ESSENTIAL CELL BIOLOGY, FOURTH EDITION

CHAPTER 4: PROTEIN STRUCTURE AND FUNCTION

© 2014 GARLAND SCIENCE PUBLISHING

The Shape and Structure of Proteins

4-1 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 A. insulin

___ motor B. carboxylase
___ storage C. rhodopsin
___ enzyme D. hemoglobin

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___ transport E. ferritin
___ structural F. myosin

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___ special purpose G. green fluorescent protein
___ receptor H. tubulin

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___ signal I. homeodomain proteins

4-2
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Indicate whether the following statements are true or false. If a statement is false, explain
why it is false.
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A. Generally, the total number of nonpolar amino acids has a greater effect on
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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
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those that form the peptide bonds.

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C. The chemical properties of amino acid side chains include charged, uncharged
polar, and nonpolar.
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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
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nonpolar side chains in the interior.

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4-3 Polypeptides are synthesized from amino acid building blocks. The condensation reaction
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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.

4-4 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.

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 (d) orientation.

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

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4-6 Use your knowledge of amino acid characteristics to order the peptides below by their
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degree of polarity. Each peptide contains eight amino acids. Use the single-letter amino
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acid designations to generate your list, placing the most polar peptide on the left and the
most nonpolar peptide on the right.
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A. SGAKKRAH
B. CATWNGQV
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C. FWGTSILA
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D. DDAEIHWA
E. SSTAMYRK
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4-7 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
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4-8 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.

4-9 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?

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(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

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protein.

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(d) The solvents create a new protein conformation.

4-10 Which of the following statements is true?

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(a) Peptide bonds are the only covalent bonds that can link together two amino acids
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in proteins.
(b) The polypeptide backbone is free to rotate about each peptide bond.
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(c) Nonpolar amino acids tend to be found in the interior of proteins.

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(d) The sequence of the atoms in the polypeptide backbone varies between different
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proteins.
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4-11 Indicate whether the following statements are true or false. If a statement is false, explain
why it is false.
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A. Van der Waals interactions and hydrophobic interactions are two ways to describe
the same type of weak forces that help proteins fold.
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B. A large number of noncovalent interactions is required to hold two regions of a

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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).

4-12 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

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4-13 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

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denatured noncovalent stable
highest renatured unstable

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4-14 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
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observed after the denaturant is removed from the protein solution?
(a) The polypeptide returns to its original conformation.
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(b) The polypeptide remains denatured.
(c) The polypeptide forms solid aggregates and precipitates out of solution.
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(d) The polypeptide adopts a new, stable conformation.

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4-15 The correct folding of proteins is necessary to maintain healthy cells and tissues.
Unfolded proteins are responsible for such neurodegenerative disorders as Alzheimer’s
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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
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proteins?
(a) They are degraded.
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(b) They bind a different target protein.

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(c) They form structured filaments.

(d) They form protein aggregates.

4-16 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.

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4-17 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.

4-18 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

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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

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remove the urea.

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C. Treat the insoluble aggregate with a protease.
D. Make the bacteria overproduce chaperone proteins in addition to protein A.
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E. Heat the protein aggregate to denature all proteins, then cool the mixture.
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4-19 The three-dimensional coordinates of atoms within a folded protein are determined
experimentally. After researchers obtain a protein’s structural details, they can use
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different techniques to highlight particular aspects of the structure. What visual model
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best displays a protein’s secondary structures (α helices and β sheets)?

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(a) ribbon
(b) space-filling
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(c) backbone
(d) wire
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4-20 Typical folded proteins have a stability ranging from 7 to 15 kcal/mole at 37°C. Stability
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is a measure of the equilibrium between the folded (F) and unfolded (U) forms of the
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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.

Figure Q4-20

4-21 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

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 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

4-22 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

4-23 Which of the following is not a feature commonly observed in β sheets?

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(a) antiparallel regions
(b) coiled-coil patterns
(c) extended polypeptide backbone

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(d) parallel regions

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4-24 Two or three α helices can sometimes wrap around each other to form coiled-coils. The
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stable wrapping of one helix around another is typically driven by ________________
interactions.
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(a) hydrophilic
(b) hydrophobic
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(c) van der Waals

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(d) ionic
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4-25 Coiled-coils are typically found in proteins that require an elongated structural
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framework. Which of the following proteins do you expect to have a coiled-coil domain?
(a) insulin
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(b) collagen
(c) myoglobin
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(d) porin
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4-26 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

4-27 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

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 (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

4-28 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.

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Figure Q4-28 gm
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4-29 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
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amino acids are italicized.

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A. Leu-Gly-Val-Leu-Ser-Leu-Phe-Ser-Gly-Leu-Met-Trp-Phe-Phe-Trp-Ile
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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
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amphipathic α helix hydrophilic β sheet

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amphipathic β sheet hydrophobic α helix

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hydrophilic α helix
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4-30 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.

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 Figure Q4-30

4-31 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.

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Figure Q4-31
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A. For panel (A) and for panel (B), indicate whether the structure is parallel or
antiparallel.
B. Draw the hydrogen bonds as dashed lines (||||||).

4-32 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

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 __________________ 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

4-33 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.

4-34 In an attempt to define the protein domains of protein X, you treat it with a protease and

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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

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new stock of chymotrypsin to arrive.

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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
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different results?
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4-35 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
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globin family. Myoglobin is a stable monomeric protein that can help carry oxygen using
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a heme molecule. Hemoglobin is stable as a tetramer. It also carries oxygen through the
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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
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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
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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
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formation of a multisubunit protein.

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4-36 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.

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 Figure Q4-36

4-37 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

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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

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explain the results in the two western blots? If not, how do you explain the difference
observed?

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Figure Q4-37
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4-38 Examine the three protein monomers in Figure Q4-38. From the arrangement of
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complementary binding surfaces, which are indicated by similarly shaped protrusions and
indentations, decide whether each monomer could assemble into a defined multimer, a
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filament, or a sheet.
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Figure Q4-38

4-39 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.

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4-40 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

4-41 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?

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(a) elastase
(b) collagen
(c) keratin

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(d) elastin

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4-42 Which of the following globular proteins is used to form filaments as an intermediate step
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to assembly into hollow tubes?
(a) tubulin
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(b) actin
(c) keratin
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(d) collagen
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4-43 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-
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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
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this result?
(a) the loss of a binding site on the mutant-protein surface through which protein Y
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normally forms dimers

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(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

4-44 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.

How Proteins Work

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4-45 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

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antibodies coiled-coils loops
antigens hundreds size-exclusion
β strands ion-exchange

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4-46 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
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interactions in the protein’s ligand-binding site. Where is the binding site typically
located in the protein structure?
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(a) on the surface of the protein
(b) inside a cavity on the protein surface
or

(c) buried in the interior of the protein

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(d) forms on the surface of the protein in the presence of ligand

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4-47 Cyclic AMP (cAMP) is a small molecule that associates with its binding site with a high
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degree of specificity. Which types of noncovalent interactions are the most important for
providing the “hand in a glove” binding of cAMP?
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(a) hydrogen bonds

(b) electrostatic interactions
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(c) van der Waals interactions

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(d) hydrophobic interactions

4-48 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.

4-49 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

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 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

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Figure Q4-49
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4-50 The process of generating monoclonal antibodies is labor-intensive and expensive. An

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alternative is to use polyclonal antibodies. A subpopulation of purified polyclonal

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antibodies that recognize a particular antigen can be isolated by chromatography. Which

type of chromatography is used for this purpose?
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(a) affinity
(b) ion-exchange
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(c) gel-filtration
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(d) any of the above

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4-51 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

4-52 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

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 (c) nuclease
(d) hydrolase

4-53 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

4-54 Studies conducted with a lysozyme mutant that contains an AspàAsn change at position
52 and a GluàGln change at position 35 exhibited almost a complete loss in enzymatic

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activity. What is the most likely explanation for the decrease in enzyme activity in the
mutant?
(a) increased affinity for substrate

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(b) absence of negative charges in the active site

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(c) change in the active-site scaffold
(d) larger amino acids in the active site decreases the affinity for substrate
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4-55 Match the general type of biochemical reaction catalyzed in the left column with the class
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of enzyme listed in the column on the right.
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___ removes a phosphate group from a molecule A. ATPase

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___ hydrolyzes ATP B. polymerase

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___ hydrolyzes bonds between nucleotides C. ligase

___ adds phosphate groups to molecules D. kinase
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___ catalyzes reactions in which one molecule is E. isomerase

oxidized and another is reduced F. nuclease
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___ hydrolyzes peptide bonds G. oxido-reductase

___ joins two ends of DNA together H. protease
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___ catalyzes the synthesis of polymers such as I. phosphatase

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RNA and DNA

___ rearranges bonds within a single molecule

4-56 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

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4-57 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

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active site isomers substrates
free energy ligand transition state
high-energy low-energy

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4-58 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
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why, although reaction-specific, the first two produce side effects, while the third does
not.
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A. Statins inhibit HMG CoA reductase to block intracellular cholesterol synthesis.
B. Methotrexate inhibits dihydrofolate reductase, which subsequently leads to
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blocked DNA replication.

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C. Gleevec® inhibits BCR, a kinase that is only produced in certain types of

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leukemia cells.
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4-59 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
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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
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state than to the undistorted substrate; in other words, if the enzyme were to have a higher
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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?

How Proteins Are Controlled

4-60 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.

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 Figure Q4-60
(a) H
(b) B
(c) C
(d) E

4-61 Which of the following statements about allostery is true?

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

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(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.

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(d) Binding of allosteric molecules usually locks an enzyme in its current
conformation, such that the enzyme cannot adopt a different conformation.

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4-62
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Indicate whether the following statements are true or false. If a statement is false, explain
why it is false.
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A. Feedback inhibition is defined as a mechanism of down-regulating enzyme
activity by the accumulation of a product earlier in the pathway.
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B. If an enzyme’s allosteric binding site is occupied, the enzyme may adopt an

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alternative conformation that is not optimal for catalysis.

C. Protein phosphorylation is another way to alter the conformation of an enzyme
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and serves exclusively as a mechanism to increase enzyme activity.

D. GTP-binding proteins typically have GTPase activity, and the hydrolysis of GTP
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transforms them to the “off” conformation.

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4-63 The Ras protein is a GTPase that functions in many growth-factor signaling pathways. In
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its active form, with GTP bound, it transmits a downstream signal that leads to cell
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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

4-64 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).
Page 16 of 29
 (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.

4-65 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

4-66 The phosphorylation of a protein is typically associated with a change in activity, the

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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 ______________.

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(a) membrane association.

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(b) protein degradation.
(c) protein secretion. gm
(d) nuclear translocation.
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4-67 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
or

ADP molecules increases, they can bind to glycolytic enzymes, which will lead to the
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production of more ATP. The best way to describe this mechanism of regulation is
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___________.
(a) feedback inhibition.
nk

(b) oxidative phosphorylation.

(c) allosteric activation.
ba

(d) substrate-level phosphorylation.

st

4-68 Some of the enzymes that oxidize sugars to yield usable cellular energy (for example,
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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.

How Proteins Are Studied

4-69 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?

Page 17 of 29
 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?

4-70 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

4-71 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

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(c) western blot analysis
(d) ion-exchange chromatography

l.c
4-72 Which of the following methods would be the most suitable to assess levels of expression

ai
of your target protein in different cell types?
(a) gel-filtration chromatography gm
(b) gel electrophoresis
(c) western blot analysis
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(d) ion-exchange chromatography
or

4-73 Which of the following methods used to study proteins is limited to proteins with a
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molecular mass of 50 kD or less?

do

(a) X-ray crystallography

(b) fingerprinting
nk

(c) nuclear magnetic resonance

(d) mass spectroscopy
ba

4-74 Determining a protein’s sequence, site of covalent modification, or entire three-

st

dimensional structure requires the careful analysis of complex data sets. Which of the
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data sets below would you have to interpret to solve the structure of a protein by using X-
ray crystallography?

Page 18 of 29
 om
l.c
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gm
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4-75 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
or

grown in specific conditions. This large-scale, systematic approach to the study of

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proteins is called _______________.

do

(a) proteomics.
(b) structural biology.
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(c) systems biology.

(d) genomics.
ba

4-76 Using the example of the p53 protein, postulate how different combinations of covalent
st

modifications can lead to a wide variety of protein functions.

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Page 19 of 29
 ANSWERS

4-1 gene regulatory __I_

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

4-2 A. False. The order in which amino acids are linked is unique for each protein and is

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the most important factor in determining overall protein structure.
B. False. Peptide bonds are planar amide bonds that are central to the polypeptide

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backbone formation. The atoms in the amino acid side chains are not considered
to be part of the backbone.

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C. True.
D. True. gm
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4-3 (a)
or

4-4 (b)
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4-5
do
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ba
st
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Page 20 of 29
4-6 E, A, D, B, C

4-7 (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.

4-8 (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.

4-9 (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

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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

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followed by renaturation of a protein does not generate a new protein fold [choice (d)].

ai
4-10 Choice (c) is the correct answer. Choice (a) is untrue, because some proteins also contain
gm
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
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the polypeptide backbone itself is always the same from protein to protein; it is the order
of the amino acid side chains that differs.
or
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4-11 A. False. Van der Waals attractions are weakly attractive forces that occur between
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all atoms. Hydrophobic interactions are only observed between nonpolar

molecules in the context of an aqueous solution.
nk

B. True.
C. False. There is a single, final fold for every polypeptide. The fold adopted is the
ba

“best” conformation, for which the free energy (G) of the molecule is at a
minimum.
st
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4-12 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

Page 21 of 29
 surface to interact with water, and the alanine is close enough to the interior of the
protein to interact with other hydrophobic side chains.

4-13 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.

4-14 (a)

4-15 (d)

4-16 (c)

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4-17 (d)

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4-18 Choices A, B, and D are all worth trying. Some proteins require molecular chaperones if

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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
gm
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
or

bacterium to fold the protein. Expressing the protein at lower levels might
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increase the amount of properly folded protein.

do

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
nk

crowded conditions, the protein should be able to refold into its proper
conformation.
ba

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
st

strongly with one another; however, chopping up the protein will also destroy its
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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.

4-19 (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.

Page 22 of 29
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.

4-21 (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.

4-22 (a)

4-23 (b)

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4-24 (b)

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4-25 (b)

ai
4-26 (d) gm
4-27 (d)
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4-28
or
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do
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ba
st
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4-29 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.

Page 23 of 29
 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.

4-30 A. The strands form an antiparallel β sheet.

Figure A4-30

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B. The prediction is that nonpolar side chains are on the inside of the barrel, and

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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

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solvent–protein hydrogen bonds. And, although the barrel seems open and
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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.
@
or

4-31 A. (A) is parallel and (B) is antiparallel.

B. See Figure A4-31.
ct
do
nk
ba
st
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Figure A4-31

4-32 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

Page 24 of 29
 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.

4-33 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.

4-34 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

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the proteases to differ. As a result, they have different substrate affinities and
cleave the bond between a different set of amino acids.

l.c
4-35 Amino acids that are found on the surface of a folded monomeric protein are the best

ai
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
gm
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
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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.
or

Nonpolar residues would interact with each other rather than the polar molecules in the
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cytosol.
do

4-36 Segments B and D. To cut the protein chain, Factor Xa and thrombin must bind to their
nk

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
ba

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
st

produced by digestion of the protein with Factor Xa, we can conclude that the enzyme
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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.

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.

Page 25 of 29
4-38 A. defined multimer with four subunits, called a tetramer
B. sheet
C. filament

4-39 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.

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4-40 A—F; B—G; C—F; D—F; E—G; F—G

4-41 (a)

l.c
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4-42 (a)

4-43
gm
(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
or

to enter the porous beads and would run through the column faster than the normal
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protein. Choice (d) is unlikely, because a small change in shape without a change in size
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would be unlikely to have a major effect on the behavior of a protein in a gel-filtration

column.
nk

4-44 (c) Choice (a) is incorrect, because S–S bonds are formed between cysteines. Choice (b)
ba

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
st

oxidation.
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4-45 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.

4-46 (b)

4-47 (a)

Page 26 of 29
4-48 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.

4-49

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l.c
ai
gm
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or

4-50 (a)
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do

4-51 (c)
nk

4-52 (d)
ba

4-53 (c)
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4-54 (b) The negatively charged amino acids aspartic acid and glutamic acid are required to
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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.

4-55 Match the general type of biochemical reaction catalyzed in the left column with the class
of enzyme listed in the column on the right.

_I_ removes a phosphate group from a molecule A. ATPase

_A_ hydrolyzes ATP B. polymerase
_F_ hydrolyzes bonds between nucleotides C. ligase
_D_ adds phosphate groups to molecules D. kinase
_G_ catalyzes reactions in which one molecule is E. isomerase
oxidized and another is reduced F. nuclease
Page 27 of 29
 _H_ hydrolyzes peptide bonds G. oxido-reductase
_C_ joins two ends of DNA together H. protease
_B_ catalyzes the synthesis of polymers such as I. phosphatase
RNA and DNA
_E_ rearranges bonds within a single molecule

4-56 (a)

4-57 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.

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4-58 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

l.c
effects can be hard to predict, and may be very specific to the patient being treated. In the

ai
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
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usefulness of this drug is limited to those CML patients that have this specific mutation.
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4-59 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
or

reactions, but with lower efficiency than genuine enzymes.

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do

4-60 (c)
nk

4-61 (a)
ba

4-62 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
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inhibitory product binds to a site on the enzyme that lowers its catalytic activity.
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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.

4-63 (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.

4-64 (d)

4-65 (a)

Page 28 of 29
4-66 (b)

4-67 (c)

4-68 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.

4-69 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

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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

l.c
the column resin, the antibodies are bound instead. The antibody will bind to your

ai
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
gm
binding surface typically recognized by the other proteins that would normally
bind to your target, reducing the number of binding partners isolated by the
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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
or

place.
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4-70 (b)
nk

4-71 (a)
ba

4-72 (c)
st

4-73 (c)
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4-74 (b)

4-75 (a)

4-76 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.

Page 29 of 29