Protein Engineering vol. 16 no. 11 pp.
799±807, 2003
DOI: 10.1093/protein/gzg101
Exploring the sequence patterns in the a-helices of proteins
Junwen Wang1,2 and Jin-An Feng1,2,3
1Department
of Chemistry and 2Center for Biotechnology,
Temple University, 1901 North 13th Street, Philadelphia, PA 19122, USA
3To
whom correspondence should be addressed.
E-mail: feng@astro.temple.edu
This paper reports an extensive sequence analysis of the
a-helices of proteins. a-Helices were extracted from the
Protein Data Bank (PDB) and were divided into groups
according to their sizes. It was found that some amino
acids had differential propensity values for adopting
helical conformation in short, medium and long a-helices.
Pro and Trp had a signi®cantly higher propensity for
helical conformation in short helices than in medium and
long helices. Trp was the strongest helix conformer in
short helices. Sequence patterns favoring helical conform-
ation were derived from a neighbor-dependent sequence
analysis of proteins, which calculated the effect of neigh-
boring amino acid type on the propensity of residues for
adopting a particular secondary structure in proteins.
This method produced an enhanced statistical signi®cance
scale that allowed us to explore the positional preference
of amino acids for a-helical conformations. It was shown
that the amino acid pair preference for a-helix had a
unique pattern and this pattern was not always predict-
able by assuming proportional contributions from the
individual propensity values of the amino acids. Our
analysis also yielded a series of amino acid dyads that
showed preference for a-helix conformation. The data
presented in this study, along with our previous study on
loop sequences of proteins, should prove useful for
developing potential `codes' for recognizing sequence
patterns that are favorable for speci®c secondary
structural elements in proteins.
Keywords: a-helix/propensity/protein structures/secondary
structure/sequence pattern
Introduction
Recognizing the sequence patterns of proteins in relation to
their structures has been one of the most important aspects of
our efforts towards the understanding the principles of protein
folding. The An®nsen experiments in the 1950s suggested that
the primary amino acid sequence contained the information
that speci®es the folded native protein structure (An®nsen,
1973). Subsequent experiments over the past few decades have
generally supported this conclusion (Dill, 1990; Baldwin and
Rose, 1999a,b; Honig 1999). Two-dimensional NMR hydrogen
exchange, coupled with stopped-¯ow pulse-labeling experi-
ments, showed that the folding intermediate(s) usually possess
secondary structures that are similar to that of the native protein
(Hughson et al., 1991; Jennings and Wright, 1993;
Chamberlain and Marquesee, 1997). These data suggested
Protein Engineering vol.16 no.11 ã Oxford University Press 2003; all rights reserved
that the formation of secondary structures largely depended on
the local amino acid sequence since the intermediate folding
states presumably did not have the established tertiary contacts
of the native structure.
Armed with this experimental evidence, computational
efforts to identify relationships between the amino acid
sequence and special local structural elements have been
intensive. Most attempts to identify such relationships have
proceeded by identifying a common structural motif, then
characterizing the frequencies of occurrence of each amino
acid at each position in that motif. One of the best examples of
Downloaded from http://peds.oxfordjournals.org/ at UCSF Library on March 19, 2015
such a study was the sequence pattern of the b-turn loop. This
four-residue b-turn has been observed in a large number of
protein structures (E®mov, 1993). It is a tight-turn loop
structure with an intra-loop hydrogen bond between the main
chain C=O(i) and the N±H(i + 3). The b-turn was later re-
classi®ed into many different sub-classes according to the
polypeptide backbone dihedral angles (Hutchinson and
Thornton, 1994). Other distinct sequence patterns include
a-helix capping residues and b-breakers. Gly is commonly
described as a helix terminator (or Ccap residue), whereas Asp,
Asn, Ser and Thr are favored at the Ncap position of a-helices
(Presta and Rose, 1988; Richardson and Richardson, 1988;
Parker and Hefford, 1997). b-Breakers are residues often found
at the b-strand initiation or termination positions of protein
structures (Colloc'h and Cohen, 1991). They include Gly, Glu,
Asp and Pro. More comprehensive approaches have involved
clustering structural segments of proteins into classes using
measures of structural similarity and then tabulating the
sequence preference for each of the classes (Unger et al.,
1989; Rooman et al., 1990; Olivea et al., 1997). In spite of
these efforts, the discovered relationship between a particular
local structure and the amino acid sequence has been insuf-
®cient for developing a rational secondary structure predictor.
We developed a method for analyzing the sequence±
structure relationship of proteins termed neighbor-dependent
sequence analysis (Crasto and Feng, 2001). This method
calculated the neighboring probability of a pair of amino acids,
in any combination, in three classes of secondary structures
(a-helix, b-strand and loop). Neighbors were de®ned as the
®rst neighbor, where the amino acids in the pair were
immediately next to each other in sequence; the second
neighbor, where the pair of amino acids was separated by one
amino acid residue in sequence; the third neighbor, where the
pair of amino acid was separated by two amino acids; or the
fourth neighbor, where the pair of amino acids was separated
by three amino acids. We applied the neighbor-dependent
sequence analysis to the residues of immediate neighbors in
loops of proteins (Crasto and Feng, 2001). A series of dyad
codes that had strong preference for loop conformation were
found. For example, it was found that Cys had a high loop
propensity in short loops when it was at a position preceding an
Arg, although both residues had low individual loop
799
J.Wang and J.-A.Feng
propensities. It was evident that the neighbor-dependent
protein sequence analysis method could reveal `hidden'
sequence codes in proteins.
a-Helices are one of the most dominant structural elements
in proteins. Extensive studies have been carried out focusing on
a-helix folding and its sequence±structure relationship. Early
studies by Chou and Fasman (Chou and Fasman, 1978) have
established a statistical scale to evaluate the likelihood of
amino acids adopting a-helix conformation. Residues with
high propensities were termed strong helix conformers, and the
residues with helix propensities slightly higher than random
distribution were termed medium helix conformers. Amino
acids having a frequency of occurrence in helices lower than
that of the random distribution were regarded as weak helix
conformers. Although no chemical±physical rationale could be
easily derived for the preference of amino acids adopting helix
conformation, such a statistical analysis has achieved limited
success in assisting our understanding of the sequence±
structure relationship of proteins, as well as in predicting
protein secondary structures (Chou and Fasman, 1978). A
recent study by Penel et al. (Penel et al., 1999) on the analysis
of side chain structures in¯uencing residues adopting a-helical
conformation showed that some amino acid residues favored
hydrogen bonding with neighboring residues via either main
chain±side chain or side chain±side chain interactions, thus
suggesting a potential neighbor-dependent preference for
residues in the a-helix. In this study, we carried out detailed
neighbor-dependent sequence analysis on a-helices in proteins.
Our results showed that amino acid pair preference for the
a-helix had a unique pattern. Based on the neighbor-dependent
propensity values, we derived a series of amino acid dyads that
had predominant preference for a-helical conformation. We
also carried out an analysis on the propensity variation of
amino acids in short, medium and long a-helices. To the best of
our knowledge, such an analysis has never been reported. Our
study showed that there were signi®cant propensity variations
for certain amino acids in helices of difference size groups.
Materials and methods
All analyses were performed using a relational database
derived from the October 2001 release of the Brookhaven
Protein Data Bank (PDB) (Bernstein et al., 1977). Sequences in
the helix and strand regions were often more conserved than
that of loop structures in homologous proteins (Benner and
Gerloff, 1990). A non-redundant set of PDB entries derived by
using PISCES (Wang and Dunbrack, 2003). Proteins with a
sequence identity of >25% were removed. Since the helical
regions of the protein structures were usually well de®ned, it
was necessary to include only structures that were determined
at high resolution. The resolution cut-off of our database was
2.5 AÊ . Based on these criteria, a total of 1430 proteins were
selected from the PDB. These PDB entries were used in the
subsequent parsing protocols.
The extraction of sequences and secondary structure
information from every PDB entry was based on two
independent secondary structure assignment strategies: assign-
ments which were experimentally observed and assignments
generated by the Kabsch and Sander DSSP algorithm (Kabsch
and Sander, 1983), which assigned secondary structure based
on an analysis of backbone dihedral angles and hydrogen
bonds. For the sake of comparison between the two methods of
assignment, we converted the more sophisticated DSSP
800
structure assignments as follows: helices, 310 helices and the
p-helices were all considered helices.
The database system used in this study was PostgreSQL
packaged in Redhat Linux 7.2. The sequence and secondary
structure information of every PDB entry were parsed into
relational tables. Two sets of tables (one was parsed based on
the author-assigned secondary structure information and the
other was parsed according to the DSSP calculation) were
compared. Considering that the authors' assignments were
experimentally observed, we decided to use such assignments
in the PDB as the standard for de®ning a-helices in the protein
structures. However, manual errors were often encountered in
the PDB. In order to avoid such issues, we applied a double-
check mechanism where every structural element in the PDB
was compared with the DSSP assignment. The a-helices that
were agreed upon by both methods were selected from the PDB
and placed in a helix library. The total number of helices
extracted was 10 643, which constituted 96.2% of the total
helices available in those PDB entries. The a-helices were
grouped according to their sizes.
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The residue helix propensity values (ea) were determined
from the ratio of the residue's frequency of occurrence in
helices versus its frequency of occurrence in the PDB
(Equation 1):
aS =nS
ea ˆ 1†
aP =nP
where aS was the number of residues of type a in the helix
library; nS was the total number of residues in the helix data
bank; aP was the number of number of residues of type a in the
PDB that contained all helices in the helix library; and nP was
the total number of residues in the PDB used in this analysis.
The ea values for residues in different helix groups were
calculated using the corresponding values.
For neighbor-dependent analysis of helices, the frequency of
occurrence of the residue type x at neighboring positions of a
helix residue (a) was calculated according to Equation 2:
‰Sx a  i†S Š=n pair†S
ex ai† ˆ 2†
‰Sx a  i†P Š=n pair†P
where Sx(a 6 i)S and Sx(a 6 i)P were the occurrences of
residue type x at the 6ith positions of the residue a in
secondary structure sequence library S (a-helix) and in our
PDB (P), respectively; n(pair)S and n(pair)P were the total number
of residue pairs in S and P, respectively. The numerator of
Equation 2 calculated the frequency of occurrence of residue x
neighbored with the residue type a in the secondary structure
(S), while the denominator of the equation calculated the
frequency of occurrence of residue x neighbored with the
residue type a in the PDB (P). The ratio of these values would
be the propensity of residue x in S when it was neighbored with
residue type a in S.
In this paper, we present the neighbor-dependent propensity
values as ex(a61). An ex(a61) value of 1.0 means that the
occurrence of the residue pair, ax (or xa), in helices is the same
as its frequency of occurrence in proteins. A value >1.0 means
the pair has an occurrence in helices higher than that in

Fig. 1. Length distribution of a-helices in the helix library.
proteins, suggesting that the pair has a preference for adopting
helix conformation. ex(a61) values lower than unity suggest less
preference for the pair in helices. For example, eP(A ± 1) = 1.52
in short helices means Pro has 50% more chance to be found in
short helices than in the proteins when it precedes Ala, i.e. Pro
at ±1 position of Ala; eP(A+1) = 0.47 suggests Pro is less likely to
be found in short helices when it follows Ala, i.e. Pro at +1
position of Ala.
Results
Length distribution of a-helices in proteins
The lengths of a-helices in proteins varied between three and
77 residues with a total of 10 643 helices in the PDB. The
population distribution of helices in the library had a mean
helical length of approximately 12.1 residues (Figure 1), which
was close to the mean helical length determined by Barlow and
Thornton (Barlow and Thornton, 1988) that included 291 a-
helices. There was a relatively large population of three-
residue helices found in proteins. Those helices were most
likely half-turn helices or irregular helices such as the 310
helices that were more frequently found in helices of less than
four residues long (Barlow and Thornton, 1988). In contrast to
the a-helix length distribution reported by Zhu and Blundell
(Zhu and Blundell, 1996), where four-residue helices had twice
the population size as that of the ®ve-residue helices, our helix
library contained more ®ve-residue helices than four-residue
helices by a ratio of 3:2 (Figure 1). There was a gradual
decrease in the helix population as the helical length increased
beyond 13 residues. Helices longer than 40 residues were rarely
found in proteins. The longest helix in our helix library had 77
residues. In fact, there were only one or two examples of each
helical length having more than 47 residues (except 50- and 51-
residue helices which had three examples of each).
In order to establish a data set that was representative of the
general helix population, we chose a subset of our helix library
containing only helices with lengths between four and 22
Sequence analysis of a-helix in proteins
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residues. This subset had a total population of 8771 helices.
Owing to the uneven distribution of different lengths of the
helices in the library where the number of short helices far
exceeded the number of medium and long helices (Figure 1), it
was likely that the helix preference patterns described for the
helix library would only re¯ect the characteristics of the short
or medium a-helices. In order to address such potential bias,
we divided the subset of helices into three groups: short helices
(four to seven residues), medium helices (eight to 13 residues)
and long helices (14 to 22 residues). Based on the selection
criteria, all three groups had approximately equal population
sizes. In the following discussions, propensities calculated
from the entire subset of four to 22 residues helices were
termed total helix propensity; propensities calculated from the
short, the medium and the long helical groups were termed
short, medium and long helix propensities, respectively.
Propensities of amino acids in a-helices of different length
groups
The helix propensities of amino acids in short helices appeared
quite different from that of the medium and long helices. Of
particular interest were Trp and Pro. Not known as a strong
helix conformer, Trp had a signi®cantly higher frequency of
occurrence in short helices (eW = 1.51) than in medium and
long helices. In fact, Trp was the strongest helix conformer in
short helices. In contrast with its presence in medium and long
helices, Pro also had a signi®cantly elevated frequency of
occurrence in short helices (eP = 0.99). A number of residues,
including Asp, Cys, Phe, Ser and Tyr, also had higher helix
propensities in short helices than their propensity values in
medium and long helices, while in the same subgroup of
helices, residues Ala, Arg, Gln, Ile, Leu, Lys, Met and Val had
slightly lower propensity values. Particularly noticeable were
residues Asp and Ala. Both residues were good helix
conformers in short helices, while their helix propensities
were quite different in the context of the overall helix
population (Figure 2). The amino acid propensities in the
801
J.Wang and J.-A.Feng
Fig. 2. A bar graph of the normalized helix propensity of amino acids in groups of different helix sizes. The propensity values of amino acids in different helix
groups are represented by different bars as indicated in the legend.
medium and the long helix subgroups were generally similar to
those of the total helix group. It appeared that the helical
composition of short helices was quite different from that of the
medium and long helices.
Neighbor-dependent sequence analysis of a-helices
In an attempt to analyze how neighboring residues affect the a-
helix conformations of amino acids, we calculated amino acid
preferences at positions immediately preceding (±1) or fol-
lowing (+1) an a-helix residue (a). The neighbor-dependent
helix propensities of 20 amino acids at the +1 and ±1 position
of a-helix residues [ex(a61)] in different groups are tabulated in
Table Ia±d. Propensity values >1.20 are in bold in the table for
ease of inspection. Based on estimated standard deviations,
most of the neighbor-dependent propensities had a comparable
level of con®dence as that of the individual amino acid
propensities (Table Ia±d) (J.Wang and J.-A.Feng, unpublished
results; Kumar and Bensal, 1996). The estimated standard
deviations were slightly higher for neighbor-dependent pro-
pensities in the short helix group than that of other helix
groups. This variation could in part be attributed to the small
population size of residue pairs in the short helix group, which
was less than one-third of the other groups.
The neighbor-dependent helix propensities of amino acids
often re¯ected the individual helix propensities of neighboring
residues. When two strong helix conformers were neighbored,
their neighbor-dependent propensity was almost invariably
high. By the same token, when two weak helix conformers
were neighbored, their neighbor-dependent propensity was
often low. Interesting patterns often occurred when a strong
helix conformer was neighbored with a weak helix conformer,
or two moderate helix conformers were neighbored.
Ala, Glu, Leu and Gln were stronger helix conformers.
Neighbor-dependent propensity calculation showed that they
802
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had a strong in¯uence on the preference of neighboring
residues adopting helical conformation. Not surprisingly,
amino acids with strong or medium individual helix propensity,
including Ala, Arg, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Trp
and Tyr, often exhibited a strong preference adopting a-helix
conformation when they were neighbored with Ala, Glu, Leu
and Gln residues. On the other hand, the neighbor-dependent
effect for stronger helix conformers positioned next to residues
with low individual helix propensity was limited. Although the
frequency of occurrence for residues with low individual helix
propensities was generally increased when they were neigh-
bored with strong helix conformers, most of the neighbor-
dependent propensities were nevertheless <1.0 (Table Ia).
Proportionally, medium helix conformers had less in¯uence on
the preference of neighboring residues adopting helix con-
formation than that of the strong helix conformers. Amino
acids Arg, Lys, Met and Trp had high neighbor-dependent
propensity when they were neighbored with each other
(Table Ia). No neighbor-dependent effect was observed when
they were positioned next to weak helix conformers.
Unique sequence patterns were observed in different helix
groups for a number of amino acids, particularly in the short
helix group. Asp had a high propensity for helix conformation
when it was neighbored with Ala, Arg and Glu in short helices,
while such a pattern was not observed in the medium and the
long helix groups. In contrast, the pairings of Ala with Val and
Ile, as well as the pairing of Arg with Leu, were less frequently
found in short helices than in other helix groups (Table Ib±d).
Similarly reduced neighbor-dependent propensity values were
also found for Arg, Ile, Lys and Met when they were
neighbored to Gln in short helices. Tyr, a weak helix
conformer, had a strong neighbor-dependent helix propensity
when it was positioned next to Met in short helices [eY(M + 1) =
1.91, eY(M ± 1) =1.82], while in other helix groups, Met had no
 Table I. Normalized neighbor-dependent helix propensity of residues in various helix groupsa

Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

(a)
Ala 1.74(4) 1.58(5) 1.04(5) 1.05(4) 1.33(10) 1.66(6) 1.68(4) 0.69(3) 1.13(7) 1.53(5) 1.84(4) 1.60(5) 1.72(7) 1.40(6) 0.42(3) 1.16(4) 0.97(4) 1.71(10) 1.35(6) 1.32(4)
Arg 1.57(5) 1.25(6) 0.98(6) 1.21(5) 1.02(11) 1.44(7) 1.63(5) 0.57(4) 1.16(9) 1.12(5) 1.38(5) 1.28(6) 1.43(10) 1.11(6) 0.31(4) 1.01(6) 0.90(6) 1.34(12) 1.12(7) 0.80(4)
Asn 1.17(5) 0.96(7) 0.51(5) 0.68(5) 0.55(9) 0.86(7) 1.13(6) 0.35(3) 0.86(9) 0.82(5) 0.95(5) 0.82(5) 1.05(10) 0.94(6) 0.42(4) 0.66(5) 0.69(5) 0.93(10) 0.77(6) 0.73(5)
Asp 1.35(4) 1.07(6) 0.63(5) 0.78(5) 0.83(9) 1.19(7) 1.23(5) 0.28(2) 0.99(8) 1.05(5) 1.17(4) 1.02(5) 1.32(9) 1.09(6) 0.50(4) 0.68(4) 0.85(5) 1.21(10) 1.12(6) 0.96(4)
Cys 1.07(9) 1.04(10) 0.78(11) 0.73(9) 0.73(15) 1.03(12) 1.13(10) 0.54(7) 0.94(13) 1.00(11) 1.3(9) 1.03(11) 1.02(19) 0.86(12) 0.21(5) 0.58(8) 0.68(9) 0.95(21) 0.56(10) 0.59(8)
Gln 1.69(5) 1.53(7) 0.91(7) 0.99(7) 0.95(13) 1.54(7) 1.51(6) 0.55(4) 1.16(10) 1.22(6) 1.64(5) 1.39(7) 1.52(11) 1.24(8) 0.29(4) 0.96(6) 0.92(6) 1.19(12) 1.27(8) 1.01(6)
Glu 1.81(4) 1.44(5) 1.01(5) 1.08(5) 1.08(11) 1.76(6) 1.65(4) 0.63(4) 1.16(8) 1.39(5) 1.61(4) 1.50(5) 1.62(8) 1.29(6) 0.35(4) 1.20(5) 1.19(5) 1.60(10) 1.2(6) 1.18(4)
Gly 0.68(3) 0.58(4) 0.31(3) 0.42(3) 0.56(7) 0.58(4) 0.60(4) 0.35(2) 0.53(5) 0.55(3) 0.71(3) 0.48(3) 0.71(6) 0.57(4) 0.45(4) 0.40(3) 0.42(3) 0.50(6) 0.49(4) 0.45(3)
His 1.17(8) 1.00(9) 0.74(9) 0.75(7) 0.60(11) 1.17(10) 1.33(9) 0.47(5) 0.62(7) 0.93(7) 1.17(6) 0.89(8) 1.10(14) 0.86(9) 0.32(5) 0.75(7) 0.82(8) 0.89(14) 0.92(10) 0.90(7)
Ile 1.49(5) 1.26(6) 0.85(5) 0.82(4) 0.86(10) 1.28(7) 1.33(5) 0.73(4) 0.94(8) 1.03(5) 1.28(5) 1.24(5) 1.43(10) 0.93(6) 0.40(4) 0.88(5) 0.68(4) 1.09(12) 0.87(6) 0.85(4)
Leu 1.74(4) 1.47(4) 1.08(5) 1.16(4) 1.19(9) 1.43(5) 1.64(4) 0.81(3) 1.25(7) 1.3(5) 1.55(4) 1.54(4) 1.55(8) 1.21(5) 0.40(3) 1.06(4) 1.04(4) 1.38(10) 1.05(5) 1.10(4)
Lys 1.61(5) 1.37(6) 0.93(5) 1.03(5) 0.87(10) 1.45(7) 1.60(5) 0.50(3) 0.99(8) 1.08(5) 1.40(4) 1.29(5) 1.40(9) 1.14(7) 0.35(4) 0.94(5) 0.96(5) 1.12(11) 1.18(6) 0.83(4)
Met 1.60(7) 1.27(9) 0.84(9) 1.00(8) 1.09(18) 1.49(11) 1.27(8) 0.70(7) 0.91(13) 1.22(9) 1.73(8) 1.50(8) 1.30(13) 1.21(11) 0.55(7) 0.95(7) 0.71(7) 1.57(21) 1.25(12) 1.08(8)
Phe 1.29(6) 1.14(7) 0.79(6) 0.84(5) 0.83(11) 1.12(8) 1.30(6) 0.68(5) 0.74(8) 0.97(6) 1.44(5) 1.09(6) 1.31(12) 0.99(8) 0.29(4) 0.76(5) 0.71(5) 1.28(13) 1.11(8) 0.89(5)
Pro 0.81(4) 0.66(6) 0.26(4) 0.52(4) 0.37(8) 0.93(6) 1.09(5) 0.28(3) 0.61(8) 0.59(5) 0.81(4) 0.54(5) 0.69(9) 0.64(6) 0.28(4) 0.57(4) 0.46(4) 0.59(9) 0.64(6) 0.44(4)
Ser 1.17(4) 1.00(5) 0.61(5) 0.76(4) 0.64(8) 1.07(6) 1.08(5) 0.39(3) 0.74(7) 1.04(5) 1.20(4) 0.97(5) 1.12(9) 0.96(6) 0.40(4) 0.65(4) 0.65(4) 1.00(9) 0.93(6) 0.82(4)
Thr 1.14(4) 0.99(6) 0.50(4) 0.53(4) 0.78(9) 1.07(7) 1.04(5) 0.49(3) 0.73(7) 0.76(5) 1.15(4) 0.95(5) 1.24(9) 0.80(5) 0.45(4) 0.65(4) 0.67(4) 0.83(9) 0.72(6) 0.73(4)
Trp 1.76(10) 1.37(11) 0.79(10) 0.96(9) 0.65(16) 1.42(12) 1.34(11) 0.76(8) 0.87(14) 1.40(12) 1.63(9) 1.16(11) 1.07(17) 1.21(13) 0.60(10) 0.88(9) 0.70(9) 1.21(21) 0.83(12) 1.04(10)
Tyr 1.31(6) 0.97(6) 0.75(6) 0.86(6) 0.81(11) 1.11(8) 1.17(7) 0.63(5) 0.75(9) 0.88(7) 1.46(6) 1.10(7) 1.36(12) 1.01(8) 0.46(5) 0.67(5) 0.56(5) 0.95(12) 0.96(8) 0.76(5)
Val 1.27(4) 1.12(5) 0.83(5) 0.79(4) 0.83(9) 1.20(6) 1.17(4) 0.57(3) 0.69(6) 0.72(4) 1.04(4) 1.12(5) 1.25(8) 0.85(5) 0.35(3) 0.70(4) 0.60(4) 0.71(9) 0.68(5) 0.66(3)

(b)
Ala 1.27(11) 1.38(17) 1.33(19) 1.47(16) 1.21(32) 1.25(18) 1.56(16) 0.76(10) 0.88(21) 0.82(13) 1.22(12) 1.53(17) 1.08(22) 1.31(19) 0.47(11) 1.34(16) 0.95(14) 1.48(34) 0.84(17) 0.82(11)
Arg 1.03(15) 0.96(18) 1.08(21) 1.47(20) 0.38(22) 1.0(21) 1.47(19) 0.72(14) 1.43(32) 0.62(13) 0.85(12) 1.34(21) 0.80(26) 1.26(22) 0.56(16) 1.15(19) 1.03(19) 1.38(43) 1.16(23) 0.56(12)
Asn 1.04(16) 1.05(22) 0.37(12) 0.75(17) 0.92(37) 0.71(19) 1.12(19) 0.37(9) 1.37(35) 0.79(16) 0.87(14) 0.67(16) 0.32(18) 0.91(21) 0.76(16) 0.94(18) 0.64(15) 1.43(40) 0.94(22) 0.78(15)
Asp 1.28(15) 1.4(21) 0.95(18) 1.12(17) 1.41(38) 1.41(25) 1.6(18) 0.46(9) 0.96(26) 1.39(18) 1.73(16) 1.34(18) 1.69(36) 1.44(21) 1.19(18) 1.07(17) 0.98(17) 2.15(43) 1.43(23) 1.34(16)
Cys 1.13(32) 1.12(35) 0.61(30) 1.30(38) 0.62(43) 0.76(33) 1.65(41) 1.00(27) 2.07(60) 0.72(29) 0.88(26) 1.00(35) 0.37(37) 1.21(45) 0.43(21) 1.14(32) 1.09(34) 2.54(109) 1.09(44) 0.54(24)
Gln 1.20(17) 1.07(22) 0.87(21) 1.00(21) 0.76(38) 1.17(23) 1.61(23) 0.8(16) 0.65(24) 0.89(19) 1.31(17) 1.08(20) 1.17(36) 1.21(26) 0.44(16) 0.80(18) 0.74(18) 1.13(39) 1.25(27) 0.96(18)
Glu 1.72(16) 1.07(16) 1.1(17) 1.56(19) 1.28(40) 1.66(24) 1.47(16) 0.70(12) 1.12(27) 1.58(18) 1.58(15) 1.34(16) 1.92(33) 1.19(20) 0.49(13) 1.5(20) 1.43(19) 2.54(45) 1.42(23) 1.19(15)
Gly 0.77(11) 0.96(15) 0.31(9) 0.63(11) 1.17(30) 0.66(15) 0.67(12) 0.44(8) 0.40(14) 0.50(10) 0.59(9) 0.57(10) 0.58(18) 0.66(13) 0.52(13) 0.61(11) 0.49(10) 0.70(23) 0.78(16) 0.41(8)
His 1.56(29) 0.72(24) 1.21(34) 0.66(22) 0.63(36) 1.23(33) 1.10(28) 0.59(17) 0.55(21) 0.77(22) 1.06(20) 0.86(25) 1.13(45) 0.90(28) 0.87(24) 0.61(20) 0.93(26) 0.95(47) 0.54(24) 0.98(23)
Ile 1.06(14) 1.26(19) 0.56(13) 0.76(13) 0.33(19) 0.75(18) 1.25(17) 0.71(13) 0.77(23) 0.70(14) 0.82(13) 0.80(14) 0.93(29) 0.96(21) 0.35(10) 0.82(14) 0.56(12) 1.53(45) 0.94(21) 0.24(8)
Leu 1.32(12) 0.98(13) 0.73(13) 1.32(14) 1.15(29) 1.17(17) 1.29(13) 0.78(11) 0.96(21) 0.94(14) 1.26(12) 1.14(13) 1.24(24) 1.54(21) 0.5(10) 0.97(12) 0.91(12) 1.50(38) 0.89(16) 0.79(11)
Lys 1.17(14) 1.26(20) 0.97(18) 1.22(17) 1.11(36) 1.15(22) 1.39(16) 0.53(11) 1.16(28) 0.80(14) 1.12(14) 1.10(15) 1.30(31) 1.16(22) 0.57(14) 1.07(17) 0.89(15) 1.34(40) 1.57(25) 0.68(12)
Met 0.83(20) 1.15(30) 0.37(18) 1.05(27) 1.05(60) 1.00(33) 1.24(28) 1.01(25) 0.19(19) 0.64(22) 0.95(21) 1.26(28) 0.90(36) 0.87(32) 0.71(25) 0.81(22) 0.62(21) 2.38(93) 1.91(49) 0.73(21)
Phe 1.15(19) 1.55(26) 0.93(20) 1.02(18) 1.96(55) 1.30(28) 1.68(24) 0.81(16) 0.45(20) 0.62(16) 1.42(19) 1.00(19) 1.17(38) 1.14(27) 0.39(14) 0.97(18) 0.93(18) 1.31(45) 0.98(24) 0.75(16)
Pro 1.52(18) 1.26(24) 0.63(16) 0.93(16) 0.85(34) 1.97(30) 2.18(22) 0.47(11) 1.10(31) 0.83(18) 1.28(17) 0.92(19) 0.92(32) 1.49(26) 0.78(18) 1.58(21) 0.93(17) 1.90(49) 1.16(25) 0.47(11)
Ser 1.19(15) 1.34(20) 0.88(17) 1.20(17) 1.34(35) 1.42(22) 1.53(19) 0.52(9) 0.89(23) 1.3(18) 1.56(16) 1.30(18) 1.17(30) 1.23(20) 0.57(14) 1.03(14) 0.44(11) 1.36(34) 1.08(21) 0.89(14)
Thr 0.73(12) 0.58(14) 0.55(14) 0.75(14) 1.09(32) 0.97(21) 0.73(14) 0.47(9) 0.81(23) 0.76(15) 0.98(12) 1.04(18) 1.10(30) 0.68(16) 0.5(12) 0.84(15) 0.70(14) 1.23(33) 1.20(23) 0.51(10)
Trp 2.27(43) 2.42(50) 0.72(29) 1.49(36) 1.20(68) 1.74(45) 2.3(48) 1.39(35) 1.94(66) 2.37(54) 1.88(35) 2.06(47) 0.31(31) 2.18(58) 0.69(34) 1.71(40) 0.88(31) 1.70(82) 1.26(47) 0.72(27)
Tyr 1.10(19) 0.80(19) 0.78(19) 1.17(21) 1.44(47) 0.78(21) 1.83(27) 1.07(19) 1.16(36) 0.48(16) 1.21(19) 1.07(22) 1.82(49) 1.06(25) 0.62(18) 0.97(19) 0.50(15) 1.44(47) 0.8(23) 0.51(14)
Val 0.90(12) 0.69(13) 0.76(15) 0.81(13) 0.66(25) 0.98(18) 0.95(13) 0.55(10) 0.54(18) 0.45(10) 0.79(11) 1.12(15) 0.93(25) 1.17(19) 0.34(9) 0.68(12) 0.47(10) 0.54(24) 0.64(16) 0.50(9)

803
Sequence analysis of a-helix in proteins

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 Table I. Continued

804
Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

(c)
Ala 1.75(7) 1.49(10) 0.97(9) 0.97(8) 1.27(18) 1.83(12) 1.64(9) 0.66(6) 1.16(13) 1.52(9) 2.10(8) 1.60(9) 1.52(14) 1.59(11) 0.39(6) 1.16(8) 0.81(7) 1.72(20) 1.63(13) 1.31(8)
Arg 1.64(10) 1.15(11) 1.03(11) 1.08(10) 1.10(21) 1.41(14) 1.68(11) 0.51(7) 1.14(16) 1.13(10) 1.42(9) 1.20(11) 1.50(20) 0.88(11) 0.30(7) 0.93(10) 0.82(10) 1.51(25) 1.06(12) 0.68(7)
J.Wang and J.-A.Feng

Asn 1.25(10) 0.90(12) 0.53(8) 0.67(9) 0.42(15) 0.82(12) 1.05(11) 0.24(4) 0.69(14) 0.81(9) 0.93(8) 0.80(10) 1.08(19) 0.95(12) 0.34(6) 0.59(8) 0.72(9) 0.90(18) 0.69(11) 0.77(8)
Asp 1.36(9) 0.93(10) 0.59(8) 0.76(8) 0.78(16) 1.13(13) 1.31(9) 0.22(4) 0.81(14) 1.05(9) 1.06(7) 0.97(9) 1.16(17) 1.20(11) 0.42(6) 0.59(7) 0.80(9) 1.41(20) 1.05(11) 1.03(8)
Cys 0.74(15) 1.04(19) 0.63(18) 0.77(17) 0.63(25) 1.14(23) 1.05(19) 0.47(11) 0.77(22) 0.91(19) 1.23(17) 1.42(23) 1.66(42) 0.71(20) 0.15(7) 0.56(13) 0.78(16) 0.50(17) 0.37(12)
Gln 1.57(11) 1.58(15) 0.85(12) 0.91(12) 0.78(22) 1.29(14) 1.41(12) 0.44(7) 1.21(19) 1.35(13) 1.66(11) 1.26(12) 1.28(21) 1.49(16) 0.36(8) 0.80(10) 0.88(11) 1.35(24) 1.16(15) 0.87(10)
Glu 1.76(9) 1.57(11) 0.88(9) 1.04(9) 1.01(20) 1.7(13) 1.93(10) 0.49(6) 0.92(14) 1.41(10) 1.72(8) 1.65(10) 1.58(17) 1.35(12) 0.40(7) 1.20(10) 1.12(9) 1.59(20) 1.32(13) 1.22(8)
Gly 0.69(6) 0.50(6) 0.36(6) 0.35(5) 0.43(11) 0.46(7) 0.57(6) 0.40(5) 0.50(9) 0.50(6) 0.72(6) 0.39(5) 0.83(12) 0.52(7) 0.41(7) 0.29(4) 0.34(5) 0.45(11) 0.45(7) 0.39(5)
His 0.91(13) 0.90(15) 0.55(13) 0.75(13) 0.87(24) 1.23(19) 1.31(17) 0.37(8) 0.59(12) 0.86(13) 1.21(12) 0.69(13) 0.77(22) 0.93(16) 0.25(8) 0.75(13) 0.74(13) 0.57(21) 0.88(17) 0.83(12)
Ile 1.55(9) 1.10(10) 0.98(10) 0.89(8) 0.79(17) 1.36(13) 1.32(10) 0.70(7) 0.86(14) 1.04(10) 1.57(10) 1.32(10) 1.44(20) 0.91(12) 0.46(7) 0.88(8) 0.77(8) 0.86(19) 0.74(11) 0.87(8)
Leu 1.92(8) 1.66(9) 1.32(10) 1.11(7) 1.21(17) 1.34(10) 1.74(9) 0.8(6) 1.17(13) 1.39(9) 1.67(7) 1.70(9) 1.55(15) 1.36(11) 0.47(5) 1.06(7) 1.06(8) 1.20(19) 1.05(10) 1.14(8)
Lys 1.65(9) 1.36(11) 0.97(10) 1.06(9) 1.02(20) 1.48(14) 1.67(10) 0.47(6) 1.12(16) 1.26(10) 1.44(9) 1.50(10) 1.29(17) 1.15(12) 0.33(6) 0.95(9) 0.89(9) 0.96(19) 1.21(12) 0.88(8)
Met 1.73(16) 1.18(17) 0.85(16) 0.98(15) 1.08(34) 1.22(20) 1.11(15) 0.50(10) 0.77(21) 1.23(17) 2.05(17) 1.42(16) 1.18(23) 1.14(21) 0.64(13) 0.81(13) 0.64(12) 1.09(36) 1.35(23) 1.09(14)
Phe 1.36(11) 0.90(12) 0.68(10) 0.90(10) 0.78(20) 1.08(14) 1.33(12) 0.71(8) 0.67(14) 1.1(12) 1.57(11) 1.37(13) 1.65(25) 1.06(15) 0.30(7) 0.67(8) 0.70(9) 1.29(25) 1.05(14) 0.96(10)
Pro 0.99(8) 0.69(10) 0.20(5) 0.55(7) 0.29(12) 0.74(11) 1.17(9) 0.27(5) 0.56(13) 0.78(10) 0.91(8) 0.56(9) 0.83(17) 0.56(9) 0.24(6) 0.49(7) 0.49(7) 0.60(16) 0.68(11) 0.49(6)
Ser 1.24(8) 0.81(9) 0.60(8) 0.63(7) 0.53(13) 0.96(11) 1.03(9) 0.36(5) 0.79(13) 1.08(9) 1.16(8) 0.87(9) 1.07(16) 0.88(10) 0.37(6) 0.56(6) 0.58(7) 1.09(17) 0.79(10) 0.80(8)
Thr 1.19(9) 1.00(11) 0.55(8) 0.51(7) 0.68(15) 0.79(11) 1.06(9) 0.41(5) 0.67(12) 0.61(8) 1.15(8) 0.94(10) 1.42(19) 0.77(10) 0.48(7) 0.64(7) 0.61(7) 0.68(14) 0.62(10) 0.76(7)
Trp 1.41(19) 0.94(18) 0.86(18) 0.80(15) 0.69(30) 1.24(22) 1.31(21) 0.60(13) 0.50(20) 1.22(22) 1.79(19) 1.14(20) 1.27(34) 1.38(26) 0.89(22) 0.93(17) 0.60(15) 1.74(46) 0.49(17) 1.33(20)
Tyr 1.28(11) 1.04(12) 0.75(11) 0.81(10) 0.88(21) 1.10(14) 1.12(12) 0.51(8) 0.36(12) 0.91(12) 1.43(11) 1.17(13) 1.15(22) 0.96(14) 0.51(9) 0.71(9) 0.67(10) 0.88(21) 0.84(13) 0.76(10)
Val 1.25(8) 1.18(9) 0.67(8) 0.82(7) 0.87(16) 1.22(11) 1.31(9) 0.51(6) 0.87(13) 0.74(7) 1.13(7) 1.19(9) 1.12(15) 0.92(10) 0.46(6) 0.66(7) 0.61(6) 0.63(15) 0.64(9) 0.71(6)

(d)
Ala 1.84(6) 1.69(8) 1.02(8) 1.00(6) 1.40(15) 1.64(9) 1.74(7) 0.69(5) 1.16(11) 1.7(8) 1.81(6) 1.63(8) 2.02(13) 1.29(9) 0.43(5) 1.11(7) 1.09(7) 1.75(16) 1.27(9) 1.45(7)
Arg 1.64(8) 1.38(10) 0.93(9) 1.23(9) 1.12(17) 1.58(12) 1.63(9) 0.58(6) 1.11(13) 1.24(9) 1.48(7) 1.33(9) 1.53(16) 1.24(10) 0.25(5) 1.03(8) 0.93(8) 1.20(18) 1.15(10) 0.94(7)
Asn 1.15(8) 0.98(10) 0.53(7) 0.67(7) 0.56(14) 0.92(10) 1.19(9) 0.43(5) 0.86(13) 0.84(8) 0.99(7) 0.87(8) 1.20(16) 0.94(10) 0.39(6) 0.64(7) 0.68(7) 0.84(15) 0.78(9) 0.69(7)
Asp 1.35(7) 1.09(9) 0.58(7) 0.71(7) 0.73(13) 1.18(11) 1.08(7) 0.28(4) 1.12(13) 0.98(7) 1.11(6) 0.98(7) 1.34(15) 0.92(8) 0.39(5) 0.66(6) 0.85(7) 0.84(13) 1.09(9) 0.83(6)
Cys 1.29(16) 1.03(16) 0.93(17) 0.57(12) 0.82(23) 1.02(18) 1.06(16) 0.48(9) 0.77(18) 1.14(17) 1.45(15) 0.76(14) 0.72(24) 0.88(18) 0.21(7) 0.46(10) 0.50(11) 1.23(36) 0.48(14) 0.76(13)
Gln 1.90(9) 1.61(12) 0.97(10) 1.04(10) 1.11(21) 1.81(13) 1.56(10) 0.57(6) 1.24(15) 1.22(10) 1.70(9) 1.55(11) 1.77(19) 1.08(12) 0.2(5) 1.11(10) 0.99(10) 1.09(18) 1.35(13) 1.12(9)
Glu 1.87(7) 1.44(8) 1.08(8) 1.00(7) 1.08(17) 1.83(11) 1.50(7) 0.71(6) 1.33(13) 1.33(8) 1.55(7) 1.43(7) 1.58(13) 1.28(10) 0.27(5) 1.12(8) 1.17(8) 1.37(15) 1.08(9) 1.16(7)
Gly 0.65(5) 0.54(5) 0.27(4) 0.43(4) 0.51(9) 0.64(7) 0.61(5) 0.29(3) 0.59(8) 0.59(5) 0.73(5) 0.52(5) 0.66(9) 0.59(6) 0.46(6) 0.43(4) 0.46(5) 0.49(9) 0.45(6) 0.50(4)
His 1.26(12) 1.13(14) 0.76(13) 0.78(11) 0.41(14) 1.12(15) 1.40(14) 0.52(8) 0.66(11) 1.02(12) 1.16(10) 1.04(13) 1.32(22) 0.81(12) 0.23(6) 0.79(11) 0.84(12) 1.10(23) 1.04(15) 0.94(11)
Ile 1.55(8) 1.37(9) 0.83(7) 0.78(6) 1.04(15) 1.35(11) 1.35(8) 0.75(6) 1.03(12) 1.10(8) 1.19(7) 1.28(8) 1.53(17) 0.94(10) 0.37(5) 0.90(7) 0.65(6) 1.15(18) 0.95(10) 0.99(7)
Leu 1.72(6) 1.46(7) 1.00(7) 1.16(6) 1.19(14) 1.56(9) 1.67(7) 0.82(5) 1.37(11) 1.32(7) 1.54(6) 1.53(7) 1.62(12) 1.01(8) 0.33(4) 1.08(6) 1.05(6) 1.48(17) 1.10(8) 1.14(6)
Lys 1.69(8) 1.40(9) 0.89(8) 0.96(7) 0.72(14) 1.51(11) 1.61(8) 0.51(5) 0.86(12) 1.02(8) 1.44(7) 1.19(7) 1.5(15) 1.13(10) 0.31(5) 0.91(7) 1.02(8) 1.18(17) 1.07(10) 0.83(6)
Met 1.68(12) 1.37(15) 0.94(14) 1.01(12) 1.10(28) 1.80(19) 1.38(13) 0.76(10) 1.18(21) 1.35(15) 1.69(13) 1.61(14) 1.48(21) 1.34(18) 0.45(9) 1.07(12) 0.77(11) 1.73(36) 1.02(17) 1.17(12)
Phe 1.28(9) 1.20(11) 0.83(9) 0.75(7) 0.59(15) 1.11(12) 1.20(9) 0.63(7) 0.87(13) 0.97(10) 1.36(8) 0.92(9) 1.10(17) 0.91(11) 0.26(5) 0.77(7) 0.67(7) 1.27(20) 1.18(12) 0.86(8)
Pro 0.52(5) 0.49(7) 0.20(5) 0.40(5) 0.31(10) 0.81(9) 0.77(6) 0.24(4) 0.53(10) 0.40(6) 0.62(6) 0.43(6) 0.53(12) 0.49(7) 0.20(4) 0.38(5) 0.33(5) 0.26(9) 0.49(8) 0.39(5)
Ser 1.11(7) 1.06(8) 0.54(7) 0.74(6) 0.56(11) 1.06(9) 1.01(7) 0.39(4) 0.68(10) 0.95(7) 1.14(6) 0.95(7) 1.14(13) 0.95(8) 0.37(5) 0.63(5) 0.75(7) 0.86(13) 0.99(9) 0.81(6)
Thr 1.20(7) 1.08(9) 0.45(6) 0.5(6) 0.77(13) 1.29(11) 1.10(8) 0.55(5) 0.75(10) 0.86(7) 1.19(6) 0.93(8) 1.14(14) 0.85(8) 0.41(5) 0.62(6) 0.70(6) 0.85(13) 0.67(8) 0.75(6)
Trp 1.88(17) 1.41(17) 0.75(14) 0.95(14) 0.49(21) 1.48(19) 1.14(16) 0.72(12) 0.88(21) 1.30(18) 1.46(14) 0.97(15) 1.12(27) 0.85(17) 0.38(12) 0.63(12) 0.72(13) 0.72(26) 0.96(19) 0.92(14)
Tyr 1.38(10) 0.95(10) 0.74(9) 0.82(8) 0.62(15) 1.20(12) 1.05(10) 0.60(7) 0.92(15) 0.96(10) 1.54(9) 1.05(10) 1.39(20) 1.03(12) 0.38(7) 0.57(7) 0.51(7) 0.89(17) 1.08(12) 0.82(8)
Val 1.38(6) 1.19(8) 0.96(8) 0.76(6) 0.84(13) 1.23(9) 1.12(7) 0.62(5) 0.60(9) 0.78(6) 1.02(6) 1.08(7) 1.42(14) 0.73(7) 0.27(4) 0.72(6) 0.61(5) 0.81(14) 0.72(8) 0.67(5)

Amino acids in the columns precede residues in the rows. Propensity values >1.20 are in bold for ease of inspection. Parts (a)±(d) list normalized propensity values derived from analysis of (a) helix library, (b) short
helices, (c) medium helices and (d) long helices. The standard deviations were estimated according the formula derived by Williams et al. (Williams et al., 1987): sd = [fij,k(1 ± fij,k)nij]1/2N/nij, where fij,k was the
frequency of occurrence of the residue pair i, j in the helix group k, nij was the total number of i, j pairs and N was the total number of residue pairs in our PDB library. The total number of residue pairs in the short
helix group was 11 101, in the medium helix group was 32 415, in the long helix group was 45 917 and in the total helix group was 89 433. The total number of residue pairs in our PDB library was 338 875. All
propensity values were represented with two decimal places in order to keep all records on a consistent basis. The residue pair Cys±Trp was not present in the medium helix group.

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 Sequence analysis of a-helix in proteins

Table II. Dyad sequence codes for different groups of helix and strand
Discussion
The large population of secondary structural sequences derived
All helices Short helices Medium helices Long helices
in this study allowed us to analyze sequence patterns in
Asp±Met Ala±Arg Arg±Met Asp±Ala different helix groups. Amino acid helix propensity values in
Met±Trp Ala±Lys Arg±Trp Asp±Met the medium and the long helix groups were quite similar to
Trp±Ile Arg±Glu Asp±Ala Phe±Leu those found in the total helix group. More signi®cant variations
Tyr±Leu Asp±Gln Asp±Trp Trp±Gln
Asp±Ile Cys±Lys Tyr±Leu
were found in short helices, particularly for residues Trp and
Asp±Phe Gln±Phe Tyr±Met Pro where both residues showed a signi®cant increase in their
Asp±Val Glu±Met frequency of occurrence (Figure 2). Such differential prefer-
Glu±Thr His±Glu ence of certain amino acids in helices of variable sizes was
His±Ala Leu±Asn
Lys±Tyr Phe±Met
found in other studies. Kumar and Bansal (Kumar and Bansal,
Phe±Glu Thr±Met 1996) have shown that long helices (helices with more than 25
Pro±Ala Trp±Leu residues) often had a higher content of residues with longer
Pro±Gln Trp±Val side chains than those in medium and short helices. It was
Pro±Glu Tyr±Leu suggested that amino acids with longer side chains could
Pro±Phe
Pro±Ser perhaps better facilitate complementary interactions with other
Ser±Gln elements of the protein structure (Kumar and Bansal, 1998).
Ser±Leu However, the analysis of our helix library failed to show a trend
that the appearance of residues with bulkier side chains was
All entries in this table were selected from Table Ia±d according to the
following criteria: (i) the dyad signatures had a propensity >1.30, whereas more favored in longer helices. It should be noted that our

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the propensity of their respective dyad pairs was <1.20, and (ii) the
propensities of the dyad signatures and their dyad pairs differed by >0.3.
Dyad pairs with an estimated standard deviation >0.3 were excluded from
the table.
in¯uence on the preference of Tyr adopting helical conform-
ation. Another noteworthy pattern was that the pairing of Met
and Ala, two strong helix conformers, showed no preference
for adopting helical conformation in short helices (Table Ib).
Such a pattern was not observed in other helix groups.
For weak helix conformers, their neighbor-dependent helix
propensities varied signi®cantly in different helix groups. In
the short helix group, Asp was a good `helix neighbor' to a
number of amino acids, including Ala, Arg, Asp, Gln, Glu, Ile,
Leu, Lys, Met, Phe, Trp, Tyr and Val, while in long helices Asp
had little effect on other amino acids adopting helical
conformation. Also in the short helix group, the pairing of
Tyr with Cys and Glu yielded strong neighbor-dependent
propensities. In medium and long helices, Tyr was found to be
favored next to Leu residues. Cys was also more frequently
found at the +1 position of Phe [eC(F + 1) = 1.96] and at the ±1
position of Trp [eC(W ± 1) = 2.54]. There were only relatively
few amino acids showing preference for the helix conformation
when neighbored to a His residue (Table Ia±c). One example
was that Cys and Trp were preferably positioned at the ±1
position of His in short helices [eW(H ± 1) = 2.07, eW(H ± 1) =
1.94]. Like that of His, residues neighboring Ser were not often
found in helical conformation. The exceptions we found were
the pairings of Ser with Trp and Glu in short helices (Table Ib).
Pro and Gly, being the residues that had the lowest
propensity for helix conformation, had the expected pattern
of weak effect on the helical propensities of their neighbors.
This pattern appeared consistent in all helix groups (Table Ia±
d). Interesting preference patterns of amino acids neighboring
Pro residues, however, were found in short helices. Pro had
high helix propensity values when it was positioned at the ±1
positions of Ala, Gln, Glu, Phe, Ser and Trp residues [eP(A ± 1) =
1.52, eP(Q ± 1) = 1.97, eP(E ± 1) = 2.18, eP(F ± 1) = 1.49, eP(S ± 1) =
1.58, eP(W ± 1) = 1.90]. On the other hand, no preference pattern
was found for residues neighboring Gly in helices.
current helix library contained only a limited number of helices
longer than 25 residues (Figure 2). It would be interesting to
revisit the helix propensities of amino acids in longer helices
when a larger population of long helices (>25 residues)
becomes available.
The differential propensity values of amino acids in different
helix groups were also re¯ected in the neighbor-dependent
sequence analysis. As a result, we found a number of sequence
patterns that were unique to speci®c helix groups. For example,
Trp had mostly comparable neighbor-dependent propensities
with other amino acids for helices in the medium, long and total
helix groups. On the other hand, in the short helix group, the
Trp was strongly favored for the helical conformation when it
was positioned at the ±1 positions of Glu, His, Lys and Ser.
However, at the +1 positions of these residues, Trp had no
preference for helical conformation (Table Ia±d). Although the
estimated standard deviations for residue pairs containing Trp
were higher than that of other residue pairs, the neighbor-
dependent propensities for Trp±Glu, Trp±His, Trp±His, Trp±
Lys and Trp±Ser were large enough to justify the statistical
signi®cance of the sequence patterns. This unsymmetrical
neighbor preference was not limited to Trp; Pro also exhibited
unusual sequence patterns in short helices. Although the
individual propensity value of Pro in the short helix group (eP =
0.95) was signi®cantly higher than its propensity value in the
medium and the long helix groups, it was nevertheless still
below the threshold of random distribution (Figure 2). Our
neighbor-dependent sequence analysis showed that Pro had
high frequencies of occurrence at the ±1 position of Ala, Gln,
Glu, Phe, Ser and Trp, with propensity values of eP(A ± 1) =
1.52, eP(Q ± 1) = 1.97, eP(E ± 1) = 2.18, eP(F ± 1) = 1.49, eP(S ± 1) =
1.58 and eP(W ± 1) = 1.90, respectively. The occurrences of Pro
at the +1 position of these amino acids in short helices, on the
other hand, were signi®cantly below that of the random
distribution (Table Ib). Earlier studies had shown that Pro was
one of the preferred residues at the Ncap position of the helices
(the position before the ®rst residue of the a-helix) (Richardson
and Richardson, 1988; Harper and Rose, 1993; Kumar and
Bansal, 1998), as well as at the ®rst position of helices (the N1
position) (Penel et al., 1999). The ®nding of high occurrence of
a Pro±X (X = Ala, Gln, Glu, Phe, Ser and Trp) pattern could
potentially be a `by-product' of Pro being favored at the N-
805
J.Wang and J.-A.Feng
terminus of helices. On the other hand, while the propensities
of amino acids, Asp and Leu, were found to be among the
highest at the second (the N2 position) and the third positions
(the N3 position) of the helix (Penel et al., 1999; Cochran and
Doig, 2001), the amino acid pairs, Pro±Asp and Pro±Leu, had
relatively low neighbor-dependent propensity values in a-heli-
ces (Table Ia±d). It appeared that the neighbor-dependent
effect played a role in determining sequence patterns in
a-helices.
While it was dif®cult to provide a physical±chemical
rationale for the sequence patterns discovered in this study,
the neighbor dependency of amino acids favoring helical
conformation appeared to be consistent with experimental
®ndings. Recent studies have shown that an amino acid
propensity value for a particular geometrical conformation is
not independent of its environment. Sequence analysis of
a-helices in proteins revealed that transitions from loop to
helix conformation required the presence of a particular group
of amino acids (Presta and Rose, 1988; Richardson and
Richardson, 1988; Parker and Hefford, 1997). The amino acid
composition at the ends of helices, where they were often more
hydrophilic in nature, was distinctly different from the
composition in the middle of the helices, where they were
often more hydrophobic in nature (Lacroix et al., 1998). These
®ndings were also supported by experimental work that
analyzed the helix propensity values of amino acids at different
positions of the synthetic peptides (Petukhov et al., 1998, 2002;
Thomas et al., 2001).
One of the applications of the knowledge on the sequence±
structure relationship of proteins is in predicting protein
secondary structures. Protein secondary structure predictions
based on statistical methods have been implemented in a
number of computer algorithms. These include the rule-based
Chou and Fasman method (Chou and Fasman, 1978), the GOR
III method which predicts the structural conformation of an
amino acid in a protein sequence based on the statistical
information of amino acids within a window surrounding that
amino acid (Gilbrat et al., 1987), and the more recent
application of the hidden Markov model (HMM) which derives
a probability for assigning a structural conformation of an
amino acid by inferring its neighboring information (Asai et al.,
1993; Schmidler et al., 2000). These studies have shown that
the accuracy of secondary structure prediction is improved as
more sophisticated implementations of sequence neighboring
sequence information are applied. Implicitly, these studies
suggest that neighboring residue type could play a role in
affecting the propensity of an amino acid adopting a particular
conformation. However, structure prediction algorithms are
usually prediction-accuracy driven; little consideration has
been placed on the nature of intermediate parameters derived
from the training data set. As demonstrated in this study, the
sequence patterns could differ signi®cantly in secondary
structures of variable lengths. It is conceivable that the
prediction ef®ciency of these algorithms could be improved
with the incorporation of knowledge learned in this study.
One of the most noticeable attributes of the neighbor-
dependent analysis method was the increased statistical
signi®cance of the residue propensities. The individual
propensity values, ea, were in the range 0.67±1.51 (Figure 2),
while the neighbor-dependent propensity values, ex(a 6 1), were
in a much greater range between 0.19 and 2.54. Because
different scale factors were applied (see Materials and
methods), the derived propensity values obviously could not
806
be directly compared. Nevertheless, the statistical signi®cance
of both methods should be comparable, i.e. a propensity value
of 1.0 represented random distribution of the amino acids in the
PDB. The expanded statistical scale of the neighbor-dependent
analysis enabled us to explore the hidden codes in the protein
sequence. Speci®cally, we were able to identify dyad signa-
tures (a±b) that were highly favorable for the helix conform-
ation, whereas their dyad pairs (i.e. b±a) had little or no
preference for their corresponding conformations. Table II lists
some of the asymmetric dyads that had a high propensity for
helix conformations. The dyads in Table II were selected from
Table Ia±d according to the following criteria: (i) all entries
had propensities >1.30, whereas the dyad pair of these entries
was <1.2, and (ii) the propensity difference of the dyad pair was
>0.3.
The existence of the dyad sequence patterns re¯ected the
dependence of the helical preference of some amino acids on
their neighbors. Such patterns were not always easily predict-
able. Short helices had by far the most diversi®ed sequence
patterns (Table II). The preference of amino acid dyads
adopting helical conformation could not be easily rationalized.
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Through structural geometrical analysis, Penel et al. (Penel
et al., 1999) suggested that neighbor-dependent sequence
preference for adopting helical conformation could arise from
side chain±main chain or side chain±side chain interactions
between neighboring residues. The sequence preference for
a-helix was not always predictable by assuming proportional
contributions from the propensity values of the individual
amino acids. For example, the occurrence of a Pro (eP = 0.55)
following a Gly (eG = 0.58), i.e. the sequence Gly±Pro, was
actually higher in helices than that of a Pro following a Gln
(Gln±Pro), even though Gln had an individual helix propensity
value of 1.22 (Table Ia). Similar examples were found in
numerous combinations of amino acid pairs. The sequence
patterns of loops were even more diversi®ed than that of the
helices discussed here (Crasto and Feng, 2001).
Residues in a-helices could have both sequence neighbors
and spatial neighbors. Spatial neighbors in helices were
residues separated by i 6 4 in sequence positions. An
intriguing question was whether neighbor-dependent effects,
such as those observed in the loop sequences, could exist
between spatial neighboring residues. For example, side chains
of the spatial neighbors could form favorable interactions,
including hydrogen bonding, polar and hydrophobic inter-
actions, thus providing stabilization energy for helical con-
formation. We examined this scenario by calculating the
neighbor-dependent propensity between residues of i 6 4 in
helices. Surprisingly, our results showed no signi®cant correl-
ations between chemical complementarity of spatial neighbors
and their neighbor-dependent propensity in a-helix (J.Wang
and J.-A.Feng, unpublished results). Spatial neighbors with
chemical complementarities apparently did not have higher
neighbor-dependent propensity than that of the spatial neigh-
bors with no chemical complementarities.
The results of this study show that the sequence patterns for
a-helices were more predictable than that of the sequence
patterns for loops. A combination of residues with high
individual propensity values for helix conformation would
usually yield a high preference for adopting a helical
conformation. On the other hand, when residues with moderate
or low helix preferences were neighbored in sequence,
unexpected sequence patterns emerged, particularly in short
helices where numerous amino acid dyads were found.
 Sequence analysis of a-helix in proteins

Considering the differential amino acid composition in helices

of different length, it was not surprising that the sequence
patterns of a-helices were size dependent.

Acknowledgements
The authors would like to thank Jennifer Davis and other members of the Feng
laboratory for helpful discussions. We also acknowledge the ®nancial support
from the National Institutes of Health (GM54630), the American Cancer
Society (PRG9926301GMC) and an appropriation from the commonwealth of
Pennsylvania.

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Received January 5, 2003; revised June 25, 2003; accepted September 4, 2003

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