molecular bio: test 1 chapter 4

Fill in the blank spaces in the table below. The first row has been completed for you. *See figure 4-5

*see figure 4-5

For each of the following, indicate whether the individual folded polypeptide chain forms a globular (G) or fibrous (F) protein molecule. A. keratin B. lysozyme C. elastin D. collagen E. hemoglobin F. actin

A—F; B—G; C—F; D—F; E—G; F—G

One way in which an enzyme can lower the activation energy required for a reaction is to bind the substrate(s) and distort its structure so that the substrate more closely resembles the transition state of the reaction. This mechanism will be facilitated if the shape and chemical properties of the enzyme's active site are more complementary to the transition state than to the undistorted substrate; in other words, if the enzyme were to have a higher affinity for the transition state than for the substrate. Knowing this, your friend looked in an organic chemistry textbook to identify a stable chemical that closely resembles the transition state of a reaction that converts X into Y. She generated an antibody against this transition-state analog and mixed the antibody with chemical X. What do you think might happen?

If your friend was lucky, she made a "catalytic antibody" that catalyzed the conversion of X into Y. Such catalytic antibodies have been isolated and shown to catalyze a variety of reactions, but with lower efficiency than genuine enzymes.

Which of the following globular proteins is used to form filaments as an intermediate step to assembly into hollow tubes? (a) tubulin (b) actin (c) keratin (d) collagen

(a) tubulin

Drawn below are segments of β sheets, which are rigid pleated structures held together by hydrogen bonds between the peptide backbones of adjacent strands (Figure Q4-31). The amino acid side chains attached to the α-carbons are omitted for clarity. *see figure 4-31 A. For panel (A) and for panel (B), indicate whether the structure is parallel or antiparallel. B. Draw the hydrogen bonds as dashed lines (||||||).

A. (A) is parallel and (B) is antiparallel. B. See Figure A4-31.

Examine the three protein monomers in Figure Q4-38. From the arrangement of complementary binding surfaces, which are indicated by similarly shaped protrusions and indentations, decide whether each monomer could assemble into a defined multimer, a filament, or a sheet. * see figure 4-38

A. defined multimer with four subunits, called a tetramer B. sheet C. filament

Protein families arise when a protein sequence that generates a stable fold diverges over many generations and acquires new functions. One example of this can be seen in the globin family. Myoglobin is a stable monomeric protein that can help carry oxygen using a heme molecule. Hemoglobin is stable as a tetramer. It also carries oxygen through the use of heme groups, but it is useful over a much more dynamic range of oxygen than myoglobin. The "globin fold" is structurally conserved across these proteins, but the ability to tetramerize arose through genetic drift and natural selection. Provide an explanation for how the globin sequence can change and still produce the same overall fold. Support your explanation by suggesting the location and type of sequence alterations that might have little effect on the overall protein fold, but may favor the formation of a multisubunit protein.

Amino acids that are found on the surface of a folded monomeric protein are the best candidates for mutations. Because they are on the surface of the protein, the side-chain interactions are not important for forming the structural core. If alternative amino acids are polar, they can interact equally well with the aqueous environment. It is likely that the sequence comparisons between myoglobin and the α/β globins will reflect changes of surface residues. Furthermore, we may predict that if the surface amino acids changed from polar amino acids to nonpolar amino acids, this would promote multimerization. Nonpolar residues would interact with each other rather than the polar molecules in the cytosol.

For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once. The α helices and β sheets are examples of protein __________________ structure. A protein such as hemoglobin, which is composed of more than one protein __________________, has __________________ structure. A protein's amino acid sequence is known as its __________________ structure. A protein __________________ is the modular unit from which many larger single-chain proteins are constructed. The three-dimensional conformation of a protein is its __________________ structure. allosteric ligand secondary domain primary subunit helix quaternary tertiary

The α helices and β sheets are examples of protein secondary structure. A protein such as hemoglobin, which is composed of more than one protein subunit, has quaternary structure. A protein's amino acid sequence is known as its primary structure. A protein domain is the modular unit from which many larger single-chain proteins are constructed. The three-dimensional conformation of a protein is its tertiary structure.

Proteins can assemble to form large complexes that work coordinately, like moving parts inside a single machine. Which of the following steps in modulating the activity of a complex protein machine is least likely to be directly affected by ATP or GTP hydrolysis? (a) translation of protein components (b) conformational change of protein components (c) complex assembly (d) complex disassembly

(a) translation of protein components

Determining a protein's sequence, site of covalent modification, or entire threedimensional structure requires the careful analysis of complex data sets. Which of the data sets below would you have to interpret to solve the structure of a protein by using X-ray crystallography? *see figure 4-74 for answer choices

(b)

Use your knowledge of amino acid characteristics to order the peptides below according to the net charge contributed by their side chains at physiological pH (~pH 7). Each peptide contains eight amino acids. Use the single-letter amino acid designations to generate your list, placing the most negatively charged peptide on the left and the most positively charged peptide on the right. In addition, for each peptide, list the total number of positive and negative charges. Remember that, at neutral pH, the amino terminus carries a positive charge and the carboxyl terminus carries a negative charge. A. YGAKKRA B. ARRKSTRK C. DERKQNST D. DDAEIYSA E. NQSTYEEG

(D) This peptide has 4 negative charges and one positive charge. The peptide in (E) has 3 negative charges and one positive charge. The peptide in (C) has 3 negative charges and 3 positive charges. The peptide in (A) has 4 positive charges and one negative charge. The peptide in (B) has 6 positive charges and one negative charge.

Which of the following statements about allostery is true? (a) Allosteric regulators are often products of other chemical reactions in the same biochemical pathway. (b) Allosteric regulation is always used for negative regulation of enzyme activity. (c) Enzymes are the only types of proteins that are subject to allosteric regulation. (d) Binding of allosteric molecules usually locks an enzyme in its current conformation, such that the enzyme cannot adopt a different conformation

(a) Allosteric regulators are often products of other chemical reactions in the same biochemical pathway.

You have two purified samples of protein Y: the wild-type (nonmutated) protein and a mutant version with a single amino acid substitution. When washed through the same gel-filtration column, mutant protein Y runs through the column more slowly than the normal protein. Which of the following changes in the mutant protein is most likely to explain this result? (a) the loss of a binding site on the mutant-protein surface through which protein Y normally forms dimers (b) a change that results in the mutant protein acquiring an overall positive instead of a negative charge (c) a change that results in the mutant protein being larger than the wild-type protein (d) a change that results in the mutant protein having a slightly different shape from the wild-type protein

(a) Dimers formed by a normal protein will run through the gel-filtration column faster than a mutant protein Y monomer. Choice (b) is unlikely, because gel-filtration columns separate proteins on the basis of size, not charge or affinity for small molecules. Choice (c) is unlikely, because if the mutant protein were larger than normal it would be less able to enter the porous beads and would run through the column faster than the normal protein. Choice (d) is unlikely, because a small change in shape without a change in size would be unlikely to have a major effect on the behavior of a protein in a gel-filtration column.

The three-dimensional coordinates of atoms within a folded protein are determined experimentally. After researchers obtain a protein's structural details, they can use different techniques to highlight particular aspects of the structure. What visual model best displays a protein's secondary structures (α helices and β sheets)? (a) ribbon (b) space-filling (c) backbone (d) wire

(a) Space-filling and wire models illustrate all atoms, which makes it difficult to see the secondary structure. Backbone models are better but not as good as the ribbon models, which are stylized and make it easy for even the untrained eye to see secondary structures.

Protein folding can be studied using a solution of purified protein and a denaturant (urea), a solvent that interferes with noncovalent interactions. Which of the following is observed after the denaturant is removed from the protein solution? (a) The polypeptide returns to its original conformation. (b) The polypeptide remains denatured. (c) The polypeptide forms solid aggregates and precipitates out of solution. (d) The polypeptide adopts a new, stable conformation.

(a) The polypeptide returns to its original conformation.

Polypeptides are synthesized from amino acid building blocks. The condensation reaction between the growing polypeptide chain and the next amino acid to be added involves the loss of ________________. (a) a water molecule. (b) an amino group. (c) a carbon atom. (d) a carboxylic acid group

(a) a water molecule.

The process of generating monoclonal antibodies is labor-intensive and expensive. An alternative is to use polyclonal antibodies. A subpopulation of purified polyclonal antibodies that recognize a particular antigen can be isolated by chromatography. Which type of chromatography is used for this purpose? (a) affinity (b) ion-exchange (c) gel-filtration (d) any of the above

(a) affinity

Globular proteins fold up into compact, spherical structures that have uneven surfaces. They tend to form multisubunit complexes, which also have a rounded shape. Fibrous proteins, in contrast, span relatively large distances within the cell and in the extracellular space. Which of the proteins below is not classified as a fibrous protein? (a) elastase (b) collagen (c) keratin (d) elastin

(a) elastase

Which of the following methods would be the most suitable to assess whether your protein exists as a monomer or in a complex? (a) gel-filtration chromatography (b) gel electrophoresis (c) western blot analysis (d) ion-exchange chromatography

(a) gel-filtration chromatography

Cyclic AMP (cAMP) is a small molecule that associates with its binding site with a high degree of specificity. Which types of noncovalent interactions are the most important for providing the "hand in a glove" binding of cAMP? (a) hydrogen bonds (b) electrostatic interactions (c) van der Waals interactions (d) hydrophobic interactions

(a) hydrogen bonds

Which of the following is not a feature commonly observed in α helices? (a) left-handedness (b) one helical turn every 3.6 amino acids (c) cylindrical shape (d) amino acid side chains that point outward

(a) left-handedness

Instead of studying one or two proteins or protein complexes present in the cell at any given time, we can now look at a snapshot of all proteins being expressed in cells being grown in specific conditions. This large-scale, systematic approach to the study of proteins is called _______________. (a) proteomics. (b) structural biology. (c) systems biology. (d) genomics.

(a) proteomics.

For some proteins, small molecules are integral to their structure and function. Enzymes can synthesize some of these small molecules, whereas others, called vitamins, must be ingested in the food we eat. Which of the following molecules is not classified as a vitamin but does require the ingestion of a vitamin for its production? (a) retinal (b) biotin (c) zinc (d) heme

(a) retinal

To study how proteins fold, scientists must be able to purify the protein of interest, use solvents to denature the folded protein, and observe the process of refolding at successive time points. What is the effect of the solvents used in the denaturation process? (a) The solvents break all covalent interactions. (b) The solvents break all noncovalent interactions. (c) The solvents break some of the noncovalent interactions, resulting in a misfolded protein. (d) The solvents create a new protein conformation.

(b) In the case of choice (b), the polypeptide is completely unfolded, allowing the complete refolding to be observed. Detergents do not break the covalent bonds of the polypeptide backbone [choice (a)]. Mild detergents that do not break all noncovalent interactions within a protein would not lead to misfolding but instead to partial unfolding [choice (c)]. Proteins fold into only one single, correct conformation. Denaturation followed by renaturation of a protein does not generate a new protein fold [choice (d)].

Studies conducted with a lysozyme mutant that contains an Asp!Asn change at position 52 and a Glu!Gln change at position 35 exhibited almost a complete loss in enzymatic activity. What is the most likely explanation for the decrease in enzyme activity in the mutant? (a) increased affinity for substrate (b) absence of negative charges in the active site (c) change in the active-site scaffold (d) larger amino acids in the active site decreases the affinity for substrate

(b) The negatively charged amino acids aspartic acid and glutamic acid are required to attack the sugar bonds being cleaved by lysozyme. Replacing these with side chains that are the same length and are polar, but uncharged, would most probably affect only the catalysis, not the binding of substrate or the stability of the protein.

Which of the following is not a feature commonly observed in β sheets? (a) antiparallel regions (b) coiled-coil patterns (c) extended polypeptide backbone (d) parallel regions

(b) coiled-coil patterns

Coiled-coils are typically found in proteins that require an elongated structural framework. Which of the following proteins do you expect to have a coiled-coil domain? (a) insulin (b) collagen (c) myoglobin (d) porin

(b) collagen

Which of the following methods would be the most suitable to assess the relative purity of a protein in a sample you have prepared? (a) gel-filtration chromatography (b) gel electrophoresis (c) western blot analysis (d) ion-exchange chromatography

(b) gel electrophoresis

Two or three α helices can sometimes wrap around each other to form coiledcoils. The stable wrapping of one helix around another is typically driven by ________________ interactions. (a) hydrophilic (b) hydrophobic (c) van der Waals (d) ionic

(b) hydrophobic

Proteins bind selectively to small-molecule targets called ligands. The selection of one ligand out of a mixture of possible ligands depends on the number of weak, noncovalent interactions in the protein's ligand-binding site. Where is the binding site typically located in the protein structure? (a) on the surface of the protein (b) inside a cavity on the protein surface (c) buried in the interior of the protein (d) forms on the surface of the protein in the presence of ligand

(b) inside a cavity on the protein surface

The phosphorylation of a protein is typically associated with a change in activity, the assembly of a protein complex, or the triggering of a downstream signaling cascade. The addition of ubiquitin, a small polypeptide, is another type of covalent modification that can affect the protein function. Ubiquitylation often results in ______________. (a) membrane association. (b) protein degradation. (c) protein secretion. (d) nuclear translocation.

(b) protein degradation.

The variations in the physical characteristics between different proteins are influenced by the overall amino acid compositions, but even more important is the unique amino acid ______________. (a) number. (b) sequence. (c) bond. (d) orientation

(b) sequence.

The biosynthetic pathway for the two amino acids E and H is shown schematically in Figure Q4-60. You are able to show that E inhibits enzyme V, and H inhibits enzyme X. Enzyme T is most likely to be subject to feedback inhibition by __________________ alone. *see figure 4-60 (a) H (b) B (c) C (d) E

(c) C

Which of the following statements is true? (a) Disulfide bonds are formed by the cross-linking of methionine residues. (b) Disulfide bonds are formed mainly in proteins that are retained within the cytosol. (c) Disulfide bonds stabilize but do not change a protein's final conformation. (d) Agents such as mercaptoethanol can break disulfide bonds through oxidation.

(c) Choice (a) is incorrect, because S-S bonds are formed between cysteines. Choice (b) is incorrect, because disulfide bonds are formed mainly in extracellular proteins. Choice (d) is incorrect; the bonds are broken by mercaptoethanol, but by reduction not by oxidation.

Fully folded proteins typically have polar side chains on their surfaces, where electrostatic attractions and hydrogen bonds can form between the polar group on the amino acid and the polar molecules in the solvent. In contrast, some proteins have a polar side chain in their hydrophobic interior. Which of the following would not occur to help accommodate an internal, polar side chain? (a) A hydrogen bond forms between two polar side chains. (b) A hydrogen bond forms between a polar side chain and the protein backbone. (c) A hydrogen bond forms between a polar side chain and an aromatic side chain. (d) Hydrogen bonds form between polar side chains and a buried water molecule.

(c) Choices (a), (b), and (d) are all common mechanisms employed to accommodate buried polar amino acids. Choice (c) is not likely to accomplish this because aromatic side chains are nonpolar, hydrophobic residues and will not interact favorably with a polar, hydrophilic side chain.

Which of the following is not true of molecular chaperones? (a) They assist polypeptide folding by helping the folding process follow the most energetically favorable pathway. (b) They can isolate proteins from other components of the cells until folding is complete. (c) They can interact with unfolded polypeptides in a way that changes the final fold of the protein. (d) They help streamline the protein-folding process by making it a more efficient and reliable process inside the cell.

(c) They can interact with unfolded polypeptides in a way that changes the final fold of the protein.

Energy required by the cell is generated in the form of ATP. ATP is hydrolyzed to power many of the cellular processes, increasing the pool of ADP. As the relative amount of ADP molecules increases, they can bind to glycolytic enzymes, which will lead to the production of more ATP. The best way to describe this mechanism of regulation is ___________. (a) feedback inhibition. (b) oxidative phosphorylation. (c) allosteric activation. (d) substrate-level phosphorylation

(c) allosteric activation.

Which of the following mechanisms best describes the manner in which lysozyme lowers the energy required for its substrate to reach its transition-state conformation? (a) by binding two molecules and orienting them in a way that favors a reaction between them (b) by altering the shape of the substrate to mimic the conformation of the transition state (c) by speeding up the rate at which water molecules collide with the substrate (d) by binding irreversibly to the substrate so that it cannot dissociate

(c) by speeding up the rate at which water molecules collide with the substrate

Which of the following methods used to study proteins is limited to proteins with a molecular mass of 50 kD or less? (a) X-ray crystallography (b) fingerprinting (c) nuclear magnetic resonance (d) mass spectroscopy

(c) nuclear magnetic resonance

Antibody production is an indispensible part of our immune response, but it is not the only defense our bodies have. Which of the following is observed during an infection that is not a result of antibody-antigen interactions? (a) B cell proliferation (b) aggregation of viral particles (c) systemic temperature increase (d) antibody secretion

(c) systemic temperature increase

Which of the following methods would be the most suitable to assess levels of expression of your target protein in different cell types? (a) gel-filtration chromatography (b) gel electrophoresis (c) western blot analysis (d) ion-exchange chromatography

(c) western blot analysis

Motor proteins use the energy in ATP to transport organelles, rearrange elements of the cytoskeleton during cell migration, and move chromosomes during cell division. Which of the following mechanisms is sufficient to ensure the unidirectional movement of a motor protein along its substrate? (a) A conformational change is coupled to the release of a phosphate (Pi). (b) The substrate on which the motor moves has a conformational polarity. (c) A conformational change is coupled to the binding of ADP. (d) A conformational change is linked to ATP hydrolysis.

(d) A conformational change is linked to ATP hydrolysis.

Although all protein structures are unique, there are common structural building blocks that are referred to as regular secondary structures. Some proteins have α helices, some have β sheets, and still others have a combination of both. What makes it possible for proteins to have these common structural elements? (a) specific amino acid sequences (b) side-chain interactions (c) the hydrophobic-core interactions (d) hydrogen bonds along the protein backbone

(d) Choices (a), (b), and (c) are factors that contribute to the uniqueness of proteins, but because the protein backbone is a repeating, identical unit, interactions between backbone atoms are something that all proteins can have in common.

The Ras protein is a GTPase that functions in many growth-factor signaling pathways. In its active form, with GTP bound, it transmits a downstream signal that leads to cell proliferation; in its inactive form, with GDP bound, the signal is not transmitted. Mutations in the gene for Ras are found in many cancers. Of the choices below, which alteration of Ras activity is most likely to contribute to the uncontrolled growth of cancer cells? (a) a change that prevents Ras from being made (b) a change that increases the affinity of Ras for GDP (c) a change that decreases the affinity of Ras for GTP (d) a change that decreases the rate of hydrolysis of GTP by Ras

(d) Ras is a proto-oncogene. When it is active, it promotes cell growth. Choice (d) is the only option that would lead to an increase in Ras activity. Choices (a), (b), and (c) would decrease its activity.

The correct folding of proteins is necessary to maintain healthy cells and tissues. Unfolded proteins are responsible for such neurodegenerative disorders as Alzheimer's disease, Huntington's disease, and Creutzfeldt-Jakob disease (the specific faulty protein is different for each disease). What is the ultimate fate of these disease-causing, unfolded proteins? (a) They are degraded. (b) They bind a different target protein. (c) They form structured filaments. (d) They form protein aggregates.

(d) They form protein aggregates.

Molecular chaperones can work by creating an "isolation chamber." What is the purpose of this chamber? (a) The chamber acts as a garbage disposal, degrading improperly folded proteins so that they do not interact with properly folded proteins. (b) This chamber is used to increase the local protein concentration, which will help speed up the folding process. (c) This chamber serves to transport unfolded proteins out of the cell. (d) This chamber serves to protect unfolded proteins from interacting with other proteins in the cytosol, until protein folding is completed.

(d) This chamber serves to protect unfolded proteins from interacting with other proteins in the cytosol, until protein folding is completed.

Protein structures have several different levels of organization. The primary structure of a protein is its amino acid sequence. The secondary and tertiary structures are more complicated. Consider the definitions below and select the one that best fits the term "protein domain." (a) a small cluster of α helices and β sheets (b) the tertiary structure of a substrate-binding pocket (c) a complex of more than one polypeptide chain (d) a protein segment that folds independently

(d) a protein segment that folds independently

Lysozyme is an enzyme that specifically recognizes bacterial polysaccharides, which renders it an effective antibacterial agent. Into what classification of enzymes does lysozyme fall? (a) isomerase (b) protease (c) nuclease (d) hydrolase

(d) hydrolase

β Sheets can participate in the formation of amyloid fibers, which are insoluble protein aggregates. What drives the formation of amyloid fibers? (a) denaturation of proteins containing β sheets (b) extension of β sheets into much longer β strands (c) formation of biofilms by infectious bacteria (d) β-sheet stabilization of abnormally folded proteins

(d) β-sheet stabilization of abnormally folded proteins

The protein structure in Figure Q4-28 contains four α helices arranged in a bundle. Label each helix by number (1 to 4) starting from the N-terminus and going to the C-terminus, both of which are labeled. List the six possible pairings of these helices, and indicate within each pair whether the helices are parallel or antiparallel. *see figure 4-28

* see figure 4-28

Fill in the blanks with the labels in the list below to identify various parts of the antibody structure in Figure Q4-49. A. constant domain of the light chain B. constant domain of the heavy chain C. antigen-binding site D. variable domain of the heavy chain E. variable domain of the light chain *see figure 4-49

*see figure 4-49

For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once. A newly synthesized protein generally folds up into a __________________ conformation. All the information required to determine a protein's conformation is contained in its amino acid __________________. On being heated, a protein molecule will become __________________ as a result of breakage of __________________ bonds. On removal of urea, an unfolded protein can become __________________. The final folded conformation adopted by a protein is that of __________________ energy. composition irreversible reversible covalent lowest sequence denatured noncovalent stable highest renatured unstable

A newly synthesized protein generally folds up into a stable conformation. All the information required to determine a protein's conformation is contained in its amino acid sequence. On being heated, a protein molecule will become denatured as a result of breakage of noncovalent bonds. On removal of urea, an unfolded protein can become renatured. The final folded conformation adopted by a protein is that of lowest energy.

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. Collagen is a protein that participates in both the cytoskeleton and the extracellular matrix. B. Collagen fibers and elastin fibers serve similar functions, which is expected because the structure of these two types of fibers is quite similar. C. The assembly of both collagen and elastin fibers requires the formation of disulfide bonds.

A. False. Collagen is not used inside the cell; it is secreted and incorporated into the existing collagen fibers in the extracellular matrix. B. False. Collagen fibers and elastin fibers are very different in structure and function. Collagen fibers are highly organized, triple-strand coiled-coils that provide strength to hold tissue together. Elastin molecules are linked together in a loose network with disulfide bonds; this allows the fibers (and tissues) to stretch without tearing. C. True.

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. Feedback inhibition is defined as a mechanism of down-regulating enzyme activity by the accumulation of a product earlier in the pathway. B. If an enzyme's allosteric binding site is occupied, the enzyme may adopt an alternative conformation that is not optimal for catalysis. C. Protein phosphorylation is another way to alter the conformation of an enzyme and serves exclusively as a mechanism to increase enzyme activity. D. GTP-binding proteins typically have GTPase activity, and the hydrolysis of GTP transforms them to the "off" conformation.

A. False. Feedback inhibition occurs when an enzyme acting early in a metabolic pathway is inhibited by the accumulation of a product late in the pathway. The inhibitory product binds to a site on the enzyme that lowers its catalytic activity. B. True. C. False. Although phosphorylation of a protein can change its conformation, this modification may be either as a positive or a negative regulator of enzyme activity, depending on the protein in question. D. True.

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. The amino acids in the interior of a protein do not interact with the ligand and do not play a role in selective binding. B. Antibodies are Y-shaped and are composed of six different polypeptide chains. C. ATPases generate ATP for the cell. D. Hexokinase recognizes and phosphorylates only one of the glucose stereoisomers.

A. False. The interior amino acids form a structural scaffold that maintains the specific orientation for those that directly interact with the ligand. Changes to these interior amino acids can change the protein shape and render it nonfunctional. B. False. Although antibodies are Y-shaped, they are composed of four, not six, polypeptide chains. There are two heavy chains and two light chains. C. False. ATPases hydrolyze ATP; they do not produce it. These enzymes enable the cell to harness the chemical energy stored in the high-energy phosphate bonds. D. True.

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. Generally, the total number of nonpolar amino acids has a greater effect on protein structure than the exact order of amino acids in a polypeptide chain. B. The "polypeptide backbone" refers to all atoms in a polypeptide chain, except for those that form the peptide bonds. C. The chemical properties of amino acid side chains include charged, uncharged polar, and nonpolar. D. The relative distribution of polar and nonpolar amino acids in a folded protein is determined largely by hydrophobic interactions, which favor the clustering of nonpolar side chains in the interior.

A. False. The order in which amino acids are linked is unique for each protein and is the most important factor in determining overall protein structure. B. False. Peptide bonds are planar amide bonds that are central to the polypeptide backbone formation. The atoms in the amino acid side chains are not considered to be part of the backbone. C. True. D. True.

Indicate whether the following statements are true or false. If a statement is false, explain why it is false. A. Van der Waals interactions and hydrophobic interactions are two ways to describe the same type of weak forces that help proteins fold. B. A large number of noncovalent interactions is required to hold two regions of a polypeptide chain together in a stable conformation. C. A single polypeptide tends to adopt 3-4 different conformations, which all have equivalent free-energy values (G).

A. False. Van der Waals attractions are weakly attractive forces that occur between all atoms. Hydrophobic interactions are only observed between nonpolar molecules in the context of an aqueous solution. B. True. C. False. There is a single, final fold for every polypeptide. The fold adopted is the "best" conformation, for which the free energy (G) of the molecule is at a minimum.

For each polypeptide sequence listed, choose from the options given below to indicate which secondary structure the sequence is most likely to form upon folding. The nonpolar amino acids are italicized. A. Leu-Gly-Val-Leu-Ser-Leu-Phe-Ser-Gly-Leu-Met-Trp-Phe-Phe-Trp-Ile B. Leu-Leu-Gln-Ser-Ile-Ala-Ser-Val-Leu-Gln-Ser-Leu-Leu-Cys-Ala-Ile C. Thr-Leu-Asn-Ile-Ser-Phe-Gln-Met-Glu-Leu-Asp-Val-Ser-Ile-Arg-Trp amphipathic α helix hydrophilic β sheet amphipathic β sheet hydrophobic α helix hydrophilic α helix

A. Hydrophobic α helix. Nearly all of the amino acid side chains in this sequence are nonpolar or hydrophobic, which favors the only hydrophobic option given in the list. B. Amphipathic α helix. In an ideal α helix, there are 3.6 residues per complete turn. Thus, an amphipathic helix with one hydrophobic side and one hydrophilic side will have, minimally, nonpolar side chains (N) repeating every third then next fourth amino acid: NxxNxxxNxxNxxxN. Polar side chains (P) will have the same pattern but shifted relative to the nonpolar side chains; for example, xxPxxxPxxPxxxPxxP. C. Amphipathic β sheet. Because of the zigzag-like structure of a β sheet, a sequence with alternating nonpolar and polar side chains may form an amphipathic β sheet that is hydrophobic on one side and hydrophilic on the other.

Figure Q4-30 shows a fatty-acid-binding protein from two different angles. Apart from its two short α helices, its structural elements are extended strands that form a curved β sheet, which is called a β barrel. A. Draw arrows on the six top strands in panel (A) (those running horizontally) to determine whether the β barrel is made up of parallel or antiparallel strands. B. Look at panel (B) and predict the relative distribution of polar and nonpolar side chains (inside the barrel or outside the barrel) and explain your answer. *see figure 4-30

A. The strands form an antiparallel β sheet. *see figure 4-30 B. The prediction is that nonpolar side chains are on the inside of the barrel, and polar side chains are distributed to the outside of the barrel. The outside of the barrel is completely exposed to the aqueous solvent and it is stabilized by solvent-protein hydrogen bonds. And, although the barrel seems open and accessible to solvent molecules, this protein binds fatty acids and would most probably do that by enclosing them inside the barrel, away from the aqueous solvent and close to nonpolar amino acid side chains lining the inside of the barrel.

Chromatography is frequently used to purify proteins from cellular extracts. There are various strategies that can be used, depending on the tools and reagents available. You are interested in isolating additional proteins that interact with your protein target, but your labmates have used all the purified protein stocks. A. Why would you need purified target protein to do this experiment? B. What other strategies/tools could you use to carry out the affinity chromatography? C. What are the limitations to the method you described in part B that would not be a concern if you could use the purified protein directly?

A. The target protein can be covalently linked to the resin used to make the affinity column. When cell extracts are applied to the column, any proteins that associate with high affinity to your target will be bound until they are eluted in the presence of high-salt buffer. B. You could instead use antibodies specific for your target protein. This is very similar to the first procedure, but instead of your protein being directly linked to the column resin, the antibodies are bound instead. The antibody will bind to your target, which should be bound to any associated proteins in the cell extracts. C. One important limitation to recognize is that the antibodies could block the binding surface typically recognized by the other proteins that would normally bind to your target, reducing the number of binding partners isolated by the affinity chromatography. You would also need to have an antibody that recognizes your target protein that could be attached to the column in the first place.

The sequences for three different tripeptides are written out below. Indicate whether you expect to find them in the inner core or on the surface of a cytosolic protein, and explain your answer. A. Serine-Threonine-Tyrosine B. Alanine-Glycine-Leucine C. Proline-Serine-Alanine

A. This tripeptide is made up of entirely polar amino acids, which means it will most likely be found on the surface of the protein, interacting with the aqueous environment of the cytosol. B. This peptide is made up of nonpolar amino acids. The side chains are most likely buried in the interior of the protein, which would promote interactions with other hydrophobic side chains and avoid unfavorable interactions with the aqueous environment of the cytosol. C. This peptide is made up of both polar and nonpolar amino acids, and one of the nonpolar amino acids is proline. Proline residues have a restricted degree of conformational freedom because the side chain is covalently linked to the backbone nitrogen as well as the α-carbon. So, a likely scenario is that the proline is at the surface of the protein, providing a structural turn between two secondary structure elements (β strands or α helices), the serine is still close enough to the surface to interact with water, and the alanine is close enough to the interior of the protein to interact with other hydrophobic side chains.

In an attempt to define the protein domains of protein X, you treat it with a protease and use polyacrylamide gel electrophoresis to analyze the peptides produced. In the past, you have used chymotrypsin to perform this experiment, but the stock of this enzyme has been used up. You find a stock of elastase and decide to use it instead of waiting for a new stock of chymotrypsin to arrive. A. Give two reasons why elastase is a good substitute for chymotrypsin in this assay. B. Why might proteolysis of the same substrate by chymotrypsin or elastase yield different results?

A. You might assume that chymotrypsin and elastase would yield the same results because (1) they are both serine proteases and (2) they have a high degree of structural similarity. B. The slight structural differences of the substrates cause the enzymatic activities of the proteases to differ. As a result, they have different substrate affinities and cleave the bond between a different set of amino acids.

For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once. Any substance that will bind to a protein is known as its __________________. Enzymes bind their __________________ at the __________________. The enzyme hexokinase is so specific that it reacts with only one of the two __________________ of glucose. Enzymes catalyze a chemical reaction by lowering the __________________, because they provide conditions favorable for the formation of a __________________ intermediate called the __________________. Once the reaction is completed, the enzyme releases the __________________ of the reaction. activation energy inhibitors products active site isomers substrates free energy ligand transition state high-energy low-energy

Any substance that will bind to a protein is known as its ligand. Enzymes bind their substrates (or inhibitors) at the active site. The enzyme hexokinase is so specific that it reacts with only one of the two isomers of glucose. Enzymes catalyze a chemical reaction by lowering the activation energy, because they provide conditions favorable for the formation of a high-energy intermediate called the transition state. Once the reaction is completed, the enzyme releases the products of the reaction.

Which of the following statements is true? (a) Peptide bonds are the only covalent bonds that can link together two amino acids in proteins. (b) The polypeptide backbone is free to rotate about each peptide bond. (c) Nonpolar amino acids tend to be found in the interior of proteins. (d) The sequence of the atoms in the polypeptide backbone varies between different proteins.

Choice (c) is the correct answer. Choice (a) is untrue, because some proteins also contain covalent disulfide bonds (-S-S- bonds) linking two amino acids. Choice (b) is untrue, because the peptide bond is rigid. Choice (d) is untrue, because the sequence of atoms in the polypeptide backbone itself is always the same from protein to protein; it is the order of the amino acid side chains that differs.

You wish to produce a human enzyme, protein A, by introducing its gene into bacteria. The genetically engineered bacteria make large amounts of protein A, but it is in the form of an insoluble aggregate with no enzymatic activity. Which of the following procedures might help you to obtain soluble, enzymatically active protein? Select all options that may be useful. Explain your reasoning. A. Make the bacteria synthesize protein A in smaller amounts. B. Dissolve the protein aggregate in urea, then dilute the solution and gradually remove the urea. C. Treat the insoluble aggregate with a protease. D. Make the bacteria overproduce chaperone proteins in addition to protein A. E. Heat the protein aggregate to denature all proteins, then cool the mixture.

Choices A, B, and D are all worth trying. Some proteins require molecular chaperones if they are to fold properly within the environment of the cell. In the absence of chaperones, a partly folded polypeptide chain has exposed amino acids that can form noncovalent bonds with other regions of the protein itself and with other proteins, thus causing nonspecific aggregation of proteins. A. Because the protein you are expressing in bacteria is being made in large quantities, it is possible that there are not enough chaperone molecules in the bacterium to fold the protein. Expressing the protein at lower levels might increase the amount of properly folded protein. B. Urea should solubilize the protein and completely unfold it. Removing the urea slowly and gradually often allows the protein to refold. Presumably, under less crowded conditions, the protein should be able to refold into its proper conformation. C. Treating the aggregate with a protease, which cleaves peptide bonds, will probably solubilize the protein by trimming it into pieces that do not interact as strongly with one another; however, chopping up the protein will also destroy its enzymatic activity. D. Overexpressing chaperone proteins might increase the amount of properly folded protein. E. Heating can lead to the partial denaturation and aggregation of proteins to form a solid gelatinous mass, as when cooking an egg white, and rarely helps solubilize proteins.

their degree of polarity. Each peptide contains eight amino acids. Use the singleletter amino acid designations to generate your list, placing the most polar peptide on the left and the most nonpolar peptide on the right. A. SGAKKRAH B. CATWNGQV C. FWGTSILA D. DDAEIHWA E. SSTAMYRK

E, A, D, B, C

Using the example of the p53 protein, postulate how different combinations of covalent modifications can lead to a wide variety of protein functions.

In a protein with a complex regulatory protein code, such as p53, the covalent attachment or removal of modifying groups can change the protein's behavior, its activity or stability, its binding partners, or its location within a cell. In the case of p53, there are at least 20 different locations (amino acids) that can be modified through such processes as phosphorylation, ubiquitylation, and acetylation.

Some of the enzymes that oxidize sugars to yield usable cellular energy (for example, ATP) are regulated by phosphorylation. For these enzymes, would you expect the inactive form to be the phosphorylated form or the dephosphorylated form? Explain your answer.

In general, the inactive form is the phosphorylated form. The main purpose of glycolysis and the citric acid cycle is to generate ATP; thus, the enzymes are inactive when the concentration of ATP is high and active when it is low. It makes sense that cells would not want to have to phosphorylate their enzymes to turn them on when ATP levels are already low, because phosphorylation requires ATP.

You have produced a monoclonal antibody that binds to the protein actin. To be sure that the antibody does not cross-react with other proteins, you test your antibody in a western blot assay on whole-cell lysates that have been subjected to electrophoresis under nondenaturing conditions (shown in Figure Q4-37A) and denaturing conditions (shown in Figure Q4-37B). Does the antibody cross-react with other proteins? If so, does this explain the results in the two western blots? If not, how do you explain the difference observed? *see figure 4-37

No, the antibody does not seem to cross-react with other proteins. In each western blot, there is only one band, indicating that only one protein is bound by the monoclonal antibody. Actin is a protein that forms long filaments. Under the nondenaturing conditions of the first gel (A), the filaments remain intact and, as a multiprotein complex, actin migrates very slowly through the polyacrylamide matrix. In the case where sodium dodecyl sulfate (SDS) is added (denaturing conditions), actin filaments dissociate into monomers. Thus, the band is lower in panel (B) because the monomers have a lower molecular weight and migrate faster through the gel.

You are digesting a protein 625 amino acids long with the enzymes Factor Xa and thrombin, which are proteases that bind to and cut proteins at particular short sequences of amino acids. You know the amino acid sequence of the protein and so can draw a map of where Factor Xa and thrombin should cut it (Figure Q4-36). You find, however, that treatment with each of these proteases for an hour results in only partial digestion of the protein, as summarized under the figure. List the segments (A-E) of the protein that are most likely to be folded into compact, stable domains. *see figure 4-36

Segments B and D. To cut the protein chain, Factor Xa and thrombin must bind to their preferred cutting sites. If these sites are folded into the interior of a stable protein domain, it will be much more difficult for the proteases to gain access to them than if they are part of a relatively unstructured part of the chain. Hence, sites that are folded inside a protein domain are protected from cleavage by a protease. From the sizes of the fragments produced by digestion of the protein with Factor Xa, we can conclude that the enzyme does not cut at the sites in regions B or D, although it does cut in region E. From the sizes of the fragments produced by thrombin, we can conclude that this enzyme cuts at the sites in A, C, and E. Therefore, the segments of the protein that are most likely to be folded into compact, stable domains are B and D.

Enzymes generally make good drug targets because a specific reaction of interest can be targeted with a high degree of selectivity. Consider the following three drugs and explain why, although reaction-specific, the first two produce side effects, while the third does not. A. Statins inhibit HMG CoA reductase to block intracellular cholesterol synthesis. B. Methotrexate inhibits dihydrofolate reductase, which subsequently leads to blocked DNA replication. C. Gleevec® inhibits BCR, a kinase that is only produced in certain types of leukemia cells.

The first two inhibitors (statins and methotrexate) are blocking reactions that are very important to all cells, not just the cells affected by the illness in question. Thus, drug side effects can be hard to predict, and may be very specific to the patient being treated. In the case of chronic myeloid leukemia (CML), the mutant enzyme is specific to the leukemia cells, and no other cells in the body are affected by this drug. This also means that the usefulness of this drug is limited to those CML patients that have this specific mutation.

For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once. The human immune system produces __________________ of different immunoglobulins, also called __________________, which enable the immune system to recognize and fight germs by specifically binding one or a few related __________________. The hypervariable structural element that forms the ligand-binding site is comprised of several __________________. Purified antibodies are useful for a variety of experimental purposes, including protein purification using __________________ chromatography. affinity billions ligands antibodies coiled-coils loops antigens hundreds size-exclusion β strands ion-exchange

The human immune system produces billions of different immunoglobulins, also called antibodies, which enable the immune system to recognize and fight germs by specifically binding one or a few related antigens. The hypervariable structural element that forms the ligand-binding site is comprised of several loops. Purified antibodies are useful for a variety of experimental purposes, including protein purification using affinity chromatography.

Typical folded proteins have a stability ranging from 7 to 15 kcal/mole at 37°C. Stability is a measure of the equilibrium between the folded (F) and unfolded (U) forms of the protein, with the unfolded form having a greater free energy (see Figure Q4-20). For a protein with a stability of 7.1 kcal/mole, calculate the fraction of protein that would be unfolded at equilibrium at 37°C. The equilibrium constant (Keq) is related to the free energy (ΔG°) by the equation Keq = 10-ΔG°/1.42. *see figure 4-20

The ΔG° of the unfolding reaction is equal to the stability of the protein, 7.1 kcal/mole. At equilibrium, the ratio of unfolded to folded protein is Keq = [U]/[F] = 10-ΔG°/1.42 = 10-7.1/1.42 = 10-5. Thus, one molecule in 100,000 is unfolded.

Knowing that there are 20 different possible amino acids that can be used at each position in a polypeptide, calculate the number of different polypeptides that could theoretically be produced for a protein that is 180 amino acids in length. Do you expect to find all of these possible protein sequences produced in living systems? Explain your answer.

There are 20180 possible sequences for a 180 amino acid polypeptide (20 different possible amino acids for each position). No, we would not expect all of the theoretically possible proteins to be made. A much smaller subset can be expected in living systems because it is not likely that all sequences would lead to a stably folded protein. Natural selection favors the retention of genes that encode proteins with stable conformations.

Match the general type of biochemical reaction catalyzed in the left column with the class of enzyme listed in the column on the right. ___ removes a phosphate group from a molecule ___ hydrolyzes ATP ___ hydrolyzes bonds between nucleotides ___ adds phosphate groups to molecules ___catalyzes reactions in which one molecule is oxidized and another is reduced ___ hydrolyzes peptide bonds ___ joins two ends of DNA together ___ catalyzes the synthesis of polymers such as RNA and DNA ___ rearranges bonds within a single molecule A. ATPase B. polymerase C. ligase D. kinase E. isomerase F. nuclease G. oxido-reductase H. protease I. phosphatase

_I_ removes a phosphate group from a molecule _A_ hydrolyzes ATP _F_ hydrolyzes bonds between nucleotides D_ adds phosphate groups to molecules _G_ catalyzes reactions in which one molecule is oxidized and another is reduced _H_ hydrolyzes peptide bonds _C_ joins two ends of DNA together _B_ catalyzes the synthesis of polymers such as RNA and DNA

Match the basic protein functions in the left column with a specific example of that type of protein in the column on the right. ___ gene regulatory ___ motor ___ storage ___enzyme ___ transport ___structural ___special purpose ___ receptor ___ signal A. insulin B. carboxylase C. rhodopsin D. hemoglobin E. ferritin F. myosin G. green fluorescent protein H. tubulin I. homeodomain proteins

gene regulatory __I_ motor __F_ storage __E_ enzyme __B_ transport __D_ structural __H_ special purpose __G_ receptor __C_ signal __A_