A direct comparison of helix propensity in proteins and peptides

Abstract

alpha-Helical secondary structure occurs widely in globular proteins and its formation is a key step in their folding. As a consequence, understanding the energetics of helix formation is crucial to understanding protein folding and stability. We have measured the helix propensities of the nonpolar amino acids for an alpha-helix in an intact protein, ribonuclease T1, and for a 17-residue peptide with a sequence identical to that of the alpha-helix in the protein. The helix propensities are in excellent agreement. This shows that when compared in the same sequence context, the helix propensities of the nonpolar amino acids are identical in helical peptides and intact proteins, and that conclusions based on studies of the helix-to-coil transitions of peptides may, in favorable cases, be directly applicable to proteins. Our helix propensities based on ribonuclease T1 are in good agreement with those from similar studies of barnase and T4 lysozyme. In contrast, our helix propensities differ substantially from those derived from studies of alanine-stabilized or salt bridge-stabilized model alpha-helical peptides.

Full text

Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 2833–2837, April 1997
Biochemistry
A direct comparison of helix propensity in proteins and peptides
JEFFREY K. MYERS,C.NICK PACE
†
, AND J. MARTIN SCHOLTZ
†
Departments of Medical Biochemistry and Genetics, and Biochemistry and Biophysics, and Center for Macromolecular Design, Texas A&M University,
College Station, TX 77843-1114
Communicated by Robert L. Baldwin, Stanford Medical Center, Stanford, CA, January 3, 1997 (received for review December 5, 1996)
ABSTRACT
a
-Helical secondary structure occurs widely
in globular proteins and its formation is a key step in their
folding. As a consequence, understanding the energetics of
helix formation is crucial to understanding protein folding
and stability. We have measured the helix propensities of the
nonpolar amino acids for an
a
-helix in an intact protein,
ribonuclease T
1
, and for a 17-residue peptide with a sequence
identical to that of the
a
-helix in the protein. The helix
propensities are in excellent agreement. This shows that when
compared in the same sequence context, the helix propensities
of the nonpolar amino acids are identical in helical peptides
and intact proteins, and that conclusions based on studies of
the helix-to-coil transitions of peptides may, in favorable
cases, be directly applicable to proteins. Our helix propensi-
ties based on ribonuclease T
1
are in good agreement with those
from similar studies of barnase and T4 lysozyme. In contrast,
our helix propensities differ substantially from those derived
from studies of alanine-stabilized or salt bridge-stabilized
model
a
-helical peptides.
Predicting the three-dimensional structure of a protein from
its amino acid sequence and gaining a detailed understanding
of the mechanism of protein folding remain two of the most
difficult, unsolved problems in biochemistry. In both cases,
understanding the many forces that contribute to the confor-
mational stability of a protein and their interplay is a major
difficulty. One approach to this problem is to uncouple the
formation of secondary structure from overall protein folding
by studying the factors that influence secondary structure
formation in model peptides.
a
-Helices are of primary interest
because they occur widely in proteins and the isolated peptides
often form helical structures in solution so that they can be
used as convenient models for protein folding and stability
(1–5). Although model
a
-helical peptides have been studied in
detail, the relevance of these models to the folding of intact
proteins has not been carefully explored. Here we present a
direct comparison of the helix propensity of the nonpolar
amino acids measured in an
a
-helix in an intact protein, and
in an
a
-helical peptide with the identical sequence.
Ribonuclease T
1
(RNase T
1
) is a small (104 residue),
monomeric protein, which has proven to be a useful model for
the study of protein folding and stability (6). RNase T
1
is an
a
1
b
class protein with several strands of
b
-sheet packed
against a relatively long (17 residues and 4.5 turns)
a
-helix,
forming a hydrophobic core (7). The sequence of the single
a
-helix in wild-type RNase T
1
is: SSDVSTAQAAGYKLHED,
which corresponds to Ser-13 through Asp-29 in the intact
protein (Fig. 1). The helical portion of the RNase T1 protein
has a near ideal site at which to measure helix propensities:
alanine 21 is in the exact center of the helix, on the solvent
exposed face, and the side chains of residues (i, i13) and (i,
i14), which could interact with residues at position 21 are all
involved in other interactions. No residues outside of the
helical region of the protein appear to be close enough to
interact with side chains at position 21. Here we compare the
differences in helix propensity for the nonpolar amino acids in
the context of the intact RNase T1 protein and in a helical
peptide derived from RNase T1.
MATERIALS AND METHODS
Mutants were constructed by the polymerase chain reaction
single-mutagenic primer technique (9) and the proteins were
purified as described (10). Purity was confirmed by SDSy
PAGE. The peptides were synthesized using an Applied
Biosystems automated peptide synthesizer (model 431A) and
standard fluorenylmethoxycarbonyl chemistry. The N and C
termini of the peptides are blocked with acetyl and carbox-
amide, respectively. The peptides were purified using reversed-
phase liquid chromatography and the identity was confirmed
using matrix-assisted laser desorption spectroscopy–time-of-
flight mass spectrometry.
The stability of the mutant proteins was determined using
urea denaturation. Usually, 22 tubes were prepared for each
denaturation curve; each sample tube contained 0.01 mgyml
21
protein in 30 mM glycine (pH 2.5), along with various con-
centrations of urea (Sigma ultrapure). A urea stock solution
was prepared fresh for each curve; the urea concentration was
measured by refractive index using a relation given previously
(11). These tubes were incubated at 25.08C for at least 16 hr
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payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
Copyright q 1997 by T
HE NATIONAL ACADEMY OF SCIENCES OF THE USA
0027-8424y97y942833-5$2.00y0
PNAS is available online at http:yywww.pnas.org.
Abbreviations: RNase T
1
, ribonuclease T
1
; CD, circular dichroism.
†
To whom reprint requests should be addressed at: Department of
Medical Biochemistry and Genetics. e-mail: pace@bioch.tamu.edu
or jm-scholtz@tamu.edu.
FIG. 1. A ribbon drawing of RNase T
1
generated from the crystal
structure (Protein Data Base entry 9RNT) determined by Martinez-
Oyanedel et al. (7). The ribbon drawing was made with MOLSCRIPT (8).
The single
a
-helix in RNase T
1
spans residues 13–29. The site of
substitution, Ala-21, is shown.
2833

before measurements were taken to ensure that the tubes had
come to equilibrium. The intrinsic fluorescence of each sample
(kept at 25.0 6 0.18C by a circulating water bath and stirred
using a magnetic stirring apparatus) was measured by exciting
at 278 nm and monitoring emission at 320 nm in a SLM AB2
fluorescence spectrometer (Aminco). Analysis of the dena-
turation curves was performed using the two-state unfolding
model and the linear extrapolation method (11). These two
methods were combined into a single equation to describe the
shape of the denaturation curve (12, 13):
Y 5
Y
f
1 m
f
@urea# 1 ~Y
u
1 m
u
@urea#! 3
exp
@ 2 ~m~D
1y2
1 @urea#!yRT!#
1 1 exp@ 2 ~m~D
1y2
1 @urea#!yRT!#
,
where Y is the observed fluorescence (after subtracting out the
intrinsic fluorescence of buffer and urea), m
f
and Y
f
are the
slope and intercept, respectively, of the pretransition baseline,
m
u
and Y
u
are the slope and intercept, respectively, of the
posttransition baseline, m is the dependence of free energy of
unfolding on urea concentration, and D
1/2
is the midpoint of
the denaturation curve. The experimental curves were fit by
the above equation using standard data analysis software. The
free energy of unfolding in the absence of denaturant (defined
as the conformational stability of the protein) is the product of
m and D
1/2
. By calculating DDG from the difference in
midpoints times an average m value, a long extrapolation back
to 0 M urea is avoided. Denaturation curves were performed
at least twice for each mutant, and two mutants were measured
four times. The standard deviations in these sets of four give
an estimated error in D
1/2
of 0.04 M and an error in m of 50
kcal mol
21
M
21
. This gives a maximum error in DDG of 0.07
kcal mol
21
.
Helicity of the peptides was measured using circular dichro-
ism (CD) on an Aviv model 62DS CD spectropolarimeter.
Samples contained 30
m
M peptide in CD buffer (1 mM each
potassium phosphate, borate, and citrate) (pH 2.5) in a 0.5-cm
pathlength cuvette, maintained at 08C by a built-in tempera-
ture controlling unit. Peptide stock solutions were made in
water and the peptide concentration was determined by the
absorbance of the single tyrosine residue at 276 nm using an
extinction coefficient of 1390 M
21
cm
21
(14). To convert the
measured CD signal at 222 nm of the peptides into a free
energy scale it is necessary to use Lifson–Roig helix–coil
theory (15). Raw CD signal (in millidegrees) was converted to
mean residue ellipticity ([
u
]
obs
) and then to fraction helix using
F
helix
5
@
u
#
obs
2 @
u
#
coil
@
u
#
helix
2 @
u
#
coil
,
where [
u
]
helix
and [
u
]
coil
represent the mean residue ellipticity
of a complete helix (242,500z(1 2 (3yn)), where n is the
number of residues in the peptide and complete random coil
(1640), respectively (16). The units of mean residue ellipticity
are degycm
2
dmol
21
.
For analysis of the peptide data we define the wt* peptide
(see below) as the ‘‘host’’ peptide and all the other peptides
contained guest residues at position 21. The helix propensities
of the host and guest residues were calculated using a version
of the Lifson–Roig model for the helix–coil transition de-
scribed previously (17). The model employs the single-
sequence approximation, meaning that only one stretch of
helical residues was allowed to exist in any one peptide
molecule in the partition function (one nucleation site per
molecule). For peptides of this length, this model is equivalent
to the full treatment of Lifson–Roig theory (17). The nucle-
ation constant, v
2
, was taken to be 0.0023 (16). The model
treats the host peptide as a homopolymer, assigning only one
propagation parameter (w
host
) to the whole peptide based on
the measured helicity. The helix propagation parameters for
the guest residues (w
guest
) are determined from the changes in
measured helicity. Relative free energy changes were calcu-
lated using DG 52RT ln (wy(1 1 v)). We define DDG 5
DG
mut
2DG
wt*
, such that a positive DDG indicates destabili-
zation of the helix.
RESULTS AND DISCUSSION
The sequence of the single
a
-helix in wild-type RNase T
1
is:
SSDVSTAQAAGYKLHED (Ser-13 through Asp-29). A pep-
tide of this sequence shows a CD spectrum characteristic of a
random coil conformation, with at most only a few percent
helix. The G23A mutation (Gly-23 underlined in the sequence
above) increases the helicity of the peptide to 30% so that it
becomes a useful model. This variant, denoted wt*, serves as
the host for our helix propensity studies. The nonpolar amino
acids are substituted at position 21 (double underlined alanine
above) in the wt* peptide and wt* protein (also containing the
G23A mutation). We have substituted six nonpolar amino
acids at position 21 in the wt* RNase T
1
protein and measured
the resulting changes in conformational stability using urea
denaturation. The data and analysis are presented in Table 1.
All mutations resulted in stable, active ribonucleases. These
types of surface substitutions are not expected to cause large
changes in the structure of the protein (18, 19). The RNase T
1
with alanine at position 21 is the most stable and that with
glycine is the least stable. Alanine is typically found to be the
best helix former and glycine the worst (excluding proline) in
studies of other peptides and proteins.
We synthesized seven 17-residue peptides with sequences
identical to the
a
-helix in wt* RNase T
1
and the six nonpolar
mutants. Our peptide model, wt*, shows a CD spectrum
characteristic of an
a
-helix, with minima at 222 nm and 208 nm,
and a maximum around 190 nm (data not shown). This helical
Table 1. Protein urea denaturation and peptide helicity data
Residue
21
D
1y2
,
M
m,
kcal mol
21
M
21
D(DG)
†
kcal mol
21
2[U]
222
,
deg cm
2
dmol
21
F
helix
D(DG)
‡
kcal mol
21
Ala 2.78 1.58 0 9,900 0.30 0
Leu 2.70 1.64 0.13 7,800 0.24 0.25
Met 2.69 1.60 0.14 8,300 0.25 0.18
Ile 2.51 1.57 0.44 6,600 0.20 0.38
Phe 2.43 1.69 0.56 5,100 0.16 0.61
Val 2.38 1.77 0.65 4,800 0.15 0.66
Gly 2.23 1.71 0.90 3,200 0.11 0.98
†
Change in conformational stability of the protein relative to wt* with alanine at position 21. Calculated
using D(DG) 5 m
avg
z(D
1y2
[wt*] 2 D
1y2
[mutant]), where m
avg
is the average m value for the seven proteins
(1.65 kcal mol
21
M
21
). Positive values of D(DG) indicate that the mutation is destabilizing. Errors in D
1y2
and m are approximately 0.04 M and 0.05 kcal mol
21
M
21
, respectively.
‡
Change in free energy for helix formation of the peptides calculated from Lifson–Roig helix–coil theory
(see Materials and Methods).
2834 Biochemistry: Myers et al. Proc. Natl. Acad. Sci. USA 94 (1997)

structure shows no dependence on peptide concentration,
suggesting that the peptide exists as a monomer. The helical
contents of the seven peptides, as measured by CD at 222 nm,
are given in Table 1. The fractional helix contents ranged from
11 to 30%. We used Lifson–Roig helix–coil theory to translate
these fractional helicities into a relative scale of free energies
as described above. The range of free energies is about 1 kcal
mol
21
, just as in the mutant proteins.
Fig. 2 compares helix propensities measured in intact RNase
T
1
with those measured in peptides with sequences identical to
those of the
a
-helices of RNase T
1
. There is excellent agree-
ment between the two systems, with a slope of unity, a
y-intercept near zero, and a correlation coefficient of 0.98. This
is an important result. It shows that results from studies of the
helix-to-coil transition of a peptide may, in favorable cases, be
directly applicable to proteins. In earlier work, we also found
excellent agreement between studies of interactions at the
carboxyl terminus of the wt* peptide and similar studies of
intact RNase T
1
(20, 21).
Helix propensities were measured earlier in two other
proteins, barnase (22) and T4 lysozyme (18, 19). These results
are compared with our RNase T
1
results in Table 2 and Fig. 3A.
The agreement between results from three proteins and the
RNase T
1
peptide suggests that the helix propensities given in
Table 1 provide a good measure of helix propensity at an
exposed site near the center of an
a
-helix in a protein.
Intrinsic helical propensities of the amino acids were first
measured systematically by Scheraga and coworkers using a
host–guest random polymer system (24). These studies sug-
gested that the helical propensities of the amino acids, with the
exception of proline, did not vary greatly, and that short
peptides (,20 residues) would not exhibit significant helix
formation in aqueous solution. However, in 1968, Klee showed
that the S-peptide of ribonuclease A does form significant
amounts of
a
-helix in aqueous solution (27, 28). This triggered
studies, first by the Baldwin laboratory and then by several
other laboratories, to find simpler model
a
-helices that could
be used to probe the determinants of
a
-helix stability. It was
found that short, monomeric helices composed mostly of
alanine exhibit significant helix formation in water (29, 30).
This helix formation occurred even without favorable side-
chain interactions, showing that the helical propensity of
alanine is higher than indicated by host–guest studies. These
alanine-stabilized peptides have been widely used to model
protein folding (1–5, 16). An important question is whether
these and other model
a
-helices give results directly applicable
Table 2. Helix propensity measured in various systems
Model system
†
(sequence)
DG(Gly) 2DG(Ala),
kcal mol
21
Intercept Slope
Correlation
coefficient
Peptides
RNase T
1
peptide 0.98 (0.00)
‡
(1.00)
‡
(1.00)
‡
(-STAQXAAYK-)
AK 1.97 0.00 1.84 0.97
(-AAKAXAAKA-)
E
4
K
4
0.74 0.06 0.70 0.97
(-KKKXXXEEE-)
EAK 1.95 20.04 1.79 0.90
(-AKEAXAKEA-)
Host–guest 0.35 20.11 0.35 0.80
(HBLP or HPLG)
AGADIR 1.10 20.04 1.02 0.96
(various)
Proteins
RNase T
1
protein-21 0.90 20.02 0.96 0.98
(-STAQXAAYK-)
Barnase-32 0.91 0.19 0.88 0.88
(-KSAQXLG-)
T4 lysozyme-44 0.96 20.13 0.92 0.93
(-QAAKXELDK-)
Coiled–coil 0.77 0.07 0.74 0.90
(-AALEXKLQA-)
†
The peptide or protein systems being compared to the RNase T
1
peptide are shown along with the
sequence near the substitution site (X), given as one-letter amino acid codes. For the peptide model
systems, the AK data are from Baldwin’s group (16), the E
4
K
4
data are from Kallenbach’s group (5),
the EAK data are from Stellwagen’s group (23) as analyzed by Chakrabartty and Baldwin (3), the
host–guest studies of Scheraga (24) and the algorithm AGADIR is from Mun˜oz and Serrano (25). HPLG
and HBLG refer to the host, hydroxypropyl- or hydroxybutyl-L-glutamine, respectively (24). The protein
models are from site 32 in barnase (22), site 44 in T4 lysozyme (18, 19) and a solvent-exposed site in
a model coiled–coil peptide (26).
‡
The intercept, slope, and correlation coefficient are derived by plotting the data from the indicated model
system against the results for the RNase T
1
peptide.
FIG. 2. Comparison of measured helix propensity in the RNase T
1
peptide and protein systems. The differences in DG are expressed
relative to alanine for the other nonpolar amino acids. Data are from
Table 1.
Biochemistry: Myers et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2835

to the
a
-helices found in proteins. With regard to the helix
propensities of nonpolar amino acids, our results suggest that
in some cases they do not.
A comparison of our peptide results with those from the
alanine-stabilized peptides studied by Baldwin and coworkers
(16) and with the salt bridge-stabilized peptides studied by
Kallenbach and coworkers (5) is shown in Fig. 3B. In both
cases, the correlation is excellent (Table 2), but there is a
sizable discrepancy in the range of propensities. The propen-
sities measured in alanine-stabilized helices are almost twice
those found with the ribonuclease T
1
helices, whereas those for
the salt bridge-stabilized helices are about 30% less (Table 2).
The propensities measured by Stellwagen’s group (23) (see
also ref. 3) with peptides stabilized by both alanine residues
and salt bridges are very similar to those measured in the
alanine-stabilized peptides (Table 2). In contrast, our peptide
data are in excellent agreement with results from the program
AGADIR developed by Mun˜oz and Serrano (23) (Table 2).
The parameters used in AGADIR were obtained from an
analysis of the measured fractional helicities of over 400
peptides. It is also obvious from Table 2 that propensities from
host–guest studies are not applicable to either proteins or
other peptides.
The major discrepancy is that the D(DG) values from the
alanine-stabilized peptides are twice as large as the those
measured in RNase T
1
and in other proteins. When the amino
acid sequences of the various systems are compared, important
differences are found (Table 2). In the alanine-stabilized
peptides, the variable residue has adjacent alanines and has
alanines at three of the four (i, i13) and (i, i14) positions. The
RNase T
1
helix has bulkier residues at some of these positions,
as do the T4 lysozyme and barnase helices. The residues in the
salt bridge-stabilized peptides of Kallenbach are even bulkier,
with glutamic acid or lysine present at most positions. The
host–guest polymers consist mainly of very large host residues
(hydroxypropyl- or hydroxybutyl-
L-glutamine). It appears that
the range of propensities scales with the size of the residues
surrounding the variable position. This suggests that the
hydration of backbone amides and carbonyls might be an
important factor (for an example, see ref. 31). The local
sequence might also change the flexibility of the backbone or
side chain and thereby influence conformational entropy. It is
surprising that these differences in sequence exert such a large
effect on the measured helix propensities. It will be interesting
to see if the theoreticians who study the helix-to-coil transition
can explain this difference in behavior.
It has been suggested that the disagreement between ala-
nine-based peptides and other systems is due to oversimplifi-
cation in applying complex helix–coil theories (32). Our pep-
tide results confirm that the problem does not lie in helix–coil
theory. One recent attempt to explain the discrepancy between
propensities measured in peptides and proteins was made by
Qian and Chan (33). In their model, protein-based systems and
isolated peptides should only give identical measures of helix
propensity when helix formation and global protein folding are
tightly coupled. The excellent agreement between our peptide
and protein data suggests that secondary and tertiary structure
formation are indeed tightly coupled in RNase T
1
.
Several explanations for the different propensities exhibited
by the amino acids with nonpolar side chains have been
proposed. These include differences in conformational en-
tropy (34), in the hydrophobic effect (19), and in hydration of
the backbone (35). The helix propensities calculated by Her-
mans et al. (32) using molecular dynamics simulations are in
remarkably good agreement with the results in Table 1. They
predict a D(DG)of'0.2 kcal mol
21
for leucine and methionine
(based on their calculations with
a
-amino-n-butyric acid), of
'0.7 kcal mol
21
for valine, and '1.2 kcal mol
21
for glycine.
They attribute the difference between alanine and glycine
entirely to differences in backbone conformational entropy,
and those between alanine and the other side chains mainly to
differences in side-chain conformational entropy. These and
other calculations (19, 34, 36, 37) suggest that the differences
in helix propensity for the nonpolar amino acids are due mainly
to differences in conformational entropy.
Recently, a structure-based thermodynamic scale for
a
-helix
propensity has been developed (38). In this approach, the
differences in helical propensity between the amino acids
cannot solely be explained by differences in side-chain con-
formational entropy, but can be faithfully calculated when
backbone conformational entropy, solvation entropy, and en-
thalpic contributions are included. Using this approach, good
agreement is found between predicted and observed helix
propensities in T4 lysozyme, barnase, and coiled-coils (see
Table 2).
The conformational stability of proteins is remarkably low,
only 5–10 kcal mol
21
. The large conformational entropy ['1.7
kcal mol
21
per residue at 300 K (36, 37, 39)] that favors the
unfolded state is barely surmounted by a large number of weak,
stabilizing interactions: '1 kcal mol
21
per -CH
2
-group buried
(40) and '1 kcal mol
21
per intramolecular hydrogen bond
formed (41). Even though the helix propensities discussed here
are similarly weak, they are important because proteins typi-
cally contain 30% of their residues in
a
-helical conformations.
Intrinsic propensities are also very important for determining
the conformational preferences of peptides. However, the
contributions from helix propensity are generally destabilizing.
As pointed out (1–5, 34), only alanine residues contribute
favorably to the stability of
a
-helices, all other amino acids are
either neutral or destabilizing and make their contributions
mainly through unfavorable conformational entropy.
In summary, one important unresolved question has been
(1–5): Do helix propensities make an equivalent energetic
FIG.3. (A) Comparison of measured helix propensity in the wt*
peptide and T4 lysozyme (18, 19) (
F
) and barnase (22) (
M
).Values
given are the change in DG of folding relative to alanine. (B)
Comparison of measured helix propensity values for the nonpolar
amino acids in the RNase T
1
wt* peptide and alanine-based peptides
(16) (
F
) and salt bridge-stabilized peptides (5) (
M
). Values are the
change in DG of helix formation relative to alanine. Slopes, intercepts,
and correlation coefficients for best-fit linear regressions of the data
in A and B are given in Table 2.
2836 Biochemistry: Myers et al. Proc. Natl. Acad. Sci. USA 94 (1997)

contribution in peptides and proteins? Our results suggest that
the answer is YES.
We thank R. L. Baldwin and the Pace and Scholtz lab groups for
helpful discussion, Geoff Horn for DNA sequencing, and Kevin Shaw
for generating Fig. 1. We acknowledge the National Institutes of
Health for financial support (Grants GM52483 to J.M.S. and
GM37039 to C.N.P. and Predoctoral Training Grant T32 GM08523 to
J.K.M.) and the Robert A. Welch Foundation (Grants A-1281 to
J.M.S. and A-1060 to C.N.P.). C.N.P. is also supported by the Tom and
Jean McMullin Professorship and J.M.S. is an American Cancer
Society Junior Faculty Research Awardee (Grant JFRA-577).
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