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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
neutralnonpolar !with side chains composed of simple carbon chains or aromatic rings",
neutralpolar !including hydro#yl, sulfhydryl, amide, and imida$ole moieties", basic !with
side chains including primary and secondary amines", and acidic !with side chains including
carbo#ylates".
The type of interaction made by each of the amino acids reflects the chemical nature of
its side chain. %or e#ample, the neutralnonpolar side chains generally ma&e hydrophobic
contacts with other molecules, and the neutralpolar side chains most commonly participate in
hydrogen bond interactions. 'imilarly, the charged !acidic and basic" side chains interact via
ionic or hydrogen bonds. %inally, all four types of side chains can ma&e van der (aals
contacts, as these depend only on the nearby presence of other atoms and not on the chemical
properties of the involved groups.
2. )rimary structure refers to the linear se*uence of amino acids within a polypeptide
chain. Accordingly, every protein has a primary structure, consisting simply of its amino acid
se*uence.
'econdary 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
heli# and the sheet.
Tertiary structure refers to the overall threedimensional structure of a polypeptide
chain. A tertiary structure includes all of the secondary structural elements present within the
polypeptide. An e#ample of a tertiary structure is shown in %igure 52+, indicating the
distinct cA,) binding and -.Abinding domains of the CA) protein.
/uarternary structure refers to the way in which multiple polypeptide subunits interact
with each other to form a protein comple#0for e#ample, when two leucine $ippercontaining
proteins dimeri$e through their helices to form a coiledcoil.
1. Two ma2or methods e#ist for determining the structure of a protein. The first of these
is 3ray crystallography, a method based on the interpretation of the diffraction pattern
formed when highly ordered crystals of pure protein are e#posed to a beam of 3rays. 3ray
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 re*uirement for highly ordered
protein crystals.
The second available method for determining protein structure is nuclear magnetic
resonance !.,4". This method e#ploits differences in the way nuclei behave in different
molecular environments to determine the relative location of atoms within a protein. .,4 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 techni*ue is better suited for smaller proteins than for
larger ones.
5. The heli# is a righthanded heli# that repeats every 1.+ amino acids, covering 5.5 6
per turn. The heli# is stabili$ed by regularly occurring hydrogen bonds formed between the
.7 and C8 groups of the polypeptide bac&bone. The bac&bone is at the interior of the
heli#, with the amino acid side chains pro2ecting outward.
The hydrogen bonding that stabili$es the heli# e#plains why this structure is so
common. %irst, this bonding is *uite energetically favorable, allowing all of the .7 or C8
groups within the bac&bone to form hydrogen bonds. Also, because these interactions only
involve atoms of the polypeptide bac&bone, there are relatively few restrictions on the identity
of the specific amino acids that can participate in the heli#.
The sheet represents a relatively flat, highly e#tended form of the polypeptide
bac&bone that includes 59+ ad2acent strands of :910 amino acids each. sheets can be
parallel, in which ad2acent strands run in the same direction, or antiparallel, in which the
strands run in opposite directions. This structure is stabili$ed by hydrogen bonds between C8
groups of one strand and .7 groups on the ad2acent strand. As with the heli#, all of the C8
and .7 groups of the polypeptide bac&bone form hydrogen bonds when present within the
sheet, e#plaining the stability and prevalence of this structure.
5. To determine if the domain really contains an heli#, you could first e#amine its
secondary structure in solution using .,4 spectroscopy or other available tools, such as
circular dichroism spectroscopy. ;ou could also indirectly assess the presence of an heli#
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 li&ely destroy its ability to bind to -.A.
+. %irst, the software can loo& for stretches of amino acids that are consistent with the
presence of helices, sheets, or other secondary structural elements. %or e#ample, because
proline residues are incompatible with the heli#, the program can conclude that any region
that contains a proline is unli&ely to contain an alpha heli# !and, conversely, any region
without any prolines may contain a heli#". The same is true for the amino acids glycine,
tyrosine, and serine, which are rarely found in helices.
The software can also scan the se*uence for regions in which particular types of amino
acids recur with a particular periodicity. %or e#ample, because the pitch of the heli# is 1.+
amino acids per turn, one might e#pect to find hydrophobic amino acids appearing every 195
amino acids if one face of an heli# faces the hydrophobic interior of a protein. A similar
strategy can be used to identify sheets, in which the side groups of ad2acent amino acids
face opposite directions. <f one face of the sheet is facing the interior of the protein, and one
face points toward the e#terior, then the sheet might contain alternating hydrophobic and
hydrophilic residues.
%inally, as the secondary and tertiary structures of many proteins have now been
determined, it is often informative to simply screen new se*uences against databases
containing structural information about other, already characteri$ed proteins or protein
domains. A close match with a se*uence that is &nown to form an heli# or a sheet
provides compelling evidence that the new structure adopts the same structure as well.
=. ''> specifically recogni$es singlestranded -.A by ma&ing nonspecific ionic or
hydrogen bond interactions with the phosphate bac&bone as well by intercalation of ring
containing side chains such as tryptophan or phenylalanine between the e#posed bases.
''> acts to stabili$e and protect singlestranded -.A in vivo. This is essential, for
e#ample, during -.A replication, where a helicase moves ahead of the replication for& to
unwind the -.A and e#pose the bases for copying. <f it weren?t for ''>, these e#posed
-.A strands would be unstable and prone to reannealing, forming internal secondary
structures, or being attac&ed by nucleases. <nstead, these possibilities are prevented by ''>,
which uses cooperative binding to rapidly and thoroughly coat the singlestranded -.A.
:. The ma2or groove is particularly well suited for protein binding for several reasons.
%irst, it has a large number of potential hydrogen bond donors and acceptors@ second, the
precise location of these donors and acceptors depends on the se*uence of the -.A@ and
third, its width and depth provide a good match for the heli#, the most common motif found
in -.Abinding proteins.
A#amples of -.Abinding proteins that use an heli# to bind to the ma2or groove
include the heli#turnheli# motif, the $inc finger motif, and the leucine $ipper -.Abinding
motif.
The TATA binding protein !T>)" uses a sheet to bind to the minor groove of the
-.A !at the TATAbo# within eu&aryotic promoters". The specificity of this interaction is
provided by a small number of hydrogen bonds and a larger number of van der (aals
contacts, between the sheet and the edges of the base pairs in the minor groove. The fact
that the TATA bo# is rich in ABT base pairs also provides specificity, because .7
2
groups
protruding from the minor groove of CBC base pairs prevent efficient van der (aals contacts
with sheets. %inally, two pairs of phenylalanine side chains intercalate between the base
pairs at either end of the recognition se*uence, causing the -.A to bend and the minor
groove to flatten. This contributes as well to the specificity because ABT base pairs are easier
to distort than CBC base pairs.
D. .onspecific interactions with the -.A bac&bone often contribute substantially to the
affinity of -.Abinding proteins for their binding sites. Typically, these interactions involve
electrostatic contacts between positivelycharged amino acid side chains and the phosphate
bac&bone of -.A.
Aven though the affinity of a typical -.Abinding protein for its specific target
se*uence is much higher than it is for nonspecific se*uences !on the order of 10
5
", the vastly
greater number of nonspecific se*uences 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.
.onspecific protein-.A interactions can be advantageous by helping to speed up the
rate at which a given -.Abinding protein finds its target site. %or e#ample, a protein can
ta&e advantage of its general affinity for -.A by randomly binding to a chromosome and
diffusing linearly along the -.A until it finds its target site. A twodimensional scan of the
-.A is much more efficient than a random, threedimensional search within the nucleus.
10. An 4.A double heli# can be recogni$ed by the presence of the 2?hydro#yl in the
sugar moiety of the nucleotide !whereas -.A has a hydrogen at the same position", and by
the fact that doublestranded 4.A forms an A form double heli# !whereas -.A primarily
assumes the > form". Also, 4.A molecules are often characteri$ed by particular
arrangements of single and doublestranded regions, which form distinctive structures such
as stemloops, hairpins, and other shapes. These particular structural motifs can themselves
be recogni$ed by 4.A binding proteins. -.A, in contrast, is essentially always double
stranded.

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