study guide 2 questions

A polypeptide in the cytoplasm of a cell has the sequence Ala-His-Trp-Ser-Gly-Leu-Arg-Pro-Gly What is the net charge of the polypeptide? Write your answer here. ____

+2

When an amino acid sequence is given, it is written and read from the N-terminus to the C-terminus, left to right. A. True B. False

A. true

Which of the following molecules would absorb the most amount of light at a wavelength of 280 nm? A. Tryptophan B. Lysine C. Thymidine D. Glycine E. Cysteine

A. tryptophan

What are the four weak (noncovalent) interactions that determine the conformation of a protein?

Hydrogen bonds, electrostatic attractions, van der Waals attractions, and the hydrophobic force.

Regular local folding patterns in a protein, including alpha helix and beta sheet.

secondary structure

Complex three-dimensional form of a folded protein.

tertiary structure

Consider a protein with 682 amino acid residues in a single polypeptide chain. What is the approximate mass of the protein in kilodaltons?

75

Bacteriorhodopsin, an integral membrane protein of Halobacterium halobium, serves as a light-driven proton pump. During its characterization, it became apparent that it spanned the membrane seven times via alpha helices, with very little of the protein extending beyond the membrane. Considering that the membrane is approximately 30 angstroms wide and there are at least three residues per turn, what is the minimum length of this protein? A. 100 residues B. 125 residues C. 150 residues D. 175 residues E. 200 residues

A. 100 residues

In the protein secondary structure, alpha helix, the hydrogen bonds: A. are roughly parallel to the axis of the helix B. are roughly perpendicular to the axis of the helix C. occur mainly between electronegative atoms of the R groups D. occur only between some of the amino acids of the helix E. occur only near the amino and carboxyl termini of the helix

A. are roughly parallel to the axis of the helix

Which of the following statements is true regarding how R groups affect alpha helix stability? A. Oppositely charged side chains can increase stability. B. Due to its small size, glycine increases stability. C. Bulky R groups create stabilizing van der Waals forces. D. Since located on the exterior of the á helix, R groups do not significantly alter stability.

A. oppositely charged side chains can increase stability

The hydrophobic effect is a result of the___. A. tendency of water to form ordered structures around nonpolar molecules B. interaction of side chains in nonpolar molecules C. random movement of water around nonpolar molecules D. tendency of water to intercalate between nonpolar molecules

A. tendency of water to form ordered structures around nonpolar molecules

Proline tends not to participate in alpha-helices because___. A. the side chain is rigid and does not fit easily B. the side chain is too bulky C. its charge repels neighboring side chains D. it is too small

A. the side chain is rigid and does not fit easily

Small proteins may have only one or two amino acid side chains that are totally inaccessible to solvent. Even in large proteins, only about 15% of the amino acids are fully buried, A list of buried side chains from a study of 12 proteins is shown in Table 1. The list is ordered by the proportion of each amino acid that is fully buried. What types of amino acids are most commonly buried? Least commonly buried? Are there any surprises on this list?

As expected, hydrophobic amino acid side chains are most frequently buried and hydrophilic side chains are least commonly buried. Perhaps the biggest surprise in this list is the high proportion of cysteine (C) side chains that are buried. Cysteine is generally grouped with polar amino acids because of its -SH group, but its hydrophobic/hydrophilic properties indicate that it is, at best, weakly polar. Tyrosine (Y) also deserves comment. Tyrosine is usually grouped with polar amino acids because of its hydroxyl moiety; however, its measured hydrophobic/hydrophilic properties are ambiguous, indicating that it is only weakly polar. By the criterion of 'buriedness,' it clearly behaves like other polar amino acids.

At pH 7, a peptide with the sequence G-M-G-V-S-D-P-D-A has the net charge of: A. -3 B. -2 C. -1 D. +1 E. +2

B. -2

What would be the most likely description of the tertiary structure of a protein with long á helix structures? A. Globular B. Filamentous C. Phosphorylated D. Charged E. Misfolded

B. Filamentous. Since the alpha helix is rigid, the protein would be long and thin and most likely, fibrous.

All of the following statements about the alpha-helix are true except that: (more than one may be correct) A. It is stabilized by intramolecular hydrogen bonds. B. It is stabilized by minimizing unfavorable R-group interactions. C. It is stabilized by hydrophobic interactions. D. It is one type of secondary structure found in some proteins. E. Prolyl and glycyl residues tend to interrupt alpha-helical structure.

C. It is stabilized by hydrophobic interactions

Trypsin is a protease that breaks peptide bonds. Trypsin interacts with the substrate at the active site. In the active site, binding to the substrate is stabilized by hydrophobic interactions between the side chains of the amino acids in the active site and the substrate. Trypsin is a monomer. Trypsin is available in high quantity in the pancreas, a glandular organ in the digestive system and endocrine system of vertebrates. Trypsin aids in digestion. Trypsin mRNA is 500 nucleotides in length. Which of the following techniques would you most likely use to obtain purified and active trypsin? A. Northern blot B. Affinity chromatography C. Southern blot D. Sanger dideoxy sequencing E. PCR

B. affinity chromatography

The transition from the double helical structure (native state) to randomly coiled single strands is called _______________. A. renaturation B. denaturation C. randomization D. relaxation

B. denaturation

A protein consisting of two identical polypeptide subunits is a heterodimer. A. True B. False

B. false

Isoleucine residues of a soluble protein are usually at the surface of the final structure of the protein. A. True B. False

B. false

Trypsin is a protease that breaks peptide bonds. Trypsin interacts with the substrate at the active site. In the active site, binding to the substrate is stabilized by hydrophobic interactions between the side chains of the amino acids in the active site and the substrate. Trypsin is a monomer. Trypsin is available in high quantity in the pancreas, a glandular organ in the digestive system and endocrine system of vertebrates. Trypsin aids in digestion. Trypsin mRNA is 500 nucleotides in length. Trypsin contains at least two polypeptides. A. True B. False

B. false

Which of the following chemical interactions or bonds requires the most energy to break or disrupt? A. hydrophobic interactions B. glycosidic bonds C. hydrogen bonds D. van der Waal's attractive forces E. van der Waal's repulsive forces

B. glycosidic bonds

All of the following statements about the alpha-helix are FALSE except that: A. It is stabilized by intramolecular disulfide bonds. B. It is stabilized by minimizing unfavorable R-group interactions. C. It is stabilized by hydrophobic interactions. D. It is one type of tertiary structure found in some proteins. E. Prolyl and glycyl residues tend to stabilize alpha-helical structure.

B. it is stabilized by minimizing unfavorable R-group interactions

Arginine, lysine, and histidine are called basic amino acids because their side groups are___. A. negatively charged B. proton acceptors C. hydroxyl groups D. very small

B. proton acceptors

Which chromatography technique uses the weak attractive interaction between nonpolar amino acid side chains and nonpolar groups attached to polysaccharide beads to retard protein migration? A. Ion exchange B. Reverse phase C. Gel filtration D. Affinity

B. reverse phase

You perform an assay on the activity of a kinase using a 20 microliter (ìl) aliquot of a lysate prepared from cells labeled for six minutes with radioactive cysteine. The total volume of the lysate is 10 milliliters (ml). In the aliquot, you measure 20 micromoles (ìmoles) of ADP created in 3 minutes. In the lysate, you measure the amount of all proteins and determine the presence of 10 micrograms (ìg) of protein in 100 microliters of lysate. What is the specific activity of the kinase in the 10 ml lysate? (Note: all numbers below have the units of ìmole/ìl/min/ìg protein) A. 0.06 B. 0.16 C. 3.3 D. 14.7 E. 69.3

C. 3.3

A 22 kDa protein you are studying includes five prolines and consists of a single polypeptide chain. One of the prolines is at the C-terminus (carboxy end of the polypeptide) and the others are spread throughout the length of the protein. In a suspension of cells the average time required to synthesize this polypeptide is 8 minutes. At time zero, radioactive proline is added to five different suspensions of cells that are already in the process of synthesizing the protein. You isolate the complete protein from individual suspensions at 2, 4, 6, 8, and 80 minutes. (Any incomplete polypeptide chains are eliminated.) With increasing time of exposure of the cells to the radioactive proline, the amount of radioactivity due to incorporation of radioactive proline at the C-terminal location, divided by the total amount of radioactivity due to incorporation of radioactive proline at the five proline amino acid sites in the in vivo synthesized polypeptides in the isolated protein should: A. Increase to a final value of 0.25 B. Remain constant at a value of 0.2 C. Decrease to a final value of 0.2 D. Remain constant at a value of 0.25 E. Increase to a final value of 0.25

C. Decrease to a final value of 0.2

Which of the following sets of three amino acids are most probably clustered in the interior of the tertiary structure of a globular protein found in the cytoplasm of E. coli? A. Asn, Gly, Lys B. Met, Asp, His C. F, V, I D. Tyr, S, K E. A, Arg, P

C. These are all amino acids with a hydrophobic nature. Hydrophobic amino acid residues tend to be buried in the interior of globular proteins found in hydrophilic environments. The cytoplasm of cells, either prokaryotic or eukaryotic, is hydrophilic.

The amino acid cysteine is likely to participate in which one of the following bonds? A. Phosphodiester B. Glycosidic C. Disulfide D. Agarose E. Lipid

C. disulfide

Which of the following atoms may participate in hydrogen bond formation in biological models? A. Carbon B. Sulfur C. Oxygen D. Phosphorus

C. oxygen

Which is an accurate conclusion derived from the Anfinsen experiment? A. Urea is required for accurate protein folding. B. Cysteine bonds direct accurate protein folding. C. Primary sequence is sufficient for accurate protein folding. D. Proteins sample all possible combinations during folding.

C. primary sequence is sufficient for accurate protein folding

Proteins with just two polypeptide chains have primary, secondary and __________ structures. A. helical B. beta sheet C. quaternary D. tortured E. double strand

C. quaternary

Christian Anfinsen performed an experiment using bovine pancreatic RNase. He treated it with urea and beta-mercaptoethanol, and then measured the recovery of enzymatic activity after the protein gradually renatured. What was the purpose of using urea in this experiment? A. To hydrolyze the peptide bonds B. To break disulfide bonds. C. To break hydrogen bonds and ionic interactions. D. To add a phosphate to the amino acids with a hydroxyl group. E. To remove phosphates from amino acids.

C. to break hydrogen bonds and ionic interactions

The main driving force behind protein folding is: A. hydrogen bonds. B. disulfide bonds. C. van der Waal forces. D. length of polypeptide. E. rate of translation.

C. van der Waal forces

______________ interactions result from weak electrostatic interactions between two polar groups, a polar group and a nonpolar group, or two nonpolar groups. A. Hydrophobic B. Hydrophilic C. Van Der Waals D. Ionic

C. van der waals

For the following 20-residue sequence in a protein, list five amino acid residues that are likely to be buried in the protein, inaccessible to water. Pick five that are good candidates for surface residues. DLKFTISVGAPVLTREQLLE

Completely buried residues are likely to be hydrophobic: L2, F4, I6, V8, V12, L13, L18, and L19 fit this description. Highly polar residues, or at least their polar groups, are likely to be on the surface, exposed to water: D1, K3, T5, S7, T14, R15, E16, Q17, and E20 fit this description. (Note that the first amino acid, at the amino terminal end on the left, is D1. The second amino acid residue is L2, etc. This numbering system tells you the amino acid and where it exists in the primary structure of the protein.)

A protein has a molecular weight of 51 kD. To the nearest whole number, how many amino acids are present in this protein? Assume that the average molecular weight of an amino acid is 120. A. 120 B. 282 C. 365 D. 425 E. 6120

D. 425

A typical amino acid will have which of the following groups around the central alpha-carbon? A. A carboxyl group, an amino group, an R group and two hydrogen atoms. B. A carboxyl group and an amino group only. C. A carboxyl group, an amino group, a nitrogenous base and a hydrogen atom. D. A carboxyl group, an amino group, an R group and a hydrogen atom. E. A hydrogen atom and three R groups.

D. a carboxyl group, an amino group, an R group, and a hydrogen atom

You want to purify a particular protein out of a nuclear extract (prepared by grinding up the nuclei), so you immobilize an antibody against that protein on beads, place the beads in a column, and allow for the extract to pass through the column. This purification procedure is called: A. gel exclusion chromatography B. western blot C. ion exchange chromatography D. affinity chromatography E. immunoflourescense

D. affinity chromatography

the polypeptide's backbone flexibility: A. allows maximum rotation of the unbranched R groups. B. allows resonance between trans and cis configurations. C. is limited by the resonance between the alpha carbon and carboxyl oxygen. D. is limited by resonance between the carboxyl oxygen and amide nitrogen. E. is determined by balance between polar and nonpolar R groups.

D. is limited by resonance between the carboxyl oxygen and amide nitrogen

the polypeptide's backbone flexibility: A. allows maximum rotation of the unbranched R groups. B. allows resonance between trans and cis configurations. C. is limited by the resonance between the alpha carbon and carboxyl oxygen. D. is limited by resonance between the carboxyl oxygen and amide nitrogen. E. is determined by balance between polar and nonpolar R groups.

D. is limited by resonance between the carboxyl oxygen and amide nitrogen. This is the peptide bond and the resonance results in a partial double bond nature preventing full rotation about the peptide bond.

A transcriptional repressor contains an amphipathic helix. Which of the following changes within the repressor would most likely disrupt its function? A. serine to threonine. B. aspartate to glutamate. C. leucine to valine. D. leucine to lysine. E. valine to methionine.

D. leucine to lysine

In a solution at pH 7.0, a polymer of lysine (polylysine) is a random coil with no secondary structure. In order for it to possibly assume an alpha-helix structure, the pH of the solution must be: A. 4 B6 C. 8 D. 10 E. 12

E. 12

Which of the following statements about quaternary structure is incorrect? A. A homotrimer consists of three identical protomers. B. Identical polypeptides can create a functional protein. C. A ribosome can be considered an oligomer. D. More DNA is required to code for a 70,000 molecular weight homodimer then a 70,000 molecular weight heterodimer. E. Less DNA is required to code for a 70,000 molecular weight homotrimer than 70,000 molecular weight homodimer.

D. more DNA is required to code for a 70,000 molecular weight homodimer than a 70,000 molecular weight deterodimer

Which one of the following amino acids would most likely NOT be found in an alpha helix? A. methionine B. lysine C. phenylalanine D. proline E. glutamine

D. proline

The following experiment is performed to identify defects in the DNA replication machinery of temperature-sensitive mutants: at the restrictive temperature, crude cellular extracts from each of the temperature-sensitive mutants were incubated with tritiated deoxythymidine triphosphate(3H-dT) for 1 minute, together with non-radioactive dNTP, and then an excess of unlabeled dTTP was added for 10 minutes. DNA was isolated from the crude extracts, denatured, and analyzed by centrifugation in a sucrose density gradient gradient. What is a general name for this type of experiment? A. Hyperchromic shift B. Denaturation C. Michaelis-Menton D. Pulse-Chase E. Electrophoresis

D. pulse-chase

Proteins with just one polypeptide chain have primary, secondary and __________ structures. A. helical B. beta sheet C. quaternary D. tertiary

D. tertiary

A peptide bond is formed by the reaction of the alpha-carbonyl group of one amino acid with___. A. the beta-carboxyl group of a second amino acid. B. the alpha-carboxyl group of a second amino acid. C. the side group of a second amino acid. D. the alpha-amino group of a second amino acid.

D. the alpha-amino group of a second amino acid

In general, a diploid cell containing the + and - alleles encoding an enzyme E has an E+ phenotype. Suppose you isolated a particular mutant that yields an E- phenotype even when the + allele is present. Which of the following is the best explanation for the observed phenomenon. A. The + allele has been mutated. B. During cell division, the + polypeptides are not passed on to daughter cells. C. The polar regions are on the ends of the protein. D. The enzyme has quaternary structure and one defective subunit impacts the activity of the entire protein. E. After one round of replication, the hybrid density alters the activity.

D. the enzyme has quaternary structure and one defective subunit impacts the activity of the entire protein

Trypsin is a protease that breaks peptide bonds. Trypsin interacts with the substrate at the active site. In the active site, binding to the substrate is stabilized by hydrophobic interactions between the side chains of the amino acids in the active site and the substrate. Trypsin is a monomer. Trypsin is available in high quantity in the pancreas, a glandular organ in the digestive system and endocrine system of vertebrates. Trypsin aids in digestion. Trypsin mRNA is 500 nucleotides in length. In a separate experiment, you test the action of the enteric trypsin inhibitor (ETI) on the pancreas trypsin isolated from X. laevis. The elevated presence of this inhibitor in humans is associated with types of digestive disorders. ETI acts as a noncompetitive inhibitor. Which one of following statements about ETI is most probably true? A. The inhibitor acts at a site other than the substrate binding site. B. The inhibitor decreases KM but has no effect on Vmax. C. The inhibitor increases KM but has no effect on Vmax. D. The inhibitor has no impact on KM but lowers the maximum velocity of trypsin. E. The inhibitor has a greater specific activity than does trypsin.

D. the inhibitor has no impact on KM but lowers the maximum velocity of trypsin

Trypsin is a protease that breaks peptide bonds. Trypsin interacts with the substrate at the active site. In the active site, binding to the substrate is stabilized by hydrophobic interactions between the side chains of the amino acids in the active site and the substrate. Trypsin is a monomer. Trypsin is available in high quantity in the pancreas, a glandular organ in the digestive system and endocrine system of vertebrates. Trypsin aids in digestion. Trypsin mRNA is 500 nucleotides in length. You measure the specific activity and Km of trypsin isolated from the pancreas and from muscle of an African horned frog, Xenopus laevis. Which one of the following statements about the Km and specific activity of trypsin from these tissues would you correctly predict to be true. (note: the genus name comes from the greek words, xeno, meaning strange, and pous, meaning foot. This is not to be confused with people who have a fear of foreigners. They are called Xenophobes. Nonetheless, you may even still dislike African horned frogs and frogs in general.) A. In measuring the Km of the muscle trypsin, you added an allosteric competitive inhibitor to the assay. B. The Km of pancreas trypsin will be measured in units of product/time. C. A double reciprocal plot of the pancreas trypsin Km will yield a hyperbolic plot. D. The specific activity of trypsin from muscle tissue will be less than the specific activity of trypsin from pancreas. E. The total amount of protein in an aliquot of muscle homogenate was measured by determining the amount of light absorbed at 260 nm.

D. the specific activity of trypsin from muscle tissue will be less than the specific activity of trypsin from the pancreas

alpha-helices and beta-sheets have the following in common___. A. they run for long stretches without interruption B. they form flat structures C. they are hydrophobic D. they are stabilized by hydrogen bonds

D. they are stabilized by hydrogen bonds

Which of the following amino acids is most likely to be found in the interior of a globular protein that circulates in the blood stream of a mammal? A. Glutamic acid B. Tyrosine C. Cytosine D. Valine E. Lysine

D. valine

Trypsin is a protease that breaks peptide bonds. Trypsin interacts with the substrate at the active site. In the active site, binding to the substrate is stabilized by hydrophobic interactions between the side chains of the amino acids in the active site and the substrate. Trypsin is a monomer. Trypsin is available in high quantity in the pancreas, a glandular organ in the digestive system and endocrine system of vertebrates. Trypsin aids in digestion. Trypsin mRNA is 500 nucleotides in length. Which one amino acid is most likely to be found on the surface of the tertiary structure of trypsin? A. Ala B. Leu C. Ile D. Val E. Asp

E. Asp

Using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins can be separated on the basis of what physical characteristic? A. Density B. Charge C. Absorbance of light at a wavelength of 280 nm D. Absorbance of light at a wavelength of 260 nm E. Size

E. size

Thyroxine is a derivative of: A. threonine B. thiamine C. tryptophan D. tyramine E. tyrosine

E. thyroxine is a derivative of tyrosine

A protein is at a near entropy minimum (point of lowest disorder, or greatest order) when it is completely stretched out like a string and when it is properly folded up.

False. In both states, either stretched like a string or properly folded, a protein has a highly ordered arrangement of its atoms. A folded protein is stable at a near entropy minimum because the entropic cost is more than balanced by the contributions of weak bonds. A stretched out protein, however, is not stable at this entropy minimum and will assume a more disordered state; that is, it will maximize its entropy.

Amino acids are commonly grouped as nonpolar (hydrophobic) or as polar (hydrophilic), based on the properties of the amino acid side chains. Quantification of these properties is important for a variety of protein structure predictions, but they cannot readily be determined from the free amino acids themselves. Why are the properties of the side chains difficult to measure using the free amino acids? How do you suppose the hydrophilicity or hydrophobicity of a molecule might be determined?

Free amino acids have an amino group and a carboxylate group, both of which are charged at neutral pH. In proteins these groups are involved in peptide bonds, which are uncharged. Thus, the hydrophobicity/hydrophilicity of a free amino acid is not the same as that of its side chain in a protein. To measure the hydrophobicity/hydrophilicity of the side chains, it is common to assess the properties of side-chain analogs. Thus, for alanine one would use methane, for threonine, ethanol, for aspartic acid, acetic acid, and so on. To assess hydrophilicity, one can measure the solubility of the side-chain analogs in water. In general, this is done by determining how the side-chain analog partitions between a vapor of the analog and water. Hydrophobicity can be measured in an analogous way by assessing how a side-chain analog partitions between water and a nonpolar solvent such as cyclohexane. One might imagine that the rank order for hydrophilicity would be the reverse of that for hydrophobicity. This is mostly true, but there are differences, most notably tyrosine (Y) and tryptophan (W).

When egg white is heated, it hardens. This cooking process cannot be reversed, but hard-boiled egg white can be dissolved by heating it in a solution containing a strong detergent (such as sodium dodecyl sulfate) together with a reducing agent, like 2-mercaptoethanol. Neither reagent alone has any effect. Why does boiling an egg white cause it to harden? Why does it require both a detergent and a reducing agent to dissolve the hard boiled egg white?

Heating egg-white proteins denatures them, allowing them to interact with one another in ways that were not possible at the lower temperature of the hen's oviduct. This process forms a tangled meshwork of polypeptide chains. In addition to these interactions, interchain disulfide bonds also form, so that hard-boiled egg white becomes one giant macromolecule. Dissolving hard-boiled egg white requires a strong detergent to overcome the noncovalent interchain interactions and mercaptoethanol to break the covalent disulfide bonds. Together, but not separately, the two reagents eliminate the bonds that hold the tangled protein chains in place. Try it for yourself sometime (most detergents will work and you can get mercaptoethanol from skunk scent - do the experiment somewhere not near me).

Comparison of the homeodomain of proteins from yeast and Drosophila shows that only 17 of 60 amino acids are identical. How is it possible for a protein to change over 70% of its amino acids and still fold in the same way?

Many different strings of amino acids can give rise to identical protein folds. The many amino acid differences between the homeodomain proteins from yeast and Drosophila are among the many possible ones that do not alter folding and function. This question could have been framed in another way; namely, how many amino acid changes are required to convert, say, an alpha helix into a beta sheet? The A2.is: surprisingly few! This question underscores the difficulty in predicting protein structures from amino acid sequences.

Hemoglobin is highly homologous to, and probably derived evolutionarily from myoglobin, which consists of a single polypeptide chain. However, at several positions, residues that are hydrophilic in myoglobin are hydrophobic in hemoglobin. How can you reconcile this information 'with the concept that hydrophobic residues fold into the interior of a protein? (Hint: consider the quaternary structure (if any) of these proteins)

Myoglobin consists of a single polypeptide. Hemoglobin is a tetramer. A residue on the outer surface of myoglobin is likely to be hydrophilic but might be hydrophobic in hemoglobin in the location where hemoglobin subunits interact with each other.

A polypeptide in the cytoplasm of a cell has the sequence Ala-His-Trp-Ser-Gly-Leu-Arg-Pro-Gly Will the R-group of the Ala residue have a charge? YES NO

NO

You are skeptical of the blanket statement that cysteines in intracellular proteins are not involved in disulfide bonds, while in extracellular proteins they are. To test this statement you carry out the following experiment. As a source of intracellular protein you use reticulocytes, which have no internal membranes and, thus, no proteins from the ER or other membrane-enclosed compartments. As examples of extracellular proteins, you use bovine serum albumin (BSA), which has 37 cysteines, and insulin, which has 6. You denature the soluble proteins from a reticulocyte lysate and the two extracellular proteins so that all cysteines are exposed. To probe the status of cysteines, you treat the proteins with N-ethylmaleimide (NGM), which reacts covalently with the -SH groups of free cysteines, but not with sulfur atoms in disulfide bonds. In the first experiment you treat the denatured proteins with radiolabeled NEM, then break disulfide bonds with dithiothreotol (DTT) and react a second time with unlabeled NEM. In the second experiment you do the reverse: you first treat the denatured proteins with unlabeled NEM, then break disulfide bonds with DTT and treat with radiolabeled NEM. The proteins are separated according to size by electrophoresis on a polyacrylamide gel. The proteins are stained to allow visualization (Figure 1). Do any cytosolic proteins have disulfide bonds? Do the extracellular proteins have any free cysteine -SH groups?

No cytosolic proteins with disulfide bonds are detected in this experiment. Treating first with radiolabeled NEM shows that many cytosolic proteins have cysteines that are not linked by disulfide bonds. Treating first with unlabeled NEM to block these sites, followed by DTT to break disulfide bonds, should expose any -SH groups that were linked by disulfide bonds. The absence of labeling indicates that no cysteines were involved in disulfide bonds. BSA and insulin are labeled extensively only after their disulfide bonds have been broken by treatment with DTT. In the absence of DTT treatment, BSA is weakly labeled. Since BSA has an odd number of cysteines, at least one cannot be involved in disulfide bonds. Structural analysis confirms that one of its 37 cysteines is not involved in a disulfide bond.

Where in the primary sequence would you predict the break between the two alpha helices to occur?

Proline and Glycine are known alpha helix breakers

In 1968 Cyrus Levinthal pointed out a complication in protein folding that is widely known as the Levinthal paradox. He argued that because there are astronomical numbers of conformations open to a protein in the denatured state, it would take a very long time for a protein to search through all the possibilities to find the correct one, even if it tested each possible conformation exceedingly rapidly. Yet denatured proteins typically take less than a second to fold inside the cell or in the test tube. How do you suppose that proteins manage to fold so quickly?

Proteins obviously can't search all possible conformations on their way to finding the correct one. Thus, there must be defined pathways to simplify the search. It is now thought that weak interactions rapidly cause the protein to collapse into a molten globule, in which bonding interactions are transient and chains maintain fluidity. Within the molten globule, very weak secondary structures form and disappear, as do tertiary interactions. The formation of small elements of correct secondary structure, stabilized by appropriate tertiary interactions, then appears to nucleate formation of the final structure. This general folding pathway represents a fight between the maximization of entropy, which tends to keep the protein as random as possible,and the minimization of enthalpy through formation of weak bonds. Molecular chaperones in the cell help to maintain appropriate interactions until the proper time in the life of the young and impressionable protein.

How are the hydrogen bonds that stabilize the secondary structure of a protein distinguishable from those that stabilize the tertiary structure?

Secondary structure: hydrogen bonds between atoms in the backbone; tertiary structure: hydrogen bonds between R groups.

The uniform arrangement of the backbone carbonyl oxygens and amide nitrogens in an á helix gives the helix a net dipole, so that it carries a partial positive charge at the amino end and a partial negative charge at the carboxyl end. Where would you expect the ends of á helices to be located in a protein? Why?

The ends of a helices, like polar amino acids, are almost always found at the surface of a protein where they can interact with polar water molecules.

Compare and contrast two aspects of the use of NMR and x-ray crystallography in protein structure determination.

There are many possible answers; any of the following will suffice. NMR uses magnets and radio-frequency irradiation; crystallography uses x rays. NMR is performed on proteins in solution; x-ray crystallography requires a protein crystal. NMR measures a nuclear event; x-ray crystallography measures events in the electron shell. In NMR, proteins emit radio waves; in x-ray crystallography, proteins emit x rays. NMR makes great use of protons; protons are largely ignored in x-ray crystallography. NMR can be applied only to small proteins; x-ray crystallography can solve large proteins and complexes. Both methods irradiate the protein sample with electromagnetic radiation (photons). Both methods yield structures with atomic resolution. Both methods make heavy use of computations.

Loops of polypeptide that protrude from the surface of a protein often form the binding sites for other molecules.

True. Chemical groups on such protruding loops can often surround a molecule, allowing the protein to bind to it with many weak bonds.

A polypeptide in the cytoplasm of a cell has the sequence Ala-His-Trp-Ser-Gly-Leu-Arg-Pro-Gly Will the R-group of the Arg residue have a charge? Circle your answer. YES NO

YES

Distinct surface regions with complementary shapes are found in the tertiary structure of a polypeptide. In and around these complementary shapes, no polar amino acids are present. Is this polypeptide likely to form quaternary structures of identical subunits? Explain your answer in one or two sentences.

Yes. The complementary shapes would allow for Van der Waals attraction increasing the likelihood of quaternary structure formation of at least, homodimers. Furthermore, the lack of polar amino acids in the area would likely mean that hydrophobic interactions could stabilize the quaternary structure.

The primary sequence of "Protein X" is: Ala-Leu-Glu-Ser-Lys-Gly-Arg-Ser-Lys-Ile-Asp-Leu. At a neutral pH, does this polypeptide have a net positive or net negative charge?

You can assume that at a neutral pH (7) that acidic amino acids will be negatively charged and basic amino acids will be positively charged. Add up the positives and negatives to determine the total charge. Three positive charges (2 Lys, 1 Arg) minus two negative charges (Glu, Asp) = 1 net positive charge.

Which of the following changes would be least likely to occur in the chicken egg proteins boiled for 5 minutes? (more than one may be correct) A. hydrolysis of the peptide bonds B. disruption of hydrogen bonds C. a warp in the alpha helix D. disruption of a disulfide bridge

a. hydrolysis of the peptide bonds. boiling temperatures are not usually sufficient to break peptide bonds

Which of the following statements is true regarding how R groups affect á helix stability? A. Oppositely charged side chains can increase stability. B. Due to its small size, glycine increases stability. C. Bulky R groups create stabilizing van der Waals forces. D. Since located on the exterior of the á helix, R groups do not significantly alter stability.

a. oppositely charged side chains can increase stability

Common folding pattern in proteins in which a linear sequence of amino acids folds into a right-handed coil stabilized by internal hydrogen bonding between backbone atoms.

alpha helix

The local folding pattern within a segment of a polypeptide chain containing neighboring residues is called its___. A. primary structure B. secondary structure C. tertiary structure D. quaternary structure

b. secondary structure

Common structural motif in proteins in which different sections of the polypeptide chain run alongside each other and are joined together by hydrogen bonding between atoms of the polypeptide backbone.

beta sheet

A region on the surface of a protein that can interact with another molecule through noncovalent bonding.

binding site

A chicken egg is placed in boiling water for 5 minutes. The egg albumin will be changed as a result of this treatment, which we could describe as:

denaturation

Which of the following chemical interactions or bonds requires the most energy to break or disrupt? A. hydrophobic interactions. B. van der Waal's repulsive forces. C. hydrogen bonds. D. van der Waal's attractive forces. E. covalent bonds.

e. covalent bonds

In a globular protein, which amino acids would probably be positioned near the surface? (more than one may be correct) A . trp B. asp C. phe D. pro E. arg F. leu

hydrophilic amino acids but not hydrophobic amino acids.

The chain of repeating carbon and nitrogen atoms, linked by peptide bonds, in a protein.

polypeptide backbone

The amino acid sequence of a protein

primary structure

Portion of a protein that has a tertiary structure of its own.

protein domain

Three-dimensional relationship of the different polypeptide chains in a multisubunit protein or protein complex.

quaternary structure

hydrophobic amino acids:

• Alanine - Ala - A • Isoleucine - Ile - I • Leucine - Leu - L • Methionine - Met - M • Phenylalanine - Phe - F • Valine - Val - V • Proline - Pro - P • Glycine - Gly - G Usually are buried in the protein core