from

cold-adapted fish • a single amino acid substitution could make the cold-adapted enzyme act (kinetically) like

the warm-adapted enzyme • for LDH from both cold-adapted and warm-adapted species, both KM and kcat increase with

increasing assay temperature (in a nearly linear fashion) • when compared at their respective operating temperatures, the two types of LDH display

very similar kinetic properties -- that is to say that the enzymes have been fine-tuned to work at the same level of catalytic efficiency even though they operate at different temperatures. At their normal operating temperatures, both types of LDH exhibit a KM of about 0.25 mM and a kcat of about 800 sec-1

Unfortunately, the authors don't provide us with any Michaelis-Menton or Lineweaver-Burk plots, so I'd like you to fill in some of the holes. Specifically, I'd like you to sketch out a Michaelis-Menton plot AND a Lineweaver-Burk plot, each of them showing four curves on the same axes: • wild-type temperate LDH at 15 oC • wild-type temperate LDH at 29 oC • mutant (tropical-like) temperate LDH at 15 oC • mutant (tropical-like) temperate LDH at 29 oC Please make sure that your four curves are clearly labeled; only relative differences between the four curves are important key points: - at their native temperatures, the two enzymes have very similar properties. Therefore the curves for the wild-type temperate LDH at 15C will be essentially superimposable with the mutant/tropical LDH at 29C. - for both enzymes, KM and kcat increase with T. Therefore, the temperate enzyme at the tropical temperature will have increased KM and Vmax relative to the paired curves. And the tropical enzyme at the temperate temperature will have decreased KM and Vmax relative to the paired curves. - this gives a graph with three curves.