Chapter 5 Answers

1. The 20 amino acids used for protein synthesis can be divided into four general

categories, based upon the chemical properties of their side chains. The four categories are

neutral-nonpolar (with side chains composed of simple carbon chains or aromatic rings),

neutral-polar (including hydroxyl, sulfhydryl, amide, and imidazole moieties), basic (with

side chains including primary and secondary amines), and acidic (with side chains including

carboxylates).

The type of interaction made by each of the amino acids reflects the chemical nature of

its side chain. For example, the neutral-nonpolar side chains generally make hydrophobic

contacts with other molecules, and the neutral-polar side chains most commonly participate in

hydrogen bond interactions. Similarly, the charged (acidic and basic) side chains interact via

ionic or hydrogen bonds. Finally, all four types of side chains can make van der Waals

contacts, as these depend only on the nearby presence of other atoms and not on the chemical

properties of the involved groups.

2. Primary structure refers to the linear sequence of amino acids within a polypeptide

chain. Accordingly, every protein has a primary structure, consisting simply of its amino acid

sequence.

Secondary structure refers to the local structures that are formed by stretches of amino

acids within a polypeptide chain. The most common secondary structural elements are the

helix and the sheet.

Tertiary structure refers to the overall three-dimensional structure of a polypeptide

chain. A tertiary structure includes all of the secondary structural elements present within the

polypeptide. An example of a tertiary structure is shown in Figure 5-26, indicating the

distinct cAMP binding and DNA-binding domains of the CAP protein.

Quarternary structure refers to the way in which multiple polypeptide subunits interact

with each other to form a protein complex—for example, when two leucine zipper-containing

proteins dimerize through their helices to form a coiled-coil.

3. Two major methods exist for determining the structure of a protein. The first of these

is X-ray crystallography, a method based on the interpretation of the diffraction pattern

formed when highly ordered crystals of pure protein are exposed to a beam of X-rays. X-ray

crystallography is an enormously powerful tool that has allowed the structural determination

of a great many proteins, including some very large polypeptides. This method, however, has

several limitations, most notably the technically difficult requirement for highly ordered

protein crystals.

The second available method for determining protein structure is nuclear magnetic

resonance (NMR). This method exploits differences in the way nuclei behave in different

molecular environments to determine the relative location of atoms within a protein. NMR is

also a very powerful method, but is itself limited by the need for high concentrations of

purified protein and by the fact that the technique is better suited for smaller proteins than for

larger ones.

4. The helix is a right-handed helix that repeats every 3.6 amino acids, covering 5.4 Å

per turn. The helix is stabilized by regularly occurring hydrogen bonds formed between the

NH and CO groups of the polypeptide backbone. The backbone is at the interior of the

helix, with the amino acid side chains projecting outward.

The hydrogen bonding that stabilizes the helix explains why this structure is so

common. First, this bonding is quite energetically favorable, allowing all of the NH or CO

groups within the backbone to form hydrogen bonds. Also, because these interactions only

involve atoms of the polypeptide backbone, there are relatively few restrictions on the identity

of the specific amino acids that can participate in the helix.

The sheet represents a relatively flat, highly extended form of the polypeptide

backbone that includes 4–6 adjacent strands of 8–10 amino acids each. sheets can be

parallel, in which adjacent strands run in the same direction, or anti-parallel, in which the

strands run in opposite directions. This structure is stabilized by hydrogen bonds between CO

groups of one strand and NH groups on the adjacent strand. As with the helix, all of the CO

and NH groups of the polypeptide backbone form hydrogen bonds when present within the

sheet, explaining the stability and prevalence of this structure.

5. To determine if the domain really contains an helix, you could first examine its

secondary structure in solution using NMR spectroscopy or other available tools, such as

circular dichroism spectroscopy. You could also indirectly assess the presence of an helix

by introducing a mutation that inserts a proline residue into the domain. As prolines are

incompatible with helices, this residue would dramatically change the structure of the

protein and likely destroy its ability to bind to DNA.

6. First, the software can look for stretches of amino acids that are consistent with the

presence of helices, sheets, or other secondary structural elements. For example, because

proline residues are incompatible with the helix, the program can conclude that any region

that contains a proline is unlikely to contain an alpha helix (and, conversely, any region

without any prolines may contain a helix). The same is true for the amino acids glycine,

tyrosine, and serine, which are rarely found in helices.

The software can also scan the sequence for regions in which particular types of amino

acids recur with a particular periodicity. For example, because the pitch of the helix is 3.6

amino acids per turn, one might expect to find hydrophobic amino acids appearing every 3–4

amino acids if one face of an helix faces the hydrophobic interior of a protein. A similar

strategy can be used to identify sheets, in which the side groups of adjacent amino acids

face opposite directions. If one face of the sheet is facing the interior of the protein, and one

face points toward the exterior, then the sheet might contain alternating hydrophobic and

hydrophilic residues.

Finally, as the secondary and tertiary structures of many proteins have now been

determined, it is often informative to simply screen new sequences against databases

containing structural information about other, already characterized proteins or protein

domains. A close match with a sequence that is known to form an helix or a sheet

provides compelling evidence that the new structure adopts the same structure as well.

7. SSB specifically recognizes single-stranded DNA by making nonspecific ionic or

hydrogen bond interactions with the phosphate backbone as well by intercalation of ring-

containing side chains such as tryptophan or phenylalanine between the exposed bases.

SSB acts to stabilize and protect single-stranded DNA in vivo. This is essential, for

example, during DNA replication, where a helicase moves ahead of the replication fork to

unwind the DNA and expose the bases for copying. If it weren’t for SSB, these exposed

DNA strands would be unstable and prone to reannealing, forming internal secondary

structures, or being attacked by nucleases. Instead, these possibilities are prevented by SSB,

which uses cooperative binding to rapidly and thoroughly coat the single-stranded DNA.

8. The major groove is particularly well suited for protein binding for several reasons.

First, it has a large number of potential hydrogen bond donors and acceptors; second, the

precise location of these donors and acceptors depends on the sequence of the DNA; and

third, its width and depth provide a good match for the helix, the most common motif found

in DNA-binding proteins.

Examples of DNA-binding proteins that use an helix to bind to the major groove

include the helix-turn-helix motif, the zinc finger motif, and the leucine zipper DNA-binding

motif.

The TATA binding protein (TBP) uses a sheet to bind to the minor groove of the

DNA (at the TATA-box within eukaryotic promoters). The specificity of this interaction is

provided by a small number of hydrogen bonds and a larger number of van der Waals

contacts, between the sheet and the edges of the base pairs in the minor groove. The fact

that the TATA box is rich in A:T base pairs also provides specificity, because NH2 groups

protruding from the minor groove of G:C base pairs prevent efficient van der Waals contacts

with sheets. Finally, two pairs of phenylalanine side chains intercalate between the base

pairs at either end of the recognition sequence, causing the DNA to bend and the minor

groove to flatten. This contributes as well to the specificity because A:T base pairs are easier

to distort than C:G base pairs.

9. Nonspecific interactions with the DNA backbone often contribute substantially to the

affinity of DNA-binding proteins for their binding sites. Typically, these interactions involve

electrostatic contacts between positively-charged amino acid side chains and the phosphate

backbone of DNA.

Even though the affinity of a typical DNA-binding protein for its specific target

sequence is much higher than it is for nonspecific sequences (on the order of 105), the vastly

greater number of nonspecific sequences in the genome still means that the protein will spend

most of its time bound to nonspecific sites. This means that the cell must produce a sufficient

number of protein molecules to ensure constant binding of the target site.

Nonspecific protein-DNA interactions can be advantageous by helping to speed up the

rate at which a given DNA-binding protein finds its target site. For example, a protein can

take advantage of its general affinity for DNA by randomly binding to a chromosome and

diffusing linearly along the DNA until it finds its target site. A two-dimensional scan of the

DNA is much more efficient than a random, three-dimensional search within the nucleus.

10. An RNA double helix can be recognized by the presence of the 2’-hydroxyl in the

sugar moiety of the nucleotide (whereas DNA has a hydrogen at the same position), and by

the fact that double-stranded RNA forms an A form double helix (whereas DNA primarily

assumes the B form). Also, RNA molecules are often characterized by particular

arrangements of single- and double-stranded regions, which form distinctive structures such

as stem-loops, hairpins, and other shapes. These particular structural motifs can themselves

be recognized by RNA binding proteins. DNA, in contrast, is essentially always double-

stranded.