From the Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
The Briefly, the argument presented in this minireview is as follows. There
are only two substantially populated conformers available to a
non-glycyl/non-prolyl peptide (12, 13), The preceding factors that promote helix formation are opposed by the
entropic cost of freezing the main chain into a single conformation. On
balance, helix formation must be energetically favorable for main chain
atoms because alanine-based peptides are helical (17). Nonetheless,
most peptides and protein segments are not helical, so unfavorable
factors usually outweigh favorable ones. The several helix-promoting
factors mentioned above all involve the peptide backbone and are
therefore common to every residue (except glycine and proline).
Consequently, helical conformation is the preferred state of the
backbone (e.g. polyalanine), while helix-disfavoring factors
must arise in the side chain.
Side chains also pay an entropic price for helix formation because the
presence of the bulky helix backbone is sterically incompatible with
some side chain conformers (18, 19). Unlike the backbone, where
helix-affecting factors are the same from residue to residue, side
chain entropy differs from one residue type to the next. For example,
helix formation largely restricts a central valine to only one of its
three possible side chain configurations because one of the The Helices are observed frequently in proteins (20), suggesting that the
This prevailing view of the 1970s came to be reversed in the 1980s,
after Bierzynski et al. (23), expanding upon earlier work by
Brown and Klee (24) demonstrated that residues 1-13 of ribonuclease,
liberated upon cyanogen bromide cleavage, contained a helix that was
stable in water at near physiological temperature. Other isolated
protein fragments were found to be structured as well (25, 26). Such
results prompted a re-evaluation; helices might function as independent
folding units after all.
What factors are responsible for helix stability (27)? Two
"textbook" features of helices are usually invoked to account for
stability, hydrogen bonding (28) (but see Ref. 29) and tight main chain
packing (14). A third implicit feature is the fact that when the chain
folds into a helix, bound water is released and returned to the bulk
phase. These helix-promoting factors are opposed by the entropic cost
of constraining the main chain to a single conformation. For backbone
atoms, there appears to be a net tendency toward helix formation
because alanine-based peptides (17, 30) are observed to be helical.
What factors are responsible for helix specificity (27, 31)? That is,
why are some peptides and protein segments helical, while others are
not? The favorable main chain contributions to helix stability are
constant from one residue to the next because all have identical
backbones (except glycine and proline). Yet, most peptides are not
helical nor are about 75% of the residues in proteins (20). Therefore,
these helix-stabilizing main chain factors that cause polyalanine to be
helical cannot be the ones that differentiate helix from non-helix.
Helix capping has been hypothesized as a general mechanism that
discriminates between helices and other conformational alternatives, including coil (22, 32). In proteins, the helix of average length
(~12 residues) has eight intrasegment hydrogen bonds between successive amide hydrogen donors and carbonyl oxygen acceptors situated
four residues previously in sequence (i.e. N-H(i) ··· O=C (i In both proteins and peptides, the chain leaving the helix tends to
occlude some of these unsatisfied donors and acceptors, hindering
access by solvent water. Provision of hydrogen bond partners for these
otherwise unsatisfied amide hydrogens and carbonyl oxygens is termed
helix capping. In addition to polar backbone groups, apolar
side chains situated near helix termini can be solvent exposed, and
therefore helix destabilizing, unless the chain folds so as to foster a
hydrophobic contact. Recently, our definition of capping has been
extended to include these hydrophobic capping interactions as
well.1
The helix capping hypothesis has been confirmed experimentally in both
peptides (34-37) and proteins (38, 39). Recurrent capping motifs such
as the capping box (40-43) and the Schellman motif (44, 45), which contribute to the stability of protein helices (39), have been shown to persist in peptides (46, 47), where
they can inhibit expected fraying (48) at helix ends.
In 1990, individual residue contributions to helix stability were
assessed in four separate host/guest systems (49-52). In such
experiments, guest residues are substituted systematically at central
positions (to avoid end effects) within a host peptide of known helical
content and the resultant effect measured. Stabilizing substitutions
increase helix content; destabilizing substitutions decrease it. An
experimental scale of helix propensities is then derived by quantifying
these effects in terms of the free energy differences
( What is the basis for these measured differences in helix propensity?
Why does a small, nondescript residue like alanine have a higher helix
propensity than valine? We hypothesized that the differences are due in
large part to the loss of side chain conformational entropy upon helix
formation (19). Physically, this effect reflects the difference between
the side chain's conformational freedom in the relatively flexible
coil state and in the more restricted helical state, with its bulky
helix backbone.
We tested this hypothesis using Monte Carlo simulations (19, 53) and
found that calculated entropy losses correlated strongly with
experimental propensities in both peptides (49-52) and proteins (54-56). Related work by other groups reached similar conclusions (57-60).
Conformational entropy is not the only proposed explanation for
differences in helix specificity. Another is the drive to segregate
polar and apolar residues on opposite helical faces, giving rise to an
amphipathic helix (61-63). While protein helices are often
amphipathic, the host/guest peptide systems (49-52) are not.
Therefore, amphipathic segregation is an unlikely explanation for the
differences in helix propensity in these peptide systems.
The electrostatic field resulting from the helix dipole has been
proposed as yet another factor that contributes to both helix stability
and specificity (64, 65). Åqvist et al. (66) demonstrated that this effect is short-ranged, and its influence is confined largely
to individual backbone dipoles localized within the first and last
turns of the helix, resulting in a formal positive charge at the helix
N terminus and formal negative charge at the C terminus. Localized
backbone charges can be stabilized by compensating side chain charges,
a fact which helps explain the early observation that the helix N
terminus tends to be enriched in acidic residues, while the C terminus
is enriched in basic residues (67, 68). These electrostatic
interactions may involve side chain-to-backbone hydrogen bonding,
thereby coupling the helix dipole to helix capping, though the two
effects can be disentangled (39).
One popular idea holds that the burial of side chain apolar surface is
an important source of helix stability in both proteins (55, 56) and
alanine-based peptides (29). Of necessity, burial would be limited to
the The hydrophobic contribution made by burial of side chain apolar
surface is proportional to the difference in area between the helix and
coil states. Although the surface area of a residue side chain in a
polyalanyl helix is easily computed, the corresponding area in the coil
state is elusive (71). Often, an extended tripeptide is used to
represent the coil state, but this model is open to question (72). As
an alternative, Creamer et al. (72) developed two limiting
cases that bracket the expected behavior of the coil between reliable
extremes. One extreme was represented by simulated hard sphere peptides
and the other by fragments excised from folded proteins. Using these
limits, it was shown that the area buried by apolar side chains upon
helix formation is considerably less than that estimated from a
tripeptide. Upon transfer from the coil to a midhelical position, an
alanine side chain loses an area between 10 and 0 Å2, and
a valine side chain exposes an area between 0 and 17 Å2.
These results underscore our intuitive expectation. In the coil, a
central residue in a peptide is partially shielded by surrounding neighbors, while in the helix, that same residue would be extruded into
a hyperexposed configuration (like a peacock's tail). Thus, loss of
side chain apolar surface from side chain-backbone interactions is an
implausible source of helix stability.
Summarizing the conclusions from these studies, the The thermodynamic hypothesis of Anfinsen asserts that the folded
state of a population of proteins corresponds to a global minimum of
free energy (73). Given that the fold is unique, the protein will have
lost all conformational entropy or nearly so. Therefore, it would
appear that an effective solution to the folding problem is to minimize
the internal energy.
However, this attractive strategy is called into question by studies of
the A clear conclusion from these studies is that side chain conformational
entropy influences whether a protein segment will be helical or
extended. This observation is of central importance to understanding
the folding problem. The protein interior is comprised almost
exclusively of residues from either Physically, loss of side chain conformational entropy measures the
effect of side chain atoms bumping into the rest of the polypeptide
chain. Side chain steric factors affect helicity markedly, and their
influence is local, realized primarily through interaction with other
atoms close in sequence (77). For illustration, Fig. 1
shows the results of two identical simulations, one of a 15-residue polyalanine and the other of a similar 15-mer where the central alanine
has been replaced by a valine. The presence of even the single valine
reduces the fractional helix population by up to 20% at an adjacent
site. Longer range steric interactions will have little influence on
helicity because, in a helix, side chain
Taken together, these studies depict a simple picture of the folding
process. Proteins, being macromolecules, are large enough to enclose a
solvent-shielded interior within which hydrophobic groups can be
sequestered. Shielded hydrophobic side chains are covalently attached
to the polar backbone, which is also shielded in most cases (78). Were
this backbone unable to form H-bonds within the molecular interior,
then hydrogen bonding would push the conformational equilibrium far
toward the unfolded state, where backbone groups could H-bond to
solvent water. Consistent with this idea, almost all backbone groups
within the interior of proteins of known structure are found to be
H-bonded (76). There are only two structures that provide ubiquitous
hydrogen bonding for interior residues, Of the two core conformations, helix is the thermodynamically preferred
state for the main chain, but some side chains lose sufficient
conformational entropy in a helix that they push the residue toward the
only other allowed region, viz. extended (i.e. Given that side chain conformational entropy is a local effect, it must
arise as an early folding event, e.g. within the
unobservable burst phase of kinetic experiments. Such events will
predispose residues to populate either
-helix is an elegantly simple structure
(1), but helix thermodynamics has proven to be complex and often
refractory. The appealing idea that helix formation is an early guiding
event in protein folding (2-6) remains controversial (7-11). We
suggest that the known facts are sufficient to resolve this controversy because they imply that side chain conformational entropy, and therefore helix propensity, must play an important organizing role
during the earliest stages of protein folding.
and
, corresponding to
regions of the
,
map near (
60°,
40°) and
(
120°,+135°), respectively. The repetition of
-values for
successive residues results in a helix, a conformation that compacts
the chain, thereby expelling water while engendering intrasegment
hydrogen bonds (1) and exquisite van der Waals contacts (14). Although
the peptide backbone has a strong tendency to leave the vapor phase and
enter water (15), the helix per se can be an even better "solvent" (16). Further, water liberated by backbone polar groups upon helix formation is released, an entropically favored event.
-carbons
"bumps" into a backbone atom in either of the other two. These side
chain steric factors predispose segments of the chain toward either
or
regions of the
,
map, and their influence would be exerted
early in folding because the interactions are local, exerted between
atoms that are close in sequence. Consecutive residues that
preferentially populate the same region, either
or
, become
candidates for further stabilization into helices or strands,
respectively. These entropically driven segments of nascent secondary
structure affect subsequent folding by favoring certain pathways and
suppressing others. Thus, helix and strand formation will be guiding
events in protein folding.
-helix (1) is a well designed structure. Its hydrogen bonds
are intrasegment and therefore self-contained, with near ideal
geometry. Backbone atoms are close packed, and all interactions are
local, confined between consecutive turns of the helix. Finally, the
pattern is completely extensible; any number of consecutive residues
can adopt a helical conformation with these favorable design
characteristics.
-helix is also a stable structure. The autonomous stability of the
helix has been a topic of keen interest for many years, motivated in
part by conjectures about protein folding. One engaging idea has been
that helices seed the folding pathway and influence later folding
events. Unfortunately, early experimental evidence indicated that the
cooperative unit for stable helix formation is ~100 residues in
length (21). This threshold exceeds the length of the average protein
helix (~12 residues (22)) by almost an order of magnitude.
Consequently, the conclusion that protein helices were far too short to
function as independent folding units seemed inescapable.
4)). Unavoidably, the initial four amide hydrogens and final
four carbonyl oxygens of the helix lack intrasegment main chain
hydrogen bonds because, upon termination, no next turn of helix exists
to provide such partners. Thus, Pauling-Corey-Branson hydrogen bonds
account for only 50% of the total in the helix of average length, with
the first four N-H groups and last four C=O groups accounting for the
remaining 50%.
G) between the stability of the host
(
Ghelix
coilhost) and
each guest
(
Ghelix
coilguest). Although
the host systems differed among these four groups, the resulting rank
order of helix propensities was remarkably similar.
-carbon in the latter case. As emphasized by these authors (29,
56), most of the stabilization energy must be contributed by side chain
to backbone interactions. Interactions among side chains are precluded
in an alanine-based peptide because the
-carbons cannot reach one
another. Although interactions between proximate side chains are
possible in peptides of heterogeneous composition, such interactions
are too infrequent and haphazard to qualify as a general explanation of
stability. Often, contacting side chains pay an entropic price that
outweighs any enthalpic gain, and their net effect is to destabilize
the molecule (69, 70).
-helix is an
energetically stabilized conformation for main chain atoms. Helix
specificity arises in the side chain, where loss of conformational entropy upon helix formation is a major determinant of the
helix-forming tendencies of residues in both peptides and proteins.
When present, helix capping contributes additional specificity.
-helix described in the previous section. Recapping, side chain
conformational entropy is a major determinant of helix propensity in
both peptides and proteins. In peptides, the reason some sequences are
helical and others are not is explained, in large part, by differences
in the entropic price that their side chains must pay to leave the coil
state and adopt a helical conformation. A parallel situation exists in
proteins where there are only two substantially populated regions in
,
space (74) that a non-prolyl/non-glycyl residue can adopt,
and
, corresponding to regions of the
,
map near
(
60°,
40°) and (
120°,+135°), respectively. Analogous to
coil,
(i.e. an extended conformation) imposes little
steric restriction on the conformation of nearby side chains. Thus,
helix propensities in proteins involve entropy differences between
extended and helical conformations.
-helices or
-strands,2 a consequence of the fact
that these two regular secondary structures are unique in their
capacity to provide buried backbone carboxamides with intramolecular
hydrogen bond partners (76). In other words, conformational entropy is
implicated in determining the structure of the protein core, and its
effect is exerted in discriminating between helix or strand.
- and
-carbons cannot
reach beyond the backbone of an adjacent helical turn.
Fig. 1.
Data were generated from Monte Carlo computer
simulations (75) of two peptides: (i)
Ac-(Ala)15-N-Me and (ii)
Ac-(Ala)7-Val-(Ala)7-N-Me. The
move set was as follows: a set of three consecutive residues was
selected at random. In all three, backbone dihedral angles were
assigned to either the -region (
=
64 ± 7°,
=
43 ± 7°) or the
-region (
=
120 ± 40°,
= +120 ± 30°). For each residue, exact values of
and
were
chosen at random from the given ranges. Either the
- or
-region
was chosen randomly with equal probability. In valine-containing
triplets, the side chain torsion (
) angle was rotated at random in
the range
180° to +180°. A hard sphere potential was used to
describe interactions between atoms. In this potential, atoms have no
attractive component, only excluded volume. United atoms were employed,
i.e. CH, CH2, and CH3 groups were
treated as single atoms with inflated radii. The atomic radii used were
scaled to 90% of their van der Waals values (33), bond lengths and
angles were fixed at standard values, and peptide units were held rigid
and planar (
= 180°).
[View Larger Version of this Image (18K GIF file)]
-helix and
-sheet (1,
79). While other interior structures are found occasionally (80), they
do not lend themselves readily to routine hydrogen bonding. Parenthetically, with only two conformational possibilities, the Levinthal paradox (81, 82) is reconciled for the protein core.
-strand). Thus, conformational entropy plays a crucial role in selecting between helix and sheet.
or
regions of
,
space preferentially. Consecutive residues that populate the same
region become candidates for further stabilization as helix or strand.
These entropically driven segments of nascent secondary structure guide
subsequent folding by favoring certain pathways and suppressing others.
It is important to emphasize that this organizing effect is due to the
entropically driven enrichment of
microstates over
microstates, or the converse, and it is exerted before stable secondary structure can be detected experimentally.