Modification of polypeptide structure

Disclosed in the present application are methods for the identification of favored and suppressed patterns of hydrophobic and nonhydrophobic amino acids in naturally occuring proteins and polypeptides. Methods are disclosed which enable protein structure alteration based on information gained from hydrophobicity pattern analysis.

BACKGROUND OF THE INVENTION 
The initial phase of the folding of amino acid copolymers (peptides, 
polypeptides, and proteins) is hypothesized to be based on the coalescence 
of hydrophobic side chains of the copolymer either within its core or 
against a hydrophobic surface. The mechanisms by which these events occur 
have not been well defined. The ability to identify, with precision, the 
roles of the hydrophobic residues placed in nonhydrophobic positions of 
motifs defined by the hydrophobic residues would lead to new avenues of 
applied research. 
SUMMARY OF THE INVENTION 
The subject invention relates to two complementary methods for establishing 
favored and suppressed patterns of hydrophobic residues from the group 
leucine, isoleucine, valine, phenylalanine, and methionine in (a) alpha 
helices of proteins and (b) throughout proteins without restriction to 
their alpha helices. 
The first of the complementary methods requires the establishment of 
template-specified positions of amino acids in a primary sequence with 
respect to application of a longitudinal, hydrophobic strip-of-helix 
template to a known or putative helix within an amino acid sequence. The 
method comprises providing an amino acid sequence comprising a known or 
putative helix and applying the longitudinal strip-of-helix template: 
EQU .quadrature..circle-solid..increment..circle-solid..quadrature..largecircle 
..largecircle..quadrature..circle-solid..increment..circle-solid..quadratur 
e..largecircle..largecircle..quadrature..circle-solid..increment..largecirc 
le. 
to the amino acid sequence of the known or putative helix to maximize the 
mean hydrophobicity of residues in .quadrature. positions. The template 
pattern is then extended to adjacent non-helical regions. The 
template-specified positions enable predictions which can be used to alter 
the structure, and thereby function, of an amino acid copolymer. For 
example, such predictions can be used to alter helical length. 
The template specified above can also be employed in connection with a 
method for predicting helices in an amino acid copolymer. The method 
comprises providing an amino acid sequence to be analyzed for the presence 
of a helix. The predictor algorithms 
.largecircle..largecircle..largecircle..quadrature..largecircle..largecirc 
le..largecircle..quadrature..largecircle..largecircle..quadrature..largecir 
cle..largecircle. and 
.largecircle..largecircle..largecircle..quadrature..largecircle..largecirc 
le..quadrature..largecircle..largecircle..largecircle..quadrature..largecir 
cle..largecircle. are applied to the sequence with a positive selection 
requiring the mean hydrophobicity of .quadrature. positions being greater 
than or equal to 3.0 on the Kyte-Doolittle scale of hydrophobicity. 
Overlapping positive selections are merged. The longitudinal strip-of-helix 
template 
.quadrature..circle-solid..increment..circle-solid..quadrature..largecircl 
e..largecircle..quadrature..circle-solid..increment..circle-solid..quadratu 
re..largecircle..largecircle..quadrature..circle-solid..increment..largecir 
cle. is then applied to the merged overlapping positive selections to 
maximize the mean hydrophobicity of residues in .quadrature. positions. 
The confidence of a prediction is then ranked based on the identification 
of a concordance of observed residues with the idealized residue-position 
assignments. 
The second complementary method for establishing favored and suppressed 
patterns of hydrophobic residues from the group Leu, Ile, Val, Phe, and 
Met (indicated .diamond-solid.) and other residues (indicated .diamond.) 
is applicable throughout the primary sequence of an amino acid copolymer 
without restriction to alpha helices. This method requires initial 
analysis of a statistically significant number of naturally occurring 
proteins. Statistically significant, as used in this context, means a 
number sufficient to reveal the pattern disclosed in Example 11 (48 
proteins were analyzed in Example 11). Templates of from 3 to 9 positions 
composed of all combinations of .diamond-solid. and .diamond. in each 
position are applied to the amino acid sequences of the naturally 
occurring proteins. The frequency with which each template is found is 
scored and the standard deviation by which a template occurs is determined 
from the expected frequency from an empirical distribution of 
.diamond-solid. and .diamond. positions based upon the natural frequencies 
of the amino acids in the naturally occurring proteins. The patterns are 
then ranked on a scale of preferred and suppressed on the basis of those 
standard deviations between observed and expected frequencies. Preferred 
patterns of hydrophobicity determined in this manner correlate with 
increased stability as determined, for example, by NMR chemical shift 
determinations.

DETAILED DESCRIPTION OF THE INVENTION 
The subject invention relates to two complementary methods for establishing 
favored and suppressed patterns of hydrophobic residues from the group 
leucine, isoleucine, valine, phenylalanine, and methionine. The first of 
the two complementary methods is based on the finding that the placement 
of hydrophobic residues (leucine, isoleucine, valine, phenylalanine and 
methionine) at positions n, n+4, n+7 and n+11 in the primary amino acid 
sequence of a protein will induce folding as a helix with a longitudinal 
hydrophobic strip, in almost all naturally occurring helices which were 
studied. Although not wishing to be bound by theory, this observation can 
be extended. It is reasonable to postulate, for example, that the 
anchoring of recurrent hydrophobic side chains along one side of a helix 
against a hydrophobic surface governs helical folding and adsorption. 
Examples of a hydrophobic surface can include, the hydrophobic core of a 
protein, the binding site of a hormone receptor, or a membrane. 
This observation led to the development of a method to identify categorical 
positions in helices based upon a template for the positions of residues 
on the surface of the cylinder of an alpha helix. Unique distributions of 
residues in those template defined positions demonstrate the role of some 
residues in determining the folding, stability and adsorption of helices 
against hydrophobic surfaces. 
The categorical positions in the helix are determined by applying the 
longitudinal, hydrophobic strip-of-helix template: 
EQU .quadrature..circle-solid..increment..circle-solid..quadrature..largecircle 
..largecircle..quadrature..circle-solid..increment..circle-solid..quadratur 
e..largecircle..largecircle..quadrature..circle-solid..increment..largecirc 
le. 
to an amino acid copolymer containing a known or putative helix. 
Conventional methods can be used to predict the presence of a helix, as 
can other methods disclosed herein. This is best accomplished using 
computer software which applies the first residue in the circular 
(infinite) template to the first residue in the helix and sums the mean 
hydrophobicity of residues in .quadrature. positions of that first 
application of the template. The mean hydrophobicity of residues in 
.quadrature. positions is called the strip-of-helix hydrophobicity index. 
The hydrophobicity of residues are given in the Kyte-Doolittle or another 
scale. The computer then applies the second residue in the template to the 
first residue in the helix in a like manner summing the mean 
hydrophobicity of residues in the .quadrature. positions. Such 
applications continued with each position in the template being applied to 
the first residue in the helix. The application with the highest 
strip-of-helix hydrophobicity index is the "best fit" of the template and 
that positioning is taken in all analyses which follow. It should be noted 
that the methods of this invention can be loosely applied to copolymers of 
small to moderate length without the need for a computer. 
There are four categories of positions in the template for the hydrophobic 
strip-of-helix. Those positions can be displayed in a linear projection of 
the template or in a sheet projection. These two alternate presentation 
formats are used conventionally. In the sheet projection, the positions in 
the primary sequence coil on the cylindrical surface of the helix, as does 
a stripe on a barber's pole, and can be segregated into longitudinal 
quadrants. Quadrant III arbitrarily holds the .quadrature. positions of 
the hydrophobic strip-of-helix and quadrants II and IV contain 
.largecircle. and .circle-solid., respectively. The .circle-solid. 
positions are close to and the .largecircle. positions are farther away 
from the longitudinal axis of quadrant III. Quadrant I contains 
.increment. positions occurring in every other cycle around the helix. 
Once the best fit of the hydrophobic strip-of-helix is determined with 
residues in the helix, the pattern of the helix can be extended to 
residues which fall beyond the helical boundaries to define .quadrature. 
and non .quadrature. residues beyond the helix. 
As discussed in greater detail in the Examples, the distribution of 
residues in template-predicted positions correlated surprising well with 
the actual pattern of residue distributions observed in known helices. The 
preferred distribution of amino acids predicted based upon 
template-specified positions is the following: 
a) leucine, isoleucine, valine, phenylalanine, or methionine in all 
.quadrature. positions in the helix; 
b) leucine, isoleucine, valine, phenylalanine, or methionine in the 
N-terminal .quadrature. position in the helix; 
c) leucine, isoleucine, valine, phenylalanine, or methionine in the 
C-terminal .quadrature. position in the helix; 
d) alanine or valine in the .quadrature. position in the helix with the 
smallest residue 
e) non(leucine, isoleucine, valine, phenylalanine, or methionine) in the 
first .quadrature. position beyond the helix at the N-terminus; 
f) non(leucine, isoleucine, valine, phenylalanine, or methionine) in the 
first .quadrature. position beyond the helix at the C-terminus; 
g) non(leucine, isoleucine, valine, phenylalanine, or methionine) in all 
non.quadrature. positions in the helix; 
h) aspartate or glutamate or asparagine in the N-terminal non .quadrature. 
positions before the N-terminal .quadrature. position; and 
i) lysine, arginine or histidine in the C-terminal .largecircle. positions 
after the C-terminal .quadrature., or in the first C-terminal .quadrature. 
position after the helix. 
The favored residues found in the template-predicted .quadrature. positions 
are the hydrophobic residues leucine, isoleucine, valine, phenylalanine 
and methionine. The distribution of these residues outside the helical 
region were found to correlate with levels expected for random 
distribution. The favored residues found in the non .quadrature. positions 
are non-hydrophobic residues. The presence of hydrophobic residues in the 
non .quadrature. positions would tend to promote the adsorption and 
folding of the protein or polypeptide to a hydrophobic surface in a manner 
which does not promote helical formation. For example, a peptide with 
alternating hydrophobic residues would adsorb as a beta-pleated sheet. 
Deviation from the template-predicted residues tends to disfavor helix 
formation. Negatively charged amino acids aspartate and glutamate were 
found more often in the non.quadrature. positions before the N-terminal 
.quadrature. position in helices. Positively charged residues were found 
more often in non.quadrature. positions after the C-terminal .quadrature. 
position in helices and in the first .quadrature. position beyond the 
C-terminus of the helix. Those residues served to stabilize the 
delta-positive charge at the N-terminus and the delta-negative charge at 
the C-terminus formed by the helix macrodipole, which is the sum of all 
the dipoles formed by the hydrogen bonds between amido protons and 
carbonyl oxygens along the peptidyl backbone. Asparagine was found more 
often in the non.quadrature. positions before the N-terminal .quadrature. 
of the helix. The side chain of asparagine hydrogen-bonded across the 
diameter of the helix to a peptidyl backbone group on the other side of 
the helix, thereby stabilizing its N-terminus. 
Furthermore, by analyzing the data generated in such experiments, it has 
become possible to discern special rules which apply to particular 
template-specified positions. These positions include, for example: 
a) the N-terminal .quadrature. position; 
b) the C-terminal .quadrature. position; 
c) the .quadrature. position in the helix with the smallest residue; 
d) the .quadrature. position residue in the helix with the least 
hydrophobic residue; 
e) non.quadrature. positions in the helix prior to the N-terminal 
.largecircle. position in the helix; and 
f) non.quadrature. positions in the helix after the C-terminal 
.largecircle. position in the helix. 
In addition, it has been possible to identify template-specified residues 
which fall outside the helix to which special rules apply. These include: 
g) the first .quadrature. position beyond the helix at the N-terminus; 
h) the first .quadrature. position beyond the helix at the C-terminus. 
As was discussed above, a strong correlation exists between 
template-predicted .quadrature. positions and hydrophobic residues. This 
correlation is particularly striking for the N- and C-terminal 
.quadrature. positions which define the extent of the helix. In addition, 
the presence of a hydrophobic residue in a terminal .quadrature. of the 
helix and the absence of a member of that group from the 
template-predicted .quadrature. falling outside the helix dictates helix 
termination. The alteration of this pattern leads to extension or 
shortening of the helix. 
It was found that helices tend to cross through their strips and most 
frequently at least one of the crossing residues was an alanine or valine 
residue. These are the smallest of the hydrophobic residues. This 
correlation suggests that crossing of strips through such a residue is a 
favored, stabile configuration. 
From the patterns which have emerged as a result of this analysis 
predictions with respect to the functional effect of structural 
alterations can be made with a high degree of certainty. Such structural 
changes can be exploited in the rational engineering of amino acid 
copolymers such as proteins and polypeptides. 
For example, the structure of an amino acid copolymer can be altered by 
first applying the strip-of-helix template to a known or putative helix 
and determining the best fit as previously described. The concordance 
between template-predicted residues to the residues which actually appear 
in the copolymer is determined. A structural change is effected by 
altering the identity of the actual residues. 
The alteration can change the identity of a residue to bring the strip into 
a greater degree of conformance with the template-predicted chemical 
nature of the residue, or the alteration can create a greater divergence. 
The former change tends to favor helix formation, whereas the latter tends 
to disfavor helix formation. 
Similar principles can also be used to alter the length of a helical 
region. The strip-of-helix template is applied to a known or putative 
helix and the best fit is established. The actual amino acid sequence at 
the N- and C-terminal .quadrature. position and the first .quadrature. 
beyond the termini of the helix is compared with the template-predictions. 
Helix length is altered by changing the identity of one or more of these 
residues in accordance with the principles previously discussed. The helix 
is lengthened by increasing the match between the actual amino acid 
residues at the identified positions and the template-predicted chemical 
identity at the positions. The helix is shortened by increasing divergence 
at these positions. 
The unique distributions of amino acids in and near known helices enables 
the prediction of helices based upon the finding of such distributions. A 
first method is based upon scoring for two templates 
.largecircle..largecircle..largecircle..quadrature..largecircle..largecirc 
le..largecircle..quadrature..largecircle..largecircle..quadrature..largecir 
cle..largecircle. and 
.largecircle..largecircle..largecircle..quadrature..largecircle..largecirc 
le..quadrature..largecircle..largecircle..largecircle..quadrature..largecir 
cle..largecircle. such that the mean hydrophobicity of residues in 
.quadrature. positions is greater than or equal to 3.0 on the 
Kyte-Doolittle scale. Those individual and merged predictions constitute 
the endpoint for the first method of predicting helices. It has a high 
sensitivity (correct predictions/number of true predictions) and 
efficiency (correct predictions/total number of predictions made). 
A second method is based upon the outcome of the first and offers a higher 
degree of efficiency at the price of moderately lower sensitivity because 
it is based also on scoring for additional stabilizing residues in 
template-defined positions. In that method, the merged, predicted helices 
of method 1, are fitted with a template 
.quadrature..circle-solid..increment..circle-solid..quadrature..largecircl 
e..largecircle..quadrature..circle-solid..increment..circle-solid..quadratu 
re..largecircle..largecircle..quadrature..largecircle..increment..largecirc 
le. and the template positions are scored for the presence of idealized 
residue-position assignments. 
The first of two complementary methods for establishing favored and 
suppressed patterns of hydrophobic residues from the group leucine, 
isoleucine, valine, phenylalanine, and methionine, and subsidiary methods 
based on template specified positions, is discussed above. The second of 
the two complementary methods for establishing favored and suppressed 
patterns of hydrophobic residues from the group leucine, isoleucine, 
valine, phenylalanine, and methionine is based on the discovery of rules 
which govern the ordering of amino acid residues throughout proteins in 
general with no restriction to the alpha helices. More specifically, it 
has been discovered that certain patterns of hydrophobic amino acid 
residue ordering are favored while other patterns are suppressed by 
natural selection. Disclosed herein are novel methods to design amino acid 
copolymers de novo or to modify natural products according to patterns of 
hydrophobic and nonhydrophobic amino acids both in the primary sequence 
and in 3-dimensional arrangements within the amino acid copolymer. These 
methods lead to the most efficiently folded and stable forms of such amino 
acid copolymers. 
Patterns of hydrophobic residues which are naturally favored or suppressed 
are determined by the analysis of patterns of such residues in a 
statistically significant number of naturally occurring proteins. More 
specifically, favored and suppressed patterns of hydrophobic and 
nonhydrophobic residues in primary amino acid sequences are found by 
assigning the hydrophobic amino acids Leu, Ile, Val, Phe, and Met (LIVFM) 
to the group .diamond-solid. and all other amino acids to the group 
.diamond.. The frequencies of patterns containing all combinations of 
.diamond-solid. and .diamond. from 3 to 12 positions are determined in a 
statistically significant number of more natural proteins (e.g., 30 or 
more). The patterns are ranked according to the magnitude of their 
deviations from empirical distributions of the amino acids 
.vertline.z.vertline..gtoreq.1.96 (p.ltoreq.0.05). For example 
.diamond..diamond-solid..diamond-solid..diamond..diamond. is favored 
(z=3.5; p&lt;0.001), while 
.diamond..diamond-solid..diamond..diamond-solid..diamond. is suppressed 
(z=-3.4; p&lt;0.001). In longer, composite patterns, 
.diamond-solid..diamond-solid. followed by .diamond..diamond. and one 
.diamond-solid. is favored 
(.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond..diamon 
d-solid..diamond..diamond., z=5.1), while conversion of the single 
hydrophobic residue to a pair is not 
(.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond..diamon 
d-solid..diamond-solid..diamond., z=0.8). Additional distributions of 
certain nonhydrophobic amino acids around .diamond. positions in strongly 
favored patterns are also favored or suppressed (Asp, Glu, Lys, Arg, Asn, 
Cys, Tyr, and Pro; for each .vertline.z.vertline.&gt;2.0). 
The natural rules governing the ordering of hydrophobic residues can be 
applied to the modification of a protein or peptide in a rational drug 
design scheme. In the design of an amino acid copolymer, the arrangements 
of hydrophobic and nonhydrophobic residues should include segments with 
such favored patterns of LIVFM versus nonLIVFM residues occurring at 
z.gtoreq.1.96 (p.ltoreq.0.05). Likewise the design should avoid segments 
with suppressed patterns of LIVFM versus nonLIVFM residues occurring at 
z.ltoreq.-1.96 (p.ltoreq.0.05). Furthermore, the placements of nonLIVFM 
residues should parallel the favored and suppressed composite patterns of 
overlapping or adjacent patterns which are identified with this method. 
That is, when .diamond..diamond-solid..diamond-solid..diamond..diamond. 
occurs it should be followed by .diamond-solid..diamond. rather than 
.diamond-solid..diamond-solid..diamond. and preceded by 
.diamond..diamond-solid. rather than by 
.diamond..diamond-solid..diamond-solid. or 
.diamond..diamond-solid..diamond-solid..diamond., according to the 
composite patterns demonstrated in this invention. Suppressed patterns may 
be employed in design of amino acid copolymers under conditions where 
ambiguity or flexibility of a segment of an amino acid copolymer is 
desired. 
It is possible that the two complementary methods described above can 
specify conflicting amino acid identifies under specific circumstances. In 
the case where modifications in amino acids within one segment of an amino 
acid copolymer are indicated by both the strip-of-helix hydrophobicity 
method (based on application of the template 
.quadrature..circle-solid..increment..circle-solid..quadrature..largecircl 
e..largecircle..quadrature..circle-solid..increment..circle-solid..quadratu 
re..largecircle..largecircle..quadrature..circle-solid..increment..largecir 
cle.) and the method derived from analysis of patterns for Leu, Ile, Val, 
Phe and Met residues (indicated .diamond-solid.) and other residues 
(indicated .diamond.), specifications by the latter method should be 
applied to residues following in .largecircle. or .circle-solid. positions 
which are identified with the strip-of-helix template method. 
The methods of the present invention are useful for the identification and 
modification of structure of amino acid copolymers which are of natural 
origin or designed/selected de novo for catalytic or therapeutic purposes. 
"Modification of structure", as used herein, specifically includes 
modifications which increase, or decrease, amphiphilic helical structure. 
Increases in amphiphilic helical structure correlate with increases in 
stability of the amino acid copolymer. The term "amino acid copolymer", as 
used herein, is intended to encompass peptides and polypeptides (amino 
acid copolymers ranging from about 8 to about 100 amino acid residues), as 
well as proteins. 
Peptides with maximal ordered amphiphilic helical structure (i.e., 
maximally stabilized amino acid copolymers) can be used for a variety of 
purposes. For example, transmembrane proteins are notoriously difficult to 
crystalize. Crystallization is a necessary prerequisite to the 
determination of atomic structure by X-ray diffraction techniques. Amino 
acid copolymers (e.g., peptides) having maximally stabilized structure can 
be used to solubilize transmembranal proteins with helices oriented around 
the hydrophobic transmembranal section in a fashion permitting formation 
of crystals which can be analyzed by x-ray diffraction methods (see 
Schafmeister, C. E., Miercke, L. J. W., and Stroud R. M. 1993, Science 
262: 734-737). The stability and thus efficacy of structural analyses of 
such peptides are enhanced by synthesis according to the method of the 
invention. 
Peptides which have maximal degrees of alpha helicity when adsorbed to 
hydrophobic surfaces, offer a unique standard for the analysis of degrees 
of helicity in other peptides or proteins by circular dichroism (CD) or 
nuclear magnetic resonance (NMR) methods. Thus, amino acid copolymers 
designed in accordance with the methods disclosed herein are useful in a 
commercial context as standards for physical measurements based on their 
high degree of ordered amphiphilic helical structure. Peptides produced 
according to the methods disclosed herein (or a series of homologous 
peptides systematically varying stability of alpha-helical structure) 
permit standardization of such correlations, for example, between several 
types of NMR chemical shifts and degree of alpha-helicity around 
individual amino acid residues. Examples of such commercial applications 
are discussed below. 
The determination of the structure of transmembranal proteins by solid 
state NMR analysis (see Tuzi, S., Naito, A., and Saito, H. 1993, Eur. J. 
Biochem. 218: 837-844; and Saito, H. and Ando, I. 1989, Ann. Rev. NMR 
Spectrosc. 21: 209-290), after dehydration of the sample, requires 
identification of cross peaks in 2D NMR spectra and quantitation of those 
cross peaks with respect to a standard, such as provided by the peptides 
designed in accordance with the methods of the invention. 
In addition, transmembranal proteins, such as multihelical ion channels or 
receptor-transducing molecules, cannot alone be crystallized for x-ray 
crystallography and are too large in vesicles for conventional NMR 
analyses. Magic angle spin solid state NMR analyses allows acquisition of 
2D NMR spectra which can be interpreted in view of spectra of peptides 
with maximal alpha-helicity (or a series of homologous peptides 
systematically varying stability of alpha-helical structure) which 
peptides are designed by the method of the invention. 
In another application for use as a commercial standard, peptides with 
ordered alpha-helical structure designed by methods of the invention allow 
standardization of CD and NMR measurements of peptide-peptide 
interactions. Specifically, the peptide PH-1.0 in 50% TFA solution creates 
a trimeric coiled coil standard to calibrate measurements of such trimeric 
coiled coil structures which are found in certain proteins. 
Peptides designed by the method of the invention for maximal alpha-helicity 
(or a series of homologous peptides systematically varying stability of 
alpha-helical structure) can be used as a standard to quantitate the low 
angle scatter measurements with shape and isotropy of other peptides which 
are studied with the intention of constructing structural changes which 
lead to novel biological or catalytic properties. 
Aside from utility of the methods disclosed herein for the production of 
commercial standards for measurement of specific physical properties, 
amino acid copolymers produced by the methods disclosed herein are 
practically useful in other contexts. For example, peptides designed 
according to the method of the invention, when attached to the solid phase 
of an affinity chromatographic system, adsorb specifically certain circa 
70,000 dalton heat shock protein-like molecules and thus permit the 
efficient purification of such molecules. Specifically, PH-1.0 and 
homologs may be used for such purifications. 
In addition, peptides designed according to the method of the invention 
demonstrate adsorption of tethered ligands to hydrophobic surfaces. They 
therefore permit the analysis of the effects of local orientation at the 
membrane surface of an electron-spin marked group which is tethered at the 
membrane surface. Adsorption of ligands to hydrophobic surfaces therefore 
improve solid phase-based methods of immunoassay, such as enzyme-linked 
immunosorption assay or radioimmunoassay. Amphiphilic helical peptides 
which incorporate catalytic functions might also be adsorbed into 
hydrophobic pores of a membrane permitting efficient catalysis of 
substrates which are filtered through such membranes. 
EXAMPLE 1 
Application of the Hydrophobic Strip-of-Helix Template to Known or Putative 
Helices to Define Categorical Positions with Functional Importance 
A circular template for a helix with a longitudinal hydrophobic strip was 
superimposed on the sequences of known or hypothesized helices in amino 
acid copolymers. The template holds 18 symbols 
.quadrature..circle-solid..increment..circle-solid..quadrature..largecircl 
e..largecircle..quadrature..circle-solid..increment..circle-solid..quadratu 
re..largecircle..largecircle..quadrature..circle-solid..increment..largecir 
cle.. This generic, helical template corresponds to a sheet projection with 
successive coils of the helix in slanting columns and with longitudinal 
quadrants in horizontal rows. By convention, quadrant III has the greatest 
strip-of-helix hydrophobicity index (SOHHI; the mean hydrophobicity in the 
Kyte-Doolittle scale of amino acids in .quadrature.). The infinite 
template was superimposed on the helical segment 18 times by attaching 
each of the 18 symbols to the first position in the segment. The overlay 
with the maximal SOHHI score is chosen. 
The distributions of amino acids in longitudinal quadrants were examined 
with respect to the assignment of residues to one quadrant with the 
greatest SOHHI (Torgerson et al., 1991, J. Biol. Chem., 266:5521-5524). 
The amino acid sequences of 247 alpha helices identified in 
crystallographic structures of 55 proteins by other investigators were 
placed in the sheet projection of an amphipathic helix to maximize the 
SOHHI score. In 89% of the 4 and 5 turn helices so examined, the alignment 
in the crystallographic structures of residues selected to be in the most 
hydrophobic longitudinal strip closely fit a straight line. The 
distributions of amino acids in all longitudinal quadrants were then 
summed over N- and C-termini, interiors, and entire helices (Tables I and 
II). Hydrophobic amino acids leucine, isoleucine, valine, and 
phenylalanine were nonrandomly distributed to quadrant III while charged 
lysine, arginine, aspartate, and glutamate residues were excluded from 
quadrant III (p&lt;0.001 for each amino acid). Selective distributions of 
other amino acids with respect to longitudinal quadrants were also seen. 
Termini began in quadrant IV with three nonhydrophobic residues preceding 
the first leucine, isoleucine, valine, phenylalanine or methionine 
occurring in the longitudinal hydrophobic strip of quadrant III (p&lt;0.001). 
The last residue of the helix fell in quadrant I with two nonhydrophobic 
residues following the last leucine, isoleucine, valine, phenylalanine or 
methionine falling in the longitudinal hydrophobic strip of quadrant III 
(p&lt;0.001). Two successive .largecircle.'s in the template were associated 
with an empty, first quadrant. These observations demonstrate a dominant 
role for the extent of the longitudinal hydrophobic strip in regulating 
the termination of helices, and lead to a refined method to predict 
termini of helices in proteins. The sharp restriction of leucine, 
isoleucine, valine and phenylalanine to one longitudinal quadrant also 
demonstrated that the longitudinal strip is quite narrow on the average 
and that hydrophobic residues in other quadrants may compete for helix 
formation. 
The distributions of individual amino acids in each quadrant were 
determined for N- and C-termini, for the interior, and for the entire 
helix. In accord with Presta and Rose (1988, Science, 240:1632-1641), the 
left terminus was defined as the first four amino acids of a helix, and 
the right terminus, the last four amino acids of a helix. The amino acids 
between the termini constituted the interior of a helix. The template 
placed the amino acids in 4 quadrants of the sheet projection with 3, 5, 
5, and 5 symbols in quadrants I, II, III, and IV of the projection, 
respectively, so that the null hypothesis distribution assigned a 
probability of 3/18 for an amino acid being in quadrant I and of 5/18 for 
being in quadrant II, III, or IV. Since each terminus had four amino 
acids, only segments with 9 or more amino acids had non-empty interiors. 
Segments with 3 or fewer amino acids were excluded from analysis. For 
segments with 4 to 7 amino acids, the N- and C-termini overlapped. The 
frequency of each amino acid was determined in each quadrant in N- and 
C-termini, interiors and the entire segment. Table I displays for each 
amino acid the standardized deviations (the observed proportion minus the 
expected proportion divided by the standard error) over the four 
quadrants. Results were separated into 3 levels of significance (p=0.05, 
0.01, and 0.001). 
TABLE I 
______________________________________ 
N-TERMINUS 
p = 0.001 
Asp Leu Ile 
______________________________________ 
I +3.5 -2.1 -2.2 
II +0.3 -3.2 -0.4 
III -3.2 +6.2 +4.9 
IV +0.1 -1.2 -2.7 
p = 0.01 Val 
______________________________________ 
I +0.3 
II -2.3 
III +3.2 
IV -1.1 
p = 0.05 Gln Glu Lys Phe 
______________________________________ 
I -1.0 +0.1 -0.2 -1.4 
II +1.9 +2.1 +0.2 -2.0 
III -2.3 -2.9 -2.8 +2.3 
IV +1.3 +0.7 +2.7 +0.9 
INTERIOR 
p = 0.001 
Asp Ile 
______________________________________ 
I +3.4 -1.2 
II +1.7 -2.2 
III -2.8 +4.4 
IV -1.8 -0.9 
p = 0.01 Thr Phe 
______________________________________ 
I +1.9 -1.6 
II +2.3 -0.7 
III -1.5 +3.5 
IV -2.7 -1.6 
p = 0.05 Asn Gln Lys Arg Leu Val 
______________________________________ 
I +0.9 +2.6 +1.5 -0.5 -2.5 -2.2 
II -0.9 -0.5 +1.1 +0.2 -0.1 -0.8 
III -2.4 -2.3 -2.8 -2.6 +2.7 +2.4 
IV +2.5 +0.7 +0.6 +2.9 +0.5 -0.2 
C-TERMINUS 
p = 0.001 
Leu Val Ile 
______________________________________ 
I -2.7 -1.9 -2.0 
II -2.2 -2.2 -1.3 
III +5.6 +5.2 +4.2 
IV -1.1 -1.6 -1.3 
p = 0.01 Asn Asp Lys Phe 
______________________________________ 
I +2.9 +3.0 +1.3 -1.2 
II +0.9 +0.4 +0.6 -1.5 
III -2.2 -2.7 -3.4 +3.9 
IV -1.0 -0.1 +1.7 -1.5 
p = 0.05 Glu Arg 
______________________________________ 
I +1.0 +2.3 
II +1.2 -0.2 
III -2.8 -2.2 
IV +0.7 +0.4 
______________________________________ 
TABLE II 
______________________________________ 
ENTIRE HELIX 
______________________________________ 
p = 0.001 
Asp Glu Lys Arg Leu Val 
______________________________________ 
I +5.5 +0.7 +1.4 +0.4 -3.9 -2.2 
II +1.2 +1.9 +1.1 +0.6 -3.4 -2.9 
III -4.8 -4.4 -5.1 -3.8 +7.7 +6.1 
IV -1.0 +1.9 +2.9 -2.8 -1.1 -1.4 
p = 0.001 
Ile Phe 
______________________________________ 
I -3.1 -2.4 
II -2.3 -2.2 
III +7.8 +5.9 
IV -2.8 -1.6 
p = 0.01 Asn Gln 
______________________________________ 
I +2.5 +1.2 
II +0.5 +1.5 
III -3.7 -4.0 
IV +1.1 +1.5 
p = 0.05 Thr Tyr 
______________________________________ 
I +1.9 +0.1 
II +1.4 +1.4 
III -2.9 -3.2 
IV -0.1 +1.7 
______________________________________ 
In Tables I and II, the observed set of frequencies for the four quadrants 
were compared to the null distribution by means of the Chi-square 
goodness-of-fit test on 3 degrees of freedom and the results were verified 
with the likelihood ratio test. The null probability distribution assigned 
3/18 to quadrant I and 5/18 to quadrants II, III, and IV. To indicate 
possible false rejection of the null hypothesis, results were 
distinguished with p-values of 0.05, 0.01, and 0.001. With p=0.001 and 50 
independent tests, a type I error occurs 5% of the time using a Bonferroni 
correction for multiple comparisons. For each quadrant within a 
statistically significant distribution of frequencies, the deviation from 
the expected proportion, p, was standardized using the quantity (observed 
frequency-p)/SE where SE was the standard error under the binomial model, 
p(1-p)/n!.sup.0.5 and n was the number of times the amino acid occurred. 
The standardization was used because the difference in proportions can be 
misleading. For example, if one amino acid appeared in quadrant I twenty 
out of forty times while a rarer amino acid appeared in quadrant I two out 
of four times, both would have observed deviations of 50-17=33%. 
EXAMPLE 2 
Comparison of the Longitudinal, Hydrophobic Strip Identified in Primary 
Sequences of Helices Against Known Helix Structures in Crystallized 
Proteins 
Predicted quadrant orientations approximated crystallographic structures 
well. Projections of the crystallographic co-ordinates of the 
.alpha.-carbon chain tracings were viewed along the helical axis, using 
the Quanta program of Polygen Corporation on a Silcon Graphics 4D/70GT 
computer system. From the geometric center of the helical projection, 
radii were drawn to quadrant III residues (.quadrature.). The maximal 
sector angle was the absolute value of the greatest angle among those 
radii. The mean sector angle was the average of the absolute values for 
the angles between the most clockwise radius and the other radii. The 
structural relevance of the template-fitting model of alpha helices was 
tested by direct examination of all 4- or 5-turn helices in 7CAT, 5CPA, 
2CYP, 4LDH, 2MBN, 1MBO, and 2SNS for alignment of the residues predicted 
to fall in the axial hydrophobic strip of quadrant III (Torgerson et al., 
1991, J. Biol. Chem. 266:5521-5524). Projections of x-ray crystallographic 
coordinates for 22 of 28 helices demonstrated a maximal sector angle among 
residues in the axial hydrophobic strip of 99.degree. and a mean sector 
angle of 61.degree. (Table III). Table III shows sectors of residues in 4- 
or 5-turn axial hydrophobic strips. The assignments of amino acid residues 
to four quadrants, based on positioning of recurrent hydrophobic residues 
in one axial strip to maximize the SOHHI, closely matched crystallographic 
measurements. 
TABLE III 
______________________________________ 
Maximum Sector 
Mean Sector 
______________________________________ 
Presta and Rose Helices 
5CPA 15-28 66 53 
5CPA 74-89 77 43 
5CPA 216-230 77 57 
1MBO 4-17 79 63 
1MBO 21-35 70 48 
1MBO 59-76 90 56 
1MBO 83-95 67 52 
1MBO 101-118 60 38 
Richardson and Richardson 
Helices 
7CAT 53-67 124 101 
7CAT 258-271 65 42 
7CAT 437-450 149 120 
7CAT 470-485 99 62 
7CAT 485-500 59 55 
5CPA 14-30 94 62 
5CPA 173-187 78 53 
2CYP 42-55 63 51 
2CYP 103-120 180 94 
2CYP 164-177 75 69 
4LDH 30-44 119 74 
4LDH 55-70 73 61 
4LDH 141-154 94 53 
4LDH 165-181 69 48 
4LDH 247-264 72 56 
2MBN 20-37 120 65 
2MBN 82-98 137 95 
2MBN 100-116 65 42 
2SNS 54-69 52 31 
2SNS 121-136 72 60 
______________________________________ 
EXAMPLE 3 
Termination of Helices with Three Nonhydrophobic Residues at the N-Terminus 
and Two Nonhydrophobic Residues at the C-Terminus 
The quadrants for the terminations of helices are presented in Table IV. A 
helix was more likely to start in quadrant IV with 3 residues preceding 
the hydrophobic one in quadrant III. The N-terminus was more likely to be 
an untethered loop. The last amino acid was more likely to be in quadrant 
I, two amino acids after the hydrophobic axial strip and was less likely 
to end in the hydrophobic strip. The standardized deviations of the 
frequencies of first and last residues of the helices are presented for 
each axial quadrant of their appearances, p.ltoreq.0.001. 
TABLE IV 
______________________________________ 
TERMINI OF HELICES 
Richardson and 
Presta and Rose 
Richardson 
______________________________________ 
Beginnings 
I -3.5 -6.6 
II -3.2 -3.6 
III -0.9 +1.7 
IV +7.0 +7.4 
Endings 
I +7.5 +11.4 
II +1.5 +2.8 
III -2.4 -4.4 
IV -5.3 -8.0 
______________________________________ 
EXAMPLE 4 
Identification of Unique Structure of Helix Termini 
An .alpha.-helix terminates when the virtual extension of its most 
hydrophobic, longitudinal strip containing leucine, isoleucine, valine, 
phenylalanine, and methionine lacks those residues. The hydrophobic 
strip-of-helix template was applied to each of 247 helices and the 
template was extended into sequences beyond the ends of the helices. 
Leucine, isoleucine, valine, phenylalanine, and methionine occurred in 
.quadrature. positions in the longitudinal strip-of-helix at an increased 
frequency (p&lt;0.001), but in the first and second .quadrature. positions 
beyond either end of each true helix, they occurred at the same frequency 
as for their empirical distribution over all the proteins. Helices 
terminate when the longitudinal hydrophobic strip is not extended. 
Frequencies of amino acids were determined in .quadrature. positions and in 
intervening loops (.largecircle., .circle-solid., and .increment. 
positions between the .quadrature.) for 247 helices and their virtual 
extensions in 55 crystallized proteins (Table V). Table 5 shows the 
distributions of amino acids in the longitudinal hydrophobic strip 
.quadrature. and in intervening loops (.largecircle., .circle-solid., and 
.increment.) in 252 .alpha.-helices (real) and in parahelical segments 
extended in virtual helical configurations (virtual). The virtual 
extensions were the template assignments in the parahelical regions 
extending the pattern assigned to the helix itself. The first and second 
virtual .quadrature. positions would have occurred in the longitudinal 
hydrophobic strip were the helix extended. In the N- and C-terminal 
.quadrature. positions of true helices, the frequencies of leucine, 
isoleucine, valine, phenylalanine, or methionine substantially exceeded 
those predicted for an empirical distribution of amino acids for all the 
proteins (p&lt;0.001). Charged amino acids were suppressed in the terminal 
.quadrature. positions of the true helices. The frequencies of leucine, 
isoleucine, valine, phenylalanine, or methionine in the first and second 
.quadrature. positions in each virtual extension of the helix fell to the 
level predicted from the null hypothesis, i.e., that the empirical 
distribution over the entire set of proteins determined the frequencies. 
The placement of leucine, isoleucine, valine, or phenylalanine in 
recurrent positions in the primary sequence determines the formation of an 
.alpha.-helix with a longitudinal strip, when the longitudinal hydrophobic 
strip can stabilize helical nucleation against a hydrophobic surface 
(Torgerson et al., 1991, J. Biol. Chem., 266:5521-5524). This finding 
demonstrates that the absence of a hydrophobic residue in the extension of 
that strip fails to anchor the next successive loop and thus dictates 
termination of the helix. In Table V, the distributions of amino acids in 
the first proximal virtual loop and second proximal virtual strip in both 
N- and C-terminal parahelical segments were uniformly at the frequency of 
the uniform distribution of amino acids in proteins and were not reported 
in the table. 
TABLE V 
______________________________________ 
N-TERMINUS C-TERMINUS 
VIRTUAL REAL REAL VIRTUAL 
STRIP LOOP STRIP STRIP LOOP STRIP 
______________________________________ 
L -- -2.1 +5.0 +9.6 -2.6 -- 
I -- -2.4 +4.2 +5.5 -3.0 -- 
V -- -3.0 +4.1 +5.6 -4.1 -- 
F -- -3.1 +2.5 +4.1 -2.8 -2.8 
M -- -2.2 +2.2 +3.1 -- -- 
D -- +4.9 -- -3.3 -- -- 
E -- +4.1 -- -3.3 -- -- 
H -- -- -- +2.0 +2.0 -- 
R -- -- -2.9 -2.6 -- +2.9 
K -- -- -2.9 -3.2 +4.5 +2.4 
A -- -- -- -- -- -- 
C -- -- -- -- -- -- 
G -- -- -- -- -- -- 
N -- +2.1 -- -3.1 -- -- 
P -- -- -- -3.2 -- -- 
Q -- -- -- -2.8 +2.1 -- 
S -- -- -- -2.1 -- -- 
T -- -2.2 -- -2.5 -- -- 
W -- -- -- -- -- -- 
Y -- -- -- -- -- -- 
______________________________________ 
EXAMPLE 5 
Definition of Helix-Stabilizing Residues and Their Unique Positions with 
Respect to the Hydrophobic Strip-of-Helix Template 
The excess frequency of several amino acids differed significantly at N- 
versus C-termini of .alpha.-helices (Table V). Aspartate and glutamate 
were increased in frequency in the N-terminal loop of the real helix. 
Lysine and histidine were increased in the C-terminal loop of the real 
helix, and arginine and histidine were increased in the C-terminal first 
virtual .quadrature. position. The distributions of these amino acids 
otherwise approximated the empirical distribution model for the remainder 
of the helix, except in .quadrature. positions where they were excluded. 
The negative residues aspartate and glutamate stabilize the helix 
N-terminal macrodipole .delta.+ charge in the native protein (Hol et al., 
1981, Nature 294:532-536). The positive residues histidine, lysine, and 
arginine stabilize the helix C-terminal macrodipole .delta.- charge. 
Asparagine was also more frequent in the N-terminal loop, forming a 
hydrogen bond to the peptidyl backbone to initiate the helix. 
EXAMPLE 6 
Structure of Residues Within the Longitudinal Hydrophobic Strip-of-Helix 
The hydrophobic strip is narrow. While most leucine, isoleucine, valine, 
phenylalanine, and methionine residues in helices fell in the longitudinal 
hydrophobic strip with the greatest strip-of-helix hydrophobicity index 
(Torgerson et al., 1991, J. Biol. Chem., 266:5521-5524), the distribution 
of these hydrophobic residues in the remainder of the helix was examined. 
The residues in the hydrophobic strip do not fall precisely in a straight 
line. Some of the positions in quadrants II and IV, the "strip 
interdigitating positions", fall closely along quadrant III. The 
distribution of leucine, isoleucine, valine, phenylalanine, and methionine 
was examined in these strip bordering positions of quadrants II and IV and 
in the remainder of quadrants I, II and IV (Table VI). Leucine, 
isoleucine, valine, phenylalanine, and methionine were restricted to 
.quadrature. and excluded from .circle-solid. as well as from 
.largecircle. and .increment.; there was no tendency for broadening of the 
longitudinal hydrophobic strip. Aspartate, glutamate, and lysine were 
found in .largecircle. and .increment. positions while histidine and 
arginine occurred in .circle-solid.. Asparagine was more often in 
.circle-solid.; proline, glutamine and threonine, in .increment.; and 
tyrosine in .circle-solid. but not .largecircle.. 
TABLE VI 
______________________________________ 
Longitudinal distributions of amino acids in .alpha.-helices. 
.quadrature. 
.circle-solid. 
.largecircle. 
.DELTA. 
______________________________________ 
L +12.9 -4.1 -4.4 -5.5 
I +10.0 -4.0 -2.8 -3.9 
V +9.7 -3.4 -3.2 -3.9 
F +8.2 -- -3.6 -3.6 
M +1.7 -- -- -- 
D -5.3 -- +4.1 -- 
E -6.1 -- +2.3 +3.0 
H -- +3.0 -- -- 
K -7.1 -- +2.6 +4.4 
R -5.1 +3.3 -- -- 
A -- -- -- -- 
C +2.2 -- -- -- 
G -2.4 -- -- -- 
N -5.1 -- +3.1 -- 
P -3.0 -- -- +2.3 
Q -4.8 -- -- +2.2 
S -2.5 -- -- -- 
T -2.8 -- -- +3.5 
W -- -- -- -- 
Y -- +4.7 -- -- 
______________________________________ 
A "hydrophobicity break" in the hydrophobic strip-of-helix was defined as 
the single amino acid in the strip with minimal hydrophobicity. The 
distributions of amino acids in that position, and in the adjacent (n-2, 
n-1, n+1, n+2) positions along the longitudinal hydrophobic strip were 
scored (Table VII). Histidine and tryptophan were frequently present in 
the "break" and both positions in the longitudinal hydrophobic strip 
adjacent to the "break" held increased frequencies of leucine, isoleucine, 
valine, and phenylalanine (each, p&lt;0.001) and methionine (p&lt;0.01). This 
demonstrates that longitudinal hydrophobic strip-of-helix sequences can be 
interrupted by a single nonhydrophobic residue. However, two successive 
non-(leucine, isoleucine, valine, phenylalanine, or methionine) residues 
in the strip-of-helix terminate the helix. This observation may help to 
locate helix termini by searching for a motif of hydrophobic residues 
occurring in terminal .quadrature. but not in the two, next virtual 
.quadrature. positions in the extension of the longitudinal hydrophobic 
strip. 
TABLE VII 
______________________________________ 
Distributions amino acids in .quadrature. positions of a longitu- 
dinal hydrophobic strip centered on the least hydrophobic residue. 
-2 -1 BREAK +1 +2 
______________________________________ 
L +4.8 +4.8 0 +7.0 +6.4 
I +4.8 +5.2 0 +6.7 +3.9 
V +3.7 +3.9 0 +5.1 +4.5 
F -- +2.9 0 +2.1 +5.9 
M -- +2.2 0 -- -- 
D -- -2.2 -- -2.7 -2.3 
E -- -2.5 -- -2.3 -2.6 
H -- -- +3.9 -- -- 
K -2.1 +2.7 -- -3.3 -2.9 
R -- -- -- -2.2 -- 
A -- -- -- -- -- 
C -- +2.1 -- +2.5 -- 
G -- -- -- -- -- 
N -- -- -2.4 -2.2 -- 
P -- -- -- -- -- 
Q -- -- -- -2.2 -- 
S -- -- -- -2.6 -- 
T -- -- -- -- -2.1 
W -- -- +2.2 -- -- 
Y -- -- -- -- -- 
______________________________________ 
*The longitudinal hydrophobic strips of 247 helices were aligned on the 
least hydrophobic residue other than leucine, isoleucine, valine, 
phenylalanine or methionine. Within the longitudinal hydrophobic strip, 
amino acids in the positions adjacent to the least hydrophobic residue 
were usually Leu, Ile, Val, Phe or Met. 
EXAMPLE 7 
Identification of Structure of Crossing Alpha-Helices and Method to 
Stabilize or Alter the Structure of Such Crossing Regions 
The orientation of crossing .alpha.-helices was tested with respect to 
their respective longitudinal strips. In crossing regions of helices, the 
circumferential breadth of the hydrophobic strip and the relative 
frequencies of both small and charged amino acids of that strip in 
crossing regions between helices were analyzed. The local organization of 
hydrophobicity or other consensus structures on a longitudinal surface of 
a helix may determine the crossing points of packed helices. For example, 
small residues in the hydrophobic strip may determine the positions of 
preferred crossing regions (as do notches in a log), and the 
hydrophilicity of charged residues may warrant exclusion from these 
hydrophobic crossing regions. 
For the analysis of crossing helices, fifteen proteins were studied with 
the Quanta program of Polygen Corp. on a Silicon Graphics 4D/70 GT 
computer system. C.alpha. backbone projections were displayed with 
coordinates and helix lengths from the Brookhaven Protein Data Bank 
(protein codes: 1ECA, 1LYZ, 1MBO, 1RHD, 1SBT, 2ACT, 2LDX, 2LZM, 2SNS, 
3C2C, 3PGK, 3TLN, 4TNC, 5CPA, 7CAT). The analysis of 13 pairs of packed 
helices previously studied by Lesk et al. (1980, J. Mol. Biol., 
136:225-270) showed that the minimal distances between C.alpha.'s of two 
crossing helices were less than 7.5 .ANG.. For all residues in crossing 
helices which were no more than 7.5 .ANG. apart, we measured distances 
from each residue's C.alpha. to the C.alpha.'s of neighboring helices. 
Such measurements were characterized with respect to residues being in 
.quadrature., .circle-solid., .largecircle., .increment., and 
.circle-solid. positions of the strip-of-helix template assignment (Reyes 
et al., 1989, J. Biol. Chem., 264:12854-12858; Torgerson et al., 1991, J. 
Biol. Chem., 266:5521-5524). 
A crossing region was defined. Two .alpha.-helices were defined as crossing 
if there was at least one pair of C.alpha.s whose interhelical distance 
was less than 7.5 .ANG.. Such a measurement was called the Limiting 
Interhelical Alpha-carbon Distance (LIAD). In parallel to the method of 
Chothia et al. (Chothia et al., J. Mol. Biol. 145:215, 1981) to measure 
multiple residue interactions at the closest point of approach between the 
two helices, the three shortest LIADs were determined without restricting 
their lengths as long as the shortest was less than 7.5 .ANG.. The pair of 
amino acids determining a LIAD was called the LIAD pair. Individual amino 
acids in a helix could occur in more than one LIAD pair. The crossing 
region was defined to include those amino acids on one helix in the 
shortest three LIAD pairs. The magnitude of the LIAD is not correlated 
with the residue volumes of the amino acids forming the shortest LIAD 
(correlation r=0.44). Single-loop helices or helices with LIAD pairs 
involving only the terminal two residues were excluded because they 
usually only approached but did not cross another helix. 86 helices in 
fifteen proteins had 74 pairs of adjacent .alpha.-helices with crossing 
regions. 
The rotational orientation between .alpha.-helices was determined with 
respect to crossing longitudinal hydrophobic strips. Amino acids in 
crossing regions frequently also lay in longitudinal hydrophobic strips 
(z=+5.1; p&lt;0.001). The frequency with which longitudinal hydrophobic 
strips in neighboring helices intersected each other, i.e., the helices 
crossed through their longitudinal hydrophobic strips (Table VIII), was 
determined. Crossing between hydrophobic strips occurred when at least one 
LIAD, termed the strip-to-strip LIAD, had both amino acids of the LIAD 
pair in hydrophobic strips of their respective helices (Table V). The 
longitudinal hydrophobic strips-of-helix frequently face each other, 
Several amino acids were significantly included in or restricted from 
crossing regions (Table VIII). Hydrophobic amino acids leucine, 
isoleucine, valine, phenylalanine, and methionine as a group occurred more 
frequently in crossing regions (z=+6.4; p&lt;0.001). In contrast, both 
negatively charged amino acids aspartate and glutamate (z=-4.2) and 
positively charged amino acids histidine, lysine, and arginine (z=-4.4) 
were excluded from crossing regions (each set, p&lt;0.001). The individual 
amino acids most often included in crossing regions were valine and 
isoleucine (each, p&lt;0.001); Ala was significant as well (p&lt;0.01). For the 
charged amino acids, lysine and aspartate were the most highly excluded 
(each, p&lt;0.001). Excepting histidine, threonine, and cysteine, our 
distributions did not differ significantly from those of Chothia et al. 
(1981; J. Mol. Biol., 145:215-250). 
The distributions of charged amino acids in the longitudinal hydrophobic 
strip was determined with respect to occurrence in crossing regions. 92% 
of aspartate, glutamate, histidine, lysine, and arginine in such strips 
did not fall in a crossing region (X.sup.2 for equal proportions=6.8; 
p&lt;0.01). The smallest amino acid was also determined the "size break", in 
each longitudinal hydrophobic strip (Table IX). Chothia et al. (1981, J. 
Mol. Biol., 145:215-220) reported generally that of smaller amino acids 
were packed along larger ones at helical crossings. In this analysis, the 
smallest residue in the strip fell in the crossing region 46% of the time, 
and 59% of the crossing regions with a strip-to-strip LIAD pair contained 
at least one "size break". Overall, 49% of the smallest amino acids in all 
longitudinal hydrophobic strips fell in crossing regions (X.sup.2 =4.8; 
p&lt;0.05). Within crossing regions, no broadening of hydrophobicity around 
the longitudinal hydrophobic strip occurred. The frequencies of 
hydrophobic residues and crossing regions involving strip interdigitating 
.circle-solid. positions were tested. There was no increase in the 
frequency of leucine, isoleucine, valine, phenylalanine, or methionine in 
.circle-solid. positions which occurred in crossing regions versus 
positions not in crossing regions (X.sup.2 =0.5; p&gt;0.05). 
TABLE VIII 
______________________________________ 
Distributions of amino acids in crossing regions of .alpha.-helices. 
# observed 
% observed 
% expected 
Z (here) 
Z (Chothia) 
______________________________________ 
L 53 11.7 9.6 -- -- 
I 47 10.4 4.8 +5.7 +4.0 
V 54 11.9 6.6 +4.6 +4.0 
P 21 4.6 4.8 -- -- 
M 14 3.1 2.5 -- -- 
D 10 2.2 6.2 -3.5 -2.2 
B 22 4.9 7.6 -2.2 -3.3 
H 4 0.9 2.7 -2.4 +2.1 
R 16 3.5 3.6 -- -- 
K 13 2.9 8.2 -4.2 -2.6 
A 71 15.7 11.5 +2.9 -- 
C 7 1.5 2.1 -- -5.1 
G 31 6.9 4.9 -- -- 
N 11 2.4 3.9 -- -- 
P 10 2.2 2.3 -- -- 
Q 11 2.4 3.7 -- -- 
S 27 6.0 5.7 -- -- 
T 10 2.2 4.9 -2.6 -- 
W 6 1.3 1.3 -- -- 
Y 14 3.1 2.9 -- -- 
______________________________________ 
Distributions of amino acids were calculated in crossing regions defined by 
the shortest three LIADs between helices when at least one LIAD was less 
than 7.5 .ANG.. 452 residues in 74 crossing regions were counted, 
including a fourth LIAD for four crossing regions which had two pairs of 
amino acids tying for third closest LIAD. The expected percentages were 
calculated from the distribution of 3661 amino acids in 257 
.alpha.-helices. Z scores were also computed from the data of Chothia et 
al. (1981, J. Mol. Biol., 145:215-250). For groups of amino acids Z scores 
were: leucine, isoleucine, valine, phenylalanine, and methionine, +6.3; 
arginine, histidine, and lysine, -4.4; aspartate and glutamate, -4.2. 
These observations demonstrate that the smallest residue, especially 
alanine or valine in a longitudinal, hydrophobic strip-of-helix is 
predictably at the crossing region between adjacent helices and that 
charged residues in the hydrophobic strip-of-helix are not in the crossing 
region. 
TABLE IX 
______________________________________ 
Distribution of amino acids in .quadrature. positions at and adjacent to 
size 
breaks within the longitudinal hydrophobic strips of crossing helices. 
-1 Break +1 
______________________________________ 
L +4.2 -- +2.9 
I +4.9 -- +5.8 
V +4.2 +4.5 +3.4 
F -- -2.1 +3.4 
M +3.3 -- -- 
D -- -- -2.1 
E -2.1 -- -2.3 
H -- -- -- 
R -- -2.8 -2.1 
K -- -- -- 
A -2.2 +6.5 -- 
C -- -- -- 
G -- -- -- 
N -- -- -- 
P -- -- -- 
Q -- -- -- 
S -- -- -- 
T -- -- -- 
W -- -- -- 
Y -- -- -- 
______________________________________ 
The 86 longitudinal hydrophobic strips were aligned on the residue with the 
smallest volume on the scale of Chothia (1975, Nature, 254:304-308). When 
identical amino acids were smallest, the selected amino acid was the one 
with the largest neighbors. The two most frequently smallest amino acids 
were alanine and valine (each p&lt;0.001). Within the longitudinal 
hydrophobic strip the positions next to the size break were usually 
leucine, isoleucine, valine, phenylalanine and methionine. 
EXAMPLE 8 
Confirmation of the Mechanism for Helix Stabilization 
To test further the idea that a narrow, longitudinal strip-of-helix 
promotes coiling of helices on hydrophobic surfaces, a series of known T 
cell-presented peptides were synthesized and their coiling on lipid 
vesicles was analyzed by circular dichroism (Lu et al. 1990, J. Immunol. 
145:899-904). There was a modest correlation between SOHHI and the degree 
of helicity (r=0.77; p=0.07). Similar studies on coiling of T 
cell-presented peptides in trifluoroethanol or sodium dodecyl sulfate have 
been reported by others, coming to the same conclusion but without a 
systematic analysis of the role of the hydrophobic strip-of-helix in such 
binding (Vita et al. 1990, Molec. Immunol. 27:291-295; Lark et al. 1989, 
Peptide Res. 2:314-321). In order to evaluate specifically the effect of 
number and placement of aliphatic, hydrophobic residues of the 
longitudinal hydrophobic strip on helical coiling, prototypic helix 
peptide PH-1.0 
(leucine-tyrosine-glutamate-leucine-glutamine-lysine-leucine-threonine-glu 
tamine-threonine-leucine-lysine) SEQ ID NO: 1) and a series of its analogs 
replacing one or two leucine residues with threonine were synthesized (Lu 
et al. 1991, J. Biol Chem. 266:10054-10057). While all of these analogs 
coiled as helices from 28 to 57% in the presence of trifluoroethanol, 
appreciable helicity in the presence of lipid vesicles was found only for 
analogs with three adjacent leucines in the longitudinal hydrophobic strip 
and for one analog with two adjacent leucines on cycles also joined by the 
salt bridge glutamate.sup.4 -lysine.sup.7. These experiments demonstrate 
that adsorption and helical coiling on lipid membranes depends upon a 
cooperative effect of hydrophobic residues forming a longitudinal 
hydrophobic strip in terms of the number and placement of hydrophobic 
residues. 
This general method of analysis, based first on a consensus pattern of 
hydrophobicity, may also be applied to the study of .beta.-sheets and 
turns. The regulating principle may be the generation of a local structure 
with a hydrophobic surface containing multiple hydrophobic residues which 
are adjacent in the folded structure but separated in the primary sequence 
in patterns which may form characteristic motifs. The strength of the 
local, secondary structure may be reflected in the mean hydrophobicity of 
the residues in the motif of the hydrophobic surface. For example, 
hydrophobic residues in particular patterns around prolines may 
characterize some .beta.-turns and the alternating hydrophobicity of 
.beta.-strands can be identified. The principles of this study may also be 
used to predict docking of secondary structural elements to form tertiary 
structures in proteins. 
TABLE X 
______________________________________ 
Helicity of PH-1.0 and its Analogs* 
TFE Solution Lipid Vesicles 
Peptide .THETA..sub.222 mm 
% Helicity .THETA..sub.222 mm 
% Helicity 
______________________________________ 
PH-1.0 -16,543 57 -9,607 
33 
PH-1.1 -10,710 37 -5,531 
19 
PH-1.2 -10,630 37 -5,531 
PH-1.3 -8,633 30 -3,760 
PH-1.4 -8,305 29 -7,903 
27 
PH-1.5 -8,206 28 -5,123 
PH-1.6 -8,726 30 -2,702 
PH-1.7 -9,894 34 -4,046 
14 
PH1.10 -8,550 29 -5,796 
PH-4.2 -9,179 32 -4,053 
14 
______________________________________ 
In Table X, percentage helicity of peptides with CD spectra of 
.alpha.-helices was calculated from -.THETA..sub.222 nm values (units: deg 
cm.sup.2 dmol.sup.-1) and peptide concentration according to Taylor and 
Kaiser (1987, Meth. Enzymol., 154: 473) using maximal values of Bradley et 
al., 1990, J. Mol. Biol. 215: 607). For analog peptides, CD spectra were 
taken at 25.degree. C. with peptides in 0.01M phosphate buffer, pH 7.0, 
with 45% TFE or at 4.degree. C. in that buffer with lipid vesicles. 
EXAMPLE 9 
Prediction of Structural Helices by Application of the Hydrophobic 
Strip-of-Helix Template 
Distribution of amino acids were determined in positions in and around 
.alpha.-helices in positions defined with the hydrophobic strip-of-helix 
template. The distributions of amino acids over .quadrature. and 
.largecircle. positions of the hydrophobic strip-of-helix template were 
determined (Vasquez et al., 1992, J. Biol. Chem. 268: 7406) and Z values 
for distributions of groups of amino acids (leucine, isoleucine, valine, 
phenylalanine and methionine; aspartate and glutamate; arginine, histidine 
and lysine; asparagine, glutamine) were calculated (Table XI). The 
distributions were so significantly different from the uniform 
distribution of the residues across the proteins that it can be concluded 
that they reflected functions of the side chains in stabilizing the 
helices. Contributions to the prediction of .alpha.-helices of the finding 
of characteristic distributions of residues in and about the 
.alpha.-helices were tested. In Table XI, absolute values of Z greater 
than 3.6 correspond to p&lt;0.0001; greater than 3.1, p&lt;0.001; greater than 
2.3, p&lt;0.01; greater than 1.6, p&lt;0.05. Values not significant at p&lt;0.05 
are indicated by -. N-loop are the N-terminal residues in the 
.alpha.-helix preceding the first .quadrature. position (N-.quadrature.) 
in the helix. C-loop are the C-terminal residues in the .alpha.-helix 
following the last .quadrature. position (C-.quadrature.) in the helix. 
C-virt. .quadrature. is the first .quadrature. position following the 
helix determined by extension of the strip-of-helix template into the 
parahelical sequence. 
TABLE XI 
__________________________________________________________________________ 
Restricted Distribution of Amino Acids in and near alpha helices in 
positions 
defined with the hydrophobic strip-of-helix template 
Amino Acids N-Loop 
N-.quadrature. 
C-.quadrature. 
C-Loop 
C-virt .quadrature. 
__________________________________________________________________________ 
Leucine, Isoleucine, Valine, Phenylalanine, 
-7.2 
+8.2 
+13.4 
-8.2 
-2.1 
Methionine 
Aspartate, Glutamate 
+6.6 
-2.1 
-4.8 
-- -- 
Lysine, Arginine -- -4.2 
-4.2 
+4.2 
+3.7 
Histidine -- -- +2.0 
+2.0 
-- 
Asparagine +2.1 
-- -3.1 
-- -- 
Glutamine -- -- -2.8 
+2.1 
-- 
__________________________________________________________________________ 
The predictor algorithm was based upon templates 
.largecircle..largecircle..largecircle..quadrature..largecircle..largecirc 
le..largecircle..quadrature..largecircle..largecircle..quadrature..largecir 
cle..largecircle. and 
.largecircle..largecircle..largecircle..quadrature..largecircle..largecirc 
le..quadrature..largecircle..largecircle..largecircle..quadrature..largecir 
cle..largecircle. which included 3 turns through the hydrophobic strip in 
an .alpha.-helical pattern. They are segments of an 18-member template 
(.quadrature..largecircle..largecircle..largecircle..quadrature..largecirc 
le..largecircle..quadrature..largecircle..largecircle..largecircle..quadrat 
ure..largecircle..largecircle..quadrature..largecircle..largecircle..largec 
ircle.) required to return an .alpha.-helical pattern to 0.degree.. That 
is, if each successive residue adds 100.degree., i.e., 100.degree., 
200.degree., 300.degree., 40.degree., 140.degree., etc., it requires 19 
residues to return to 0.degree. exactly. 
.largecircle..largecircle..largecircle. was added to the N-terminus and 
.largecircle..largecircle. to the C-terminus because those additional 
positions were usually found in .alpha.-helices; appended to the 
respective terminal, .quadrature. is in the longitudinal strip-of-helix 
(p&lt;0.001) (Torgerson et al., 1991, J. Biol. Chem., 266:5521-5524). That 
is, helices did not end in .largecircle. positions in the longitudinal 
strip but instead with three .largecircle. positions at the N-terminus and 
two .largecircle. positions at the C-terminus, on the average. Template 
assignments scored positive when the mean hydrophobicity of residues in 
.quadrature. positions was greater than 3.0 on the Kyte-Doolittle scale. 
Practically, this usually required all three .quadrature. positions to 
come from the group of amino acids (with hydrophobicity indices): leucine 
(3.8), isoleucine (4.5), valine (4.2), phenylalanine (2.8), and methionine 
(1.9). The effect of lowering the hydrophobicity threshold for positive 
scoring of the template is seen in Table XII. Overlapping predictions were 
merged when the presence of amino acids in restricted distributions 
described in Table XI was scored. When a region of overlapping template 
predictions was found, the entire sequence was taken from a putative helix 
and evaluated with the method described in Example I. 
The sensitivity and efficiency of .alpha.-helix predictions were determined 
with only the hydrophobic strip-of-helix template and with addition of 
certain residue-position patterns (Table XIII) (Vasquez et al., 1992, J. 
Biol. Chem. 268: 7406). The sensitivity and efficiency of the predictor 
algorithm based on 
.largecircle..largecircle..largecircle..quadrature..largecircle..largecirc 
le..largecircle..quadrature..largecircle..largecircle..quadrature..largecir 
cle..largecircle. and 
.largecircle..largecircle..largecircle..quadrature..largecircle..largecirc 
le..quadrature..largecircle..largecircle..largecircle..quadrature..largecir 
cle..largecircle. templates was better than with a previous 4-turn template 
(.quadrature..largecircle..largecircle..largecircle..quadrature..largecirc 
le..largecircle..quadrature..largecircle..largecircle..largecircle..quadrat 
ure.) in which the pattern terminated with .quadrature. positions, and 
alternate placements of .largecircle..largecircle..largecircle. vs 
.largecircle..largecircle. loops within the template was not an option 
(Reyes et al., 1989, J. Biol. Chem., 264:13854-12858). The current method 
was also superior to the Chou-Fasman and Garnier-Osguthrope-Robson methods 
embodied in a DNA* program. Addition of the requirement for finding of 
aspartate or glutamate in N-terminal .largecircle. positions; or 
histidine, lysine, arginine at C-terminal .largecircle. or first 
parahelical .quadrature. positions had a marginal (not significant) 
improvement in efficiency and a loss in sensitivity. The loss in 
sensitivity (number helical identified/total number of helices) was 
expected since only a fraction of the helices had such 
macrodipole-stabilizing residues. This program scored the presence of 
helix-stabilizing residues in certain positions, and constituted the most 
sensitive and efficient method available to predict .alpha.-helices from 
primary sequence. It is slso an essential step toward the development of 
docking algorithms which will permit better predictions in the future 
based upon analysis of fittings of locally ordered structures identified 
with this invention. 
TABLE XII 
______________________________________ 
Sensitivity (Sens.) and Efficiency (Eff.) of Template Predictions 
Helices .gtoreq. 
Mean Hydrophobicity 
All Helices 8 Residues 
of .quadrature. positions, .ltoreq. 
Sens. Eff. Sens. 
Eff. 
______________________________________ 
3.5 0.26 0.37 0.30 0.37 
3.0 0.36 0.42 0.42 0.42 
2.5 0.29 0.32 0.34 0.32 
2.0 0.16 0.20 0.18 0.20 
1.5 0.06 0.11 0.07 0.11 
______________________________________ 
TABLE XIII 
______________________________________ 
Categorical Positions in and around alpha helices as 
defined with the hydrophobic strip-of-helix template 
Sensitivity 
Efficiency 
______________________________________ 
Template Only .36 .42 
Template and D, E at N-term. 
.15 .47 
Template and H, K, R at C-term. 
.22 .49 
Template and N at N-term. 
.05 .41 
Template and Q at C-term. 
.02 .15 
Template and D, E, at N-term. or H, K, R at 
.28 .49 
C-term. 
D, E, at N-term. and H, K, R at C-term. 
.09 .48 
E at N-term. or H, K, R at C-term. or N at 
.34 .49 
N-term. 
______________________________________ 
EXAMPLE 10 
Demonstration that Positions in the Strip-of-Helix are Sensitive to 
Function-Loss Mutation 
The sensitivity of bacteriophage T4 lysozyme function to amino acid 
substitutions at defined positions in and around the longitudinal, 
hydrophobic strips of 9 .alpha.-helices was assessed after systematic 
replacement of each residue in the protein with a series of 13 amino 
acids. The hydrophobic strips were defined by identifying the longitudinal 
sectors in the helices with the highest mean residue hydrophobicities. 
Sensitivity to mutation (the percentage of replacements leading to loss of 
function) was calculated for each residue in the following positions: 
whole protein, helices, hydrophobic strips, other positions within the 
helices, and various positions within the hydrophobic strips as well as 
their extensions beyond the helices. 
Application of the hydrophobic strip-of-helix-identifying algorithm to the 
9 .alpha.-helices of T4 lysozyme demonstrated sensitivities to amino acid 
replacements in certain positions identified with the strip-of-helix 
hydrophobicity template (Rennell et al., 1992, J. Biol. Chem., 267: 
17748-17752). Residues of the hydrophobic strip .quadrature. positions 
were generally sensitive; the C-terminal strip residues were particularly 
sensitive. The sensitivities to substitutions of groups of residues in T4 
lysozyme, including those occupying the structural positions described 
above, are compared in Table XIV. The protein as a whole scored 16; that 
is, 16% (328/2015) of substitutions tested were found to be deleterious. 
Buried residues, as a group, were more sensitive to substitutions. Loss of 
function resulted from 38% of substitutions for residues with side chains 
which have less than 12% of their surface areas accessible to solvent; 
this sensitivity increased to 42% if the group was restricted to those 
residues with completely inaccessible side chains. These observations 
establish a criterion for the performance of a scheme to pick out critical 
residues. 
This example demonstrates the minimal structural change to destroy function 
of a molecule by altering a residue in the longitudinal hydrophobic strip, 
most specificially at the C-terminus, from a leucine, isoleucine, valine, 
phenylalanine, or methionine to another residue not in that group. The 
altered molecule would have a primary sequence close to the wild type but 
no biological activity. Such changes may be applied to vaccine development 
against toxins from various sources as isolated proteins or in the context 
of site-specific mutation of a pathogen to destroy a virulence factor in 
an otherwise infectable particle. This is a new approach to the rational 
design of attenuated pathogens for vaccination purposes. 
TABLE XIV 
__________________________________________________________________________ 
Sensitivities of positions in T4 lysozyme to amino acid substitutions 
No. of 
Substitutions 
Residues Expected 
Group residues 
(n) Score 
represented 
score 
Difference 
P 
__________________________________________________________________________ 
All residues 
163 2015 16 all (16) 0 -- 
&gt;88% Buried 
50 621 38 ACDFGILMNRSTVWY 
20 18 &lt;.001 
100% Buried 
24 302 42 ACDFGILMNTVY 
19 23 &lt;.001 
Conserved 
14 172 47 ADEGHLTWY 22 25 &lt;.001 
.alpha.-Helical 
97 1200 16 all but H 15 1 -- 
Hydrophobic strip 
25 307 26 ACFIKLVW 19 7 &lt;.001 
N-terminal 
9 110 26 AIKLW 18 8 &lt;.05 
C-terminal 
9 111 44 FLIVW 24 20 &lt;.0001 
Smallest 9 111 31 AILVW 18 13 &lt;.001 
N-Shoulders 
3 36 14 KL 16 -2 -- 
C-Shoulders 
7 86 31 CFIL 20 11 &lt;.01 
Virtual N-terminal 
8 97 10 FGKLRST 15 -5 -- 
Virtual C-terminal 
9 112 31 EGILNRST 18 13 &lt;.001 
Non-strip 
72 880 13 all but C & H 
15 -2 &lt;.05 
__________________________________________________________________________ 
EXAMPLE 11 
Discovery of Favored and Suppressed Patterns of Hydrophobic and 
Nonhydrophobic Amino Acids in Protein Sequences 
Ultraearly events in protein folding may be influenced by short sequence 
motifs which collapse with the least ambiguity into the molten globule 
nucleations which initiate secondary structure (Kauzmann, W. (1959) Adv. 
Prot. Chem., 14, 1-63; Dill, K. A. (1989) Biochemistry, 24, 1501-1509; 
Kuwajima, K. (1989) Proteins, 6, 87-103; Dill, K. A., et al., (1993) Proc. 
Natl. Acad. Sci. USA, 90, 1942-1946). Since hydrophobic residues Leu, Ile, 
Val, Phe, and Met (LIVFM) are the largest subset of residues with 
chemically similar side chains (combined frequency 0.2525 in the proteins 
studied), they dominant such local motifs, as reflected in their 
distribution as not-too-many and not-too-few together. Too many adjacent 
hydrophobic amino acids in a short segment may lead to many folding 
pathways of comparable energy levels, leading to multiple, malfunctional 
final forms, and thus be selected against on an evolutionary basis. In 
contrast, too few hydrophobic residues without other strong, 
structure-determining motifs may lack the initial restriction on forms 
afforded by selective placement of hydrophobic residues and consequently 
also lead to multiple, final forms. An optimal frequency of hydrophobic 
(or conversely nonhydrophobic) runs would be created by these competing 
tendencies. To test this hypothesis that hydrophobic residues might not be 
distributed in a random fashion, certain 5-residue patterns were examined 
for preferential or suppressed occurance. A correlation between preferred 
patterns and associated secondary structures was then identified in mature 
proteins. The frequencies of runs of hydrophobic LIVFM residues and of 
nonLIVFM residues were determined in 48 proteins (8024 amino acids) (Table 
XV). Those frequencies were compared to the expected frequencies for the 
random placement of such residues in proteins. While the frequencies of 
single or two adjacent hydrophobic residues approximated the respective 
random expectations, the observed frequency of runs of three or more 
hydrophobic residues were less than expected (p&lt;0.01). The frequency of 
runs of nonhydrophobic residues was not suppressed to a similar degree in 
shorter runs; only at the level of 6 nonhydrophobic residues did z=-2.1 
(corresponding to p&lt;0.05). 
The counts of the number of occurrences of a template within a protein (or 
of a pattern within a template) were treated as binomial random variables 
with N possible positions and the proportion, P, equal to the empirical 
frequency of the pattern over all proteins under study. An empirical 
distribution is that subset of random distributions of residues when the 
frequency of each type of residue in the distribution is equal to the 
frequency in the overall population. When the term random distribution is 
used by others, they usually mean empirical distribution. The observed 
counts, X, had mean, NP, and variance, NP(1-P). The observed proportion, 
X/N, had variance, P(1-P)/N. With a value of P.sub.o under the null 
hypothesis, the z score was then (X/N-P.sub.o)/.sqroot.P(1-P)/N where z 
was approximately normally distributed. To avoid misinterpretations 
arising from multiple testings (type I statistical errors), statistical 
significance was restricted to one-tailed probabilities (p-values) of 
p&lt;0.01 for .vertline.z.vertline.=2.6 and p&lt;0.001 for 
.vertline.z.vertline.=3.3. 
Analyses were completed with 48 proteins with known crystallographic 
structure and the following Brookhaven file codes: 1BP2, 1CPV, 1CRN, 1CRO, 
1ECA, 1EDC, 1EMQ, 1GP1, 1INS,C, 1INS,D, 1LZ1, 1MBO, 1PPT, 1RHD, 1SBT, 
1SN3, 2ACT, 2APP, 2AZA, 2BSC, 2CAB, 2CYP, 2CCY, 2CDV, 2LHB, 2LZM, 2MBN, 
2MLT, 2OVO, 2PAB, 2SNS, 2SSI, 2STV, 3ADK, 3C2C, 3CYT, 3GRS, 3TLN, 351C, 
4DFR, 4FXN, 4LDH, 5CPA, 5RSA, 5PTI, 6ADH, 7CAT, 7LYZ. 
TABLE XV 
______________________________________ 
Divergence between observed and expected frequencies of run 
of hydrophobic and nonhydrophobic residues 
Hydrophobic Nonhydrophobic 
Nested Exclusive Nested Exclusive 
Ob- z Ob- z Ob- z Ob- z 
Runs served score served 
score 
served 
score 
served 
score 
______________________________________ 
1 2046 0.0 1098 -0.8 5998 0.0 337 -2.0 
2 513 0.2 335 3.4 4464 0.2 333 3.3 
3 98 -2.6 64 -0.6 3267 -1.0 224 1.1 
4 18 -2.5 14 -0.8 2403 -1.4 163 0.6 
5 2 -2.1 2 -1.1 1763 -1.7 128 1.2 
6 0 -1.4 0 -1.0 1286 -2.1 89 0.3 
7 937 -2.4 80 2.0 
8 677 -2.8 48 0.0 
9 497 -2.6 40 0.7 
10 365 -2.5 29 0.5 
11 273 -2.0 18 -0.4 
12* 210 -1.2 10 -1.2 
______________________________________ 
*All subsequent runs had a frequency of .ltoreq. 8. 
EXAMPLE 12 
Favored and Suppressed Patterns of Hydrophobic Residues 
Five-position templates (with all combinations of LIVFM and nonLIVFM 
residues) were scored across the 48 proteins (Table XVI). Z scores are 
presented for the patterns arranged in a binomial progression (Table XVIA) 
or to emphasize dependence on spacing (Table XVIB). Z scores for selected 
combinations of favored motifs and contrasting patterns, are presented in 
Table XVII. Certain combinations of 
.diamond..diamond-solid..diamond-solid..diamond..diamond. and 
.diamond-solid..diamond..diamond..diamond-solid..diamond. patterns are 
significantly preferred. 
TABLE XVIA 
______________________________________ 
Patterns of LIVFM .diamond-solid. and nonLIVFM .diamond. residues and z 
scores 
for differences between frequencies which were observed 
versus expected under an hypothesis for an empirical 
distribution of residues 
______________________________________ 
.diamond..diamond..diamond..diamond..diamond. 
1.7* .diamond..diamond-solid..diamond..diamond-solid..diamond-s 
olid. -0.1 .diamond-solid..diamond..diamond-solid..di 
amond-solid..diamond. 
2.2 
.diamond..diamond..diamond..diamond..diamond-solid. 
-0.1 .diamond..diamond-solid..diamond-solid..diamond..diamond. 
3.5 .diamond-solid..diamond..diamond-solid..di 
amond-solid..diamond-solid. 
-1.6 
.diamond..diamond..diamond..diamond-solid..diamond-solid. 
-0.4 .diamond..diamond-solid..diamond-solid..diamond..diamond-s 
olid. 0.3 .diamond-solid..diamond-solid..diamond..di 
amond..diamond. 
1.6 
.diamond..diamond..diamond..diamond-solid..diamond-solid. 
0.8 .diamond..diamond-solid..diamond-solid..diamond-solid..dia 
mond. -0.8 .diamond-solid..diamond-solid..diamond..di 
amond..diamond-solid. 
2.5 
.diamond..diamond..diamond-solid..diamond..diamond. 
2.1 .diamond..diamond-solid..diamond-solid..diamond-solid..dia 
mond-solid. 
-1.6 .diamond-solid..diamond-solid..diamond..di 
amond-solid..diamond. 
-1.6 
.diamond..diamond..diamond-solid..diamond..diamond-solid. 
1.7 .diamond-solid..diamond..diamond..diamond..diamond. 
0.2 .diamond-solid..diamond-solid..diamond..di 
amond-solid..diamond-solid. 
1.9 
.diamond..diamond..diamond-solid..diamond-solid..diamond. 
2.1 .diamond-solid..diamond..diamond..diamond..diamond-solid. 
0.5 .diamond-solid..diamond-solid..diamond-sol 
id..diamond..diamond. 
-1.0 
.diamond..diamond..diamond-solid..diamond-solid..diamond-solid. 
-0.8 .diamond-solid..diamond..diamond..diamond-solid..diamond. 
2.4 .diamond-solid..diamond-solid..diamond-sol 
id..diamond..diamond-solid. 
-1.2 
.diamond..diamond-solid..diamond..diamond..diamond. 
-0.6 .diamond-solid..diamond..diamond..diamond-solid..diamond-s 
olid. 1.4 .diamond-solid..diamond-solid..diamond-sol 
id..diamond-solid..diamond. 
-1.6 
.diamond..diamond-solid..diamond..diamond..diamond-solid. 
1.9 .diamond-solid..diamond..diamond-solid..diamond..diamond. 
-2.7 .diamond-solid..diamond-solid..diamond-sol 
id..diamond-solid..diamond-solid. 
-2.1 
.diamond..diamond-solid..diamond..diamond-solid..diamond. 
-3.4 .diamond-solid..diamond..diamond-solid..diamond..diamond-s 
olid. -3.1 
______________________________________ 
*At a p &lt; 0.01, one would expect to find 0.3 of the 32 patterns with an 
absolute value of z &gt; 2.6. 
TABLE XVIB 
______________________________________ 
Patterns of LIVFM (.diamond-solid.) and nonLIVFM (.diamond.) residues, 
with z scores, 
arranged to emphasize dependence on spacing 
.diamond-solid..diamond-solid..diamond..diamond..diamond. 
1.6 
.diamond-solid..diamond..diamond-solid..diamond..diamond. 
-2.7 
.diamond-solid..diamond..diamond..diamond-solid..diamond. 
2.4 
.diamond-solid..diamond..diamond..diamond..diamond-solid. 
0.5 
.diamond-solid..diamond-solid..diamond..diamond..diamond. 
1.6 
.diamond..diamond-solid..diamond-solid..diamond..diamond. 
3.5 
.diamond..diamond..diamond-solid..diamond-solid..diamond. 
2.1 
.diamond..diamond..diamond..diamond-solid..diamond-solid. 
0.8 
.diamond-solid..diamond..diamond-solid..diamond..diamond-solid. 
-3.1 
.diamond..diamond-solid..diamond..diamond-solid..diamond. 
-3.4 
______________________________________ 
TABLE XVII 
______________________________________ 
Patterns of LIVFM (.diamond-solid.) and nonLIVFM (.diamond.) residues 
arranged to 
emphasize combinations of some favored and suppressed patterns of 
Table XVIA. 
.diamond..diamond-solid..diamond-solid..diamond. 
3.2 
.diamond..diamond..diamond-solid..diamond-solid..diamond. 
2.1 
.diamond..diamond-solid..diamond-solid..diamond..diamond. 
3.5 
.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond. 
2.1 
.diamond-solid..diamond..diamond..diamond-solid..diamond-solid..diamon 
d..diamond. 3.1 
.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond..d 
iamond-solid. 3.4 
.diamond-solid..diamond..diamond..diamond-solid..diamond-solid..diamon 
d..diamond..diamond-solid. 
3.0 
.diamond..diamond-solid..diamond..diamond..diamond-solid..diamond-solid 
..diamond..diamond..diamond-solid. 
3.6 
.diamond-solid..diamond..diamond..diamond-solid..diamond-solid..diamon 
d..diamond..diamond-solid..diamond. 
3.2 
.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond..d 
iamond-solid..diamond. 
4.7 
.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond..d 
iamond-solid..diamond..diamond. 
5.1 
.diamond..diamond-solid..diamond-solid..diamond..diamond..diamond-s 
olid..diamond..diamond..diamond-solid. 
4.7 
.diamond..diamond-solid..diamond-solid..diamond..diamond..diamond-s 
olid..diamond..diamond..diamond-solid..diamond. 
4.1 
.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond..d 
iamond-solid..diamond..diamond..diamond-solid..diamond. 
5.0 
.diamond..diamond-solid..diamond..diamond..diamond-solid..diamond-solid 
..diamond..diamond..diamond-solid..diamond. 
3.7 
.diamond..diamond..diamond-solid..diamond..diamond..diamond-solid..diamo 
nd-solid..diamond..diamond..diamond-solid..diamond. 
4.5 
.diamond..diamond..diamond..diamond-solid..diamond..diamond..diamond-soli 
d..diamond-solid..diamond..diamond..diamond-solid..diamond. 
3.5 
.diamond..diamond..diamond..diamond..diamond-solid..diamond..diamond..diam 
ond-solid..diamond-solid..diamond..diamond..diamond-solid..diamond. 
3.9 
.diamond..diamond..diamond..diamond..diamond-solid..diamond..diamond..diam 
ond-solid..diamond-solid..diamond..diamond..diamond-solid..diamond..diamon 
d. 3.4 
.diamond-solid..diamond-solid..diamond..diamond-solid..diamond..di 
amond-solid..diamond..diamond-solid..diamond. 
-2.5 
.diamond..diamond-solid..diamond..diamond..diamond-solid..diamond. 
1.1 
.diamond..diamond-solid..diamond..diamond..diamond-solid..diamond..diam 
ond..diamond-solid..diamond. 
0.8 
.diamond..diamond-solid..diamond..diamond-solid..diamond..diamond..diam 
ond-solid..diamond. -0.5 
.diamond..diamond-solid..diamond-solid..diamond..diamond..diamond-sol 
id..diamond-solid..diamond. 
0.4 
.diamond..diamond-solid..diamond-solid..diamond..diamond-solid..diamond-so 
lid..diamond..diamond..diamond-solid..diamond-solid..diamond. 
-0.6 
______________________________________ 
EXAMPLE 13 
Distributions of Nonhydrophobic Residues within Preferred Motifs 
The distributions of all non Leu, Ile, Val, Phe, Met (nonLIVFM) residues in 
each .diamond. position of the templates were counted and z scores were 
calculated for the differences between observed frequencies and the 
frequencies of those residues in positions not occupied by LIVFM. Scores 
for .vertline.z.vertline.&gt;1.96 (p&lt;0.05) are given. Table XVIII reports 
distributions of amino acids around two sequential hydrophobic residues. 
The nonLIVFM position immediately preceding two hydrophobic residues 
showed an increased frequency of Arg and decreased frequencies of Asn and 
Tyr. The frequency of Glu was increased in the nonLIVFM position 2 
residues before the first hydrophobic position. A polarity was seen in 
distributions of nonhydrophobic residues about the two hydrophobic 
residues: negatively charged residues were favored before but not after 
the pair of hydrophobic amino acids. Such patterns of preferred pairings 
of amino acids among positions of oligopeptide sequences has been observed 
by others (Cserzo, M. et al., (1989) Int. J. Peptide Protein Res., 34, 
184-195; Klapper, M. H. (1977) Biochem. & Biophys. Res. Comm., 78, 
1018-1024). 
Table XIX shows the distributions of nonLIVFM residues about a single 
hydrophobic residue. Cys was suppressed in the position before the single 
LIVFM. Glu was increased in the second position preceding the hydrophobic 
residue, and Gly, Pro, and Tyr were suppressed in that position. Gly was 
suppressed in the position after the single hydrophobic residue and Lys 
was favored in the first position following the single hydrophobic 
residue. Table XX presents patterns with single .diamond-solid. or double 
.diamond-solid..diamond-solid. hydrophobic residues when such combinations 
were favored in the hydrophobic templates of Table XVI. In general, the 
patterns of amino acids in .diamond. positions of these combinations were 
what would be expected from combination of individual patterns. 
TABLE XVIII 
______________________________________ 
Distributions of nonLIVFM residues in .diamond. positions 
around .diamond-solid..diamond-solid. positions in certain templates 
.diamond. 
.diamond-solid. 
.diamond-solid. 
.diamond. 
N -2.1 
R 3.1 
Y -2.6 
--.sup.a 
2.8 
.diamond. .diamond. 
.diamond-solid. 
.diamond-solid. 
.diamond. 
E 3.1 N -2.2 
R 2.6 
Y -2.3 
.diamond. 
.diamond-solid. 
.diamond-solid. 
.diamond. 
.diamond. 
K 3.0 
N -2.2 
P -2.1 P -2.2 
R 2.2 
Y -2.2 
-- 3.4 
.diamond. .diamond. 
.diamond-solid. 
.diamond-solid. 
.diamond. 
.diamond. 
N -2.0 
P -2.1 
-- 2.1 
______________________________________ 
.sup.a -- = D or E 
TABLE XIX 
______________________________________ 
Distributions of nonLIVFM residues in .diamond. positions 
around .diamond-solid. positions in certain templates 
______________________________________ 
.diamond. 
.diamond-solid. 
.diamond. 
C -2.3 
.diamond. .diamond. 
.diamond-solid. 
.diamond. 
C -2.4 
E 2.1 
G -2.2 G -2.1 
P -2.2 
--.sup.a 2.3 
.diamond. 
.diamond-solid. 
.diamond. 
.diamond. 
C -2.4 E -2.4 
.diamond. .diamond. 
.diamond-solid. 
.diamond. 
.diamond. 
A 2.3 
C -2.4 
E 2.5 E -3.0 
G -2.1 G -2.7 
K 2.0 
P -2.6 
W 2.0 
Y -2.1 
---2.4 
______________________________________ 
.sup.a -- = D or E 
TABLE XX 
______________________________________ 
Distributions of nonLIVFM residues in .diamond. positions around 
.diamond-solid..diamond-solid. 
and .diamond-solid. positions in certain templates 
______________________________________ 
.diamond. 
.diamond. 
.diamond-solid. 
.diamond-solid. 
.diamond. 
.diamond. 
.diamond-solid. 
D 3.0 
E 2.4 
T 2.4 
--.sup.a 4.0 
.diamond. 
.diamond. 
.diamond-solid. 
.diamond-solid. 
.diamond. 
.diamond. 
.diamond-solid. 
.diamond. 
D 2.9 
E 2.2 
-- 3.8 
T 2.3 K 2.7 
Y 2.1 
.diamond-solid. 
.diamond. 
.diamond. 
.diamond-solid. 
.diamond-solid. 
.diamond. 
.diamond. 
.diamond-solid. 
.diamond. 
E 2.0 
G -2.7 
-- 2.7 
K -2.0 
R 2.6 
______________________________________ 
--.sup.a = D or E 
EXAMPLE 14 
Distributions of .phi. and .psi. at Each Position of the Preferred Pattern 
.diamond..diamond-solid..diamond-solid..diamond..diamond. 
In order to test whether a favored pattern of hydrophobic and 
nonhydrophobic residues associates with secondary structures, Ramachandran 
plots were made for .phi. versus .psi. angles at residues in each position 
of sequences fitting the 
.diamond..diamond-solid..diamond-solid..diamond..diamond. pattern. 
Percentages of residues with .phi. and .psi. angles falling within 
.alpha.-helix and .beta.-sheet regions of Ramachandran plots for positions 
1 through 5 of the template 
.diamond..diamond-solid..diamond-solid..diamond..diamond. are presented in 
Table XXI. For the subset of sequences with .phi., .psi. angles at 
position 2 within limits for .alpha.-helices, the percentages at each 
template position of residues with .phi., .psi. angles within limits for 
.alpha.-helices were: 1-89%, 2-100%, 3-87%, 4-76%, 5-66%. Comparably, for 
the subset of sequences with .phi., .psi. angles at position 2 within 
limits for .beta.-strands, the percentages at each template position of 
residues with .phi., .psi. angles within limits for .beta.-strand were: 
1-70%, 2-100%, 3-83%, 4-78%, 5-43%. That is, residues around position 2 
usually demonstrated the same .alpha.-helical or .beta.-strand 
conformation found at position 2. 
TABLE XXI 
______________________________________ 
Percentage of residues at each position in the pattern .diamond..diamond-s 
olid..diamond-solid..diamond..diamond. with 
C.sub..alpha. .phi., .psi. values within the indicated limits 
.alpha.-helix 
.beta.-sheet 
______________________________________ 
.phi. limits -65, -15 +90, +180 
.psi. limits -45, -100 
-45, -165 
Position: 
1 57 26 
2 57 29 
3 55 34 
4 48 31 
5 46 23 
______________________________________ 
EXAMPLE 15 
Association of the 
.diamond..diamond-solid..diamond-solid..diamond..diamond. Motif with 
.alpha.-Helical or .beta.-Strand Configurations 
The favored pattern 
.diamond..diamond-solid..diamond-solid..diamond..diamond. occurs in 
sequences which have .alpha.-helical, .beta.-strand, or other 
conformations (Table XXII). However, the association of that pattern is 
greater when the second position of the sequence has .phi., .psi. angles 
within .alpha.-helical limits (z=+3.6), than when the second position of 
the sequence has .beta.-strand conformation (z=-1.0). Recalculation of an 
expected frequency of LIVFM in the 
.diamond..diamond-solid..diamond-solid..diamond..diamond. to weight at 0.4 
(the frequency of LIVFM amino acids in the pattern) the fraction of 
residues with .phi., .psi. angles of .alpha.-helices, did not alter the 
significance of the association of 
.diamond..diamond-solid..diamond-solid..diamond..diamond. with sequences 
with .alpha.-helical .phi., .psi. angles at position 2. 
The distributions of nonLIVFM amino acids was determined in .diamond. 
positions sequences fitting the 
.diamond..diamond-solid..diamond-solid..diamond..diamond. patterns, when 
the second position had .phi., .psi. angles of either .alpha.-helices or 
.beta.-strands. In the 94 sequences with second positions having 
.alpha.-helical .phi., .psi. angles, the only associations at p&lt;0.01 were 
position 1: Arg, z=3.4; position 5: Gly, z=-2.1. In the 46 sequences with 
second positions having .beta.-strand .phi., .psi. angles, the only 
associations at p&lt;0.01 were position 1: Arg, z=2.5, and Trp, z=2.2; 
position 3: Gly, z=-1.9, Thr, z=2.4, and Tyr, z=1.8. There was no pattern 
obviously distinguishing .alpha.-helical from .beta.-strand sequences 
fitting the .diamond..diamond-solid..diamond-solid..diamond..diamond. 
template. 
TABLE XXII 
______________________________________ 
Frequencies of .phi., .psi. angles within limits of .alpha.-helices or 
.beta.-strands 
for all, .diamond-solid. or .diamond. residues and second positions 
.diamond..diamond-solid..diamond-solid..diamond..diamond. 
all .diamond-solid. 
.diamond. 
pattern n.sup.a 
z.sup.b 
______________________________________ 
.alpha.-helix 
.41 .45 .39 0.58 94 +3.6 
.beta.-strand 
.33 .40 .31 0.28 46 -1.0 
other .26 .15 .30 0.14 31 -0.5 
total 1.00 1.00 1.00 1.00 161 
______________________________________ 
.sup.a number of patterns with second position .phi., .psi. angles within 
indicated limits 
.sup.b z score of pattern frequencies compared to frequencies over all 
positions 
EXAMPLE 16 
Distributions of LL in .quadrature. Positions of Hydrophobic 
Strips-of-Helix 
The occurrence of two sequential amino acids from the group Leu, Ile, Val, 
Phe, Met with one residue in a .quadrature. position was termed LL. The 
frequency of such LL events in N-terminal, C-terminal, and middle (between 
N- and C-terminal positions) .quadrature. positions in the longitudinal 
strip-of-helix were determined (Table XXIII). LL occurred more often in 
middle .quadrature. positions of the helix and were suppressed at the 
N-terminus. Skewing of LL .quadrature. toward one end or the other of the 
strip was determined in terms of the relative distance of each LL 
.quadrature. as a fraction of the number of .quadrature. positions along 
the helix from the N-terminus. For example, a LL in .quadrature. position 
4 of a strip with five .quadrature. positions, occurred at 0.75. The mean 
distribution of LL was 0.60. The mean distribution of LL was not 
significantly removed from the middle of the strip. 
TABLE XXIII 
______________________________________ 
Distributions of LL in .quadrature. positions of helical hydrophobic 
strips. 
Distribution* 
.quadrature. positions 
.quadrature. positions 
total with LL Z score 
______________________________________ 
N-termini 
211 22 -4.6 
middle 371 112 3.7 
C-termini 
211 52 0.4 
total 793 186 
______________________________________ 
*In this analysis 48 proteins, containing 8024 residues, 2827 of which 
were in helices were scored for LL. LL were more frequently found in 
helices that expected for an empirical distribution over the entire 
protein sequence (z score = 3.5). 
EXAMPLE 17 
Distributions of Hydrophobic Amino Acids at N- and C-termini of Alpha 
Helices 
Most residues in .largecircle. positions between the terminal .quadrature. 
position in the longitudinal, hydrophobic strip and the first virtual 
.quadrature. position tend to fold as part of the .alpha.-helix. The 
actual frequency of the distribution of intervening .largecircle. 
positions in 227 helices and their adjacent sequences in 48 proteins was 
compared to the hypothesis that the helices would terminate with equal 
frequencies in the terminal strip .quadrature. and intervening 
.largecircle. positions (Table XXIV). The preference for intervening 
.largecircle. positions to fold in the .alpha.-helices was reflected in 
cumulative z-scores: N-termini 1.8, C-termini 3.8, both termini 4.0 
(p&lt;0.001). 
TABLE XXIV 
______________________________________ 
Distributions of amino acids at N- and C-termini of .alpha.-helices 
showing that helices tend to start and terminate with 
the .largecircle. position nearest the first virtual .quadrature. 
position 
beyond the N- and C-termini, respectively 
Prediction 
Observed Expected z score 
______________________________________ 
N-terminus 
+++.quadrature..sup.1 
56 38 +3.2 
-++.quadrature. 
34 38 -- 
--+.quadrature. 
38 38 -- 
---.quadrature. 
30 38 -- 
C-terminus 
.quadrature.+++ 
62 38 +4.3 
.quadrature.++- 
13 38 -4.4 
.quadrature.+-- 
41 38 -- 
.quadrature.--- 
22 38 -2.8 
.quadrature.++ 
27 25 -- 
.quadrature.+- 
25 25 -- 
.quadrature.-- 
38 25 +2.9 
______________________________________ 
.sup.1 .quadrature. = terminal .quadrature. position, with the residue in 
this position always being in the helix by definition. 
+ = distal .largecircle. position before the first virtual .largecircle. 
position, with the residue in this .largecircle. position being in the 
helix. 
- = distal .largecircle. position before the first virtual .largecircle. 
position, with the residue in this .largecircle. position not being in th 
helix. 
EXAMPLE 18 
Demonstration of Strucutral Effects of Design Characteristic Specified by 
the Method of the Invention 
Materials and Methods 
Peptide synthesis and analysis. PH-1.0 (FIG. 1) was synthesized by the 
Merrifield solid state synthesis method with t-BOC reagents. It was 
dissolved in 0.1% trifluoroacetic acid in water, desalted by adsorption to 
Sep-Pak C18 cartridges, and eluted with 30% acetonitrile in 0.1% 
trifluoroacetic acid. The peptide was purified by reverse phase HPLC on a 
C18 column with an acetonitrile gradient in 0.1% trifluoroacetic acid. 
Peptide composition was verified by amino acid analysis and confirmed by 
1D .sup.1 H NMR spectroscopy. 
Sample preparation. NMR samples were prepared by dissolving peptides in 700 
.mu.l of 90% H.sub.2 O/10% D.sub.2 O or 50% trifluoroethanol-d.sub.2 
/H.sub.2 O to a final concentration of 1-5 mM. The pH of the solutions was 
adjusted with 0.01M NaOH or HCl directly in the NMR tube and measured with 
a glass electrode without correcting for isotope effects. Micelle 
solutions were prepared by dissolving 180 mg of SDS-d.sub.25 (Cambridge 
Isotopes, Woburn, Mass.) in 500 .mu.l of 90% H.sub.2 O/10% D.sub.2 O, and 
sonicating to assure dissolution. 200 .mu.l of a concentrated peptide 
solution in 90% H.sub.2 O/10% D.sub.2 O were added to the micelles 
yielding about 6.5 mM peptide, and the pH was adjusted. The concentration 
of SDS-d.sub.25 was well above the micelle critical concentration with an 
estimated peptide:micelle ratio of 1:1.4, thus minimizing the 
concentration of unbound peptide without sacrificing the sensitivity of 
the NMR signals. Sodium 3-(trimethylsilyl)propionate-2,2,3,3-d.sub.4 was 
used as the internal reference in all cases. 
NMR experiments. Proton NMR spectra were acquired on a Varian UNITY series 
500 MHz spectrometer with the use of the .sup.1 H channel of a triple 
resonance probe (.sup.1 H/.sup.13 C/.sup.15 N) (Varian Analytical 
Instruments, Palo Alto, Calif.). Spectra were processed using VnmrS v4.1 
and VnmrX software on SUN 4-65 and 4-60 computers. 
1D .sup.1 H spectra were acquired at 5.degree. C. intervals from -5.degree. 
C. to 55.degree. C. Water suppression was accomplished by continuous wave, 
low power irradiation of the water resonance through the transmitter 
channel during 1 sec prior to the 90.degree. pulse, a feature incorporated 
in all the pulse sequences. A polynomial baseline correction and an 
exponential line broadening were used in the processing of these spectra. 
The center of transmitter frequency was set at the water resonance for all 
experiments. 
2D NMR experiments were acquired in the phase sensitive mode using the 
States-Haberkorn hypercomplex method. Water suppression was accomplished 
as above where the presaturation period (0.5 sec) was incorporated into 
the 2D pulse sequence through the transmitter. 2D spectra were apodized 
using a gaussian window function in the t2 and t1 dimensions. 
2D .sup.1 H TOCSY experiments were acquired with 2048 data points in the t2 
dimension and 2.times.256 t1 increments, with 32 scans per t1 value, a 
pulse delay of 0.1 sec and a MLEV16 mixing period of 80 or 120 ms. The 
final 2D spectra were processed with zero-filling to a final spectrum size 
of 2048.times.2048 data points. 
2D .sup.1 H NOESY experiments were acquired with 4096 data points in the t2 
dimension and 2.times.512 t1 increments, with 64 scans per t1 value, a 
pulse delay of 0.1 sec, and a mixing time of 200 ms. The final 2D spectra 
were processed with zero-filling to a final spectrum size of 
4096.times.4096 data points. With the use of NOE buildup rates, which were 
determined by varying the mixing times from 40-300 ms, it was determined 
that spin diffusion effects did not occur at mixing times below 250 ms. 
3D structure. Interproton distance constraints were derived from the NOESY 
spectra. NOESY crosspeaks were assigned and classified according to their 
intensities as strong, medium and weak with the use of their volume 
integrals. Distances associated with these ranges were calibrated using 
the intensity of the crosspeak corresponding to known fixed interproton 
distances, i.e. the ortho and meta aromatic protons of Tyr (2.5 .ANG.). 
The following constraint ranges were used: 1.8-2.7 .ANG. for strong, 
1.8-3.5 .ANG. for medium and 1.8-5.0 .ANG. for weak crosspeaks, 
respectively. The bounds of the constraint distances were modified for 
methyl groups, degenerate aromatic ring resonances, and methylene protons 
without stereospecific assignments, according to the pseudo-atom approach. 
RESULTS 
Testing the Stabilizing Function of Two Sequential LIVFM Residues Crossing 
the Longitudinal Hydrophobic Strip-of-Helix 
Analysis of amino acid distributions about .alpha.-helices, which are 
compared after justification on their longitudinal, hydrophobic 
strips-of-helix, leads to the recognition of highly restricted placements 
of certain amino acids in and about .alpha.-helices. Those placements 
appear to reflect the roles of those amino acids in helix formation and 
stability. The longitudinal hydrophobic strip-of-helix is found by 
application of a template 
(.quadrature..circle-solid..increment..circle-solid..quadrature..largecirc 
le..largecircle..quadrature..circle-solid..increment..circle-solid..quadrat 
ure..largecircle..largecircle..quadrature..circle-solid..increment..largeci 
rcle., joined in a circle) to the primary sequence of an .alpha.-helix in a 
protein to maximize the mean hydrophobicity of residues in .quadrature. 
positions. The frequency of LL (occupancy of an .quadrature. position with 
an amino acid of the group LIVFM when the preceding or the following 
position in the primary sequence was also from the group LIVFM) was 
determined in N-terminal, C-terminal, and middle (between N- and 
C-terminal) .quadrature. positions in the longitudinal strip-of-helix for 
48 proteins. LL occurred more often in middle .quadrature. positions of 
the helix and were suppressed at the N-terminus. Skewing of LL toward one 
end or the other of the hydrophobic strip was tested in terms of the 
relative distance of each LL .quadrature. as a fraction of the number of 
.quadrature. positions along the helix from the N-terminus. For example, a 
LL in the fourth .quadrature. position of a strip with five .quadrature. 
positions, occurred at 0.75. The mean distribution of LL was 0.60 and was 
not significantly removed from the middle of the strip. 
In order to demonstrate that the presence of LL at a .quadrature. position 
between the distal N- and C-terminal .quadrature. positions in the 
longitudinal hydrophobic strip led to greater stability of a peptidyl 
sequence, the conformations and stability of PH-1.0 was compared with that 
of PH-1,12 which had a LL at the third .quadrature. position resulting 
from a Thr.sup.9 .fwdarw.Leu substitution. Relative to PH-1.0, PH-1.12 
(FIG. 1) demonstrated increased chemical shifts at C.sup..alpha. of 
Gln.sup.6, Gln.sup.10, and Lys.sup.13 which lay in the helical loops 
adjacent to the LL structure. There was also an increase in the chemical 
shift at the C.sup..alpha. of Leu.sup.8, but no alteration was seen in the 
chemical shifts at the C.sup..alpha. H of Leu.sup.5, Leu.sup.9, and 
Leu.sup.12. In contrast, in 50% TFE/H.sub.2 O the chemical shifts at each 
residue position in all three peptides were comparable, indicating the 
specificity of the variations seen in the SDS micelle-adsorbed 
conformations. 
Testing the Hypothesis that Favored and Disfavored Motifs of LIVFM Residues 
Correlate to Degree of Order in Micelle-Adsorbed Peptides 
Hydrophobic amino acids of the group LIVFM are distributed in favored or 
suppressed patterns within protein sequences. The frequencies of all 
5-position combinations of .diamond-solid.=LIVFM and .diamond.=nonLIVFM 
residues were analyzed in 48 proteins of known crystallographic structure. 
Some motifs were strongly preferred or dispreferred, e.g. 
.diamond..diamond-solid..diamond-solid..diamond..diamond. was favored 
(z=3.5), while .diamond..diamond-solid..diamond..diamond-solid..diamond. 
was not (z=-3.4). In longer patterns, .diamond-solid..diamond-solid. 
followed by .diamond..diamond. and one .diamond-solid. was favored 
(.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond..diamon 
d-solid..diamond..diamond., z=5.1), while conversion of the single 
hydrophobic residue to a pair was not 
(.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond..diamon 
d-solid..diamond-solid..diamond., z=0.8). While the strongly favored 
pattern .diamond..diamond-solid..diamond-solid..diamond..diamond. was 
found in both .alpha.-helical and .beta.-strand sequences, it associated 
significantly with .alpha.-helices (z=3.6 for the second position .phi., 
.psi. angles of .alpha.-helices), but was also not significantly 
suppressed in .beta.-strands (z=-1.1). Such selections for certain motifs 
of LIVFM and non LIVFM residues might occur if they lead efficiently to 
the local nucleations hypothesized to characterize molten globule 
intermediates in the folding of proteins, regardless of the final local 
secondary structure in which those motifs appear. 
In order to demonstrate that favored or disfavored motifs of Leu, Ile, Val, 
Phe, and Met residues leads to greater or lesser stability, respectively, 
of adsorbed amphiphilic .alpha.-helices, the conformations and stability 
of PH-1.0 was compared with that of PH-1.13 (with substitution of 
Gln.sup.10 .fwdarw.Leu). The patterns of these peptides were: PH-1.0 
.diamond-solid..diamond..diamond..diamond..diamond-solid..diamond..diamond 
..diamond-solid..diamond..diamond..diamond..arrow-down dbl..diamond. and 
PH-1.13 
.diamond-solid..diamond..diamond..diamond..diamond-solid..diamond..diamond 
..diamond-solid..diamond..diamond-solid..diamond..diamond-solid..diamond.. 
The homolog LYQELQKLTLTLK (SEQ ID NO: 3) (with a disfavored pattern of 
three alternating Leu residues, z=-3.4) demonstrated decreased chemical 
shift at the C.sup..alpha. H of each residue from Gln.sup.6 to Lys.sup.13. 
The chemical shifts of C.sup..alpha. H of residues from Leu.sup.1 through 
Leu.sup.5 were comparable. 
In addition to demonstrating destabilization of local structure in the 
comparison of PH-1.13 relative to PH-1.0, increased stabilization of 
PH-1.12 
(.diamond-solid..diamond..diamond..diamond..diamond-solid..diamond..diamon 
d..diamond-solid..diamond-solid..diamond..diamond..diamond-solid..diamond.) 
was found relative to PH-1.0 by introduction of the favored 
.diamond..diamond..diamond-solid..diamond-solid..diamond..diamond..diamond 
-solid..diamond..diamond., z=5.1. The LL-hydrophobic strip and `binary 
motif` correlations with local stability reflect a basic geometric 
mechanism regulating stabilization of peptidyl sequences folding against 
hydrophobic regions, for example, in the initial phase of protein folding 
or in the binding of a hormone to its receptor. 
In FIG. 1, the difference between the experimentally observed (exp) and 
random coil (rc) values of the chemical shifts for the C.sup..alpha. H at 
each residue position in three peptides is plotted from NMR analyses in 
50% trifluorethanol/water (TFE) and on sodium dodecylsulfate micelles 
(SDS). The value presented at each residue position, n, is the mean of 
values observed at n-1, n, and n+1. The chemical shifts reflect the degree 
of order in structure about the C.sup..alpha. in these peptides for which 
alpha helical conformation was demonstrated by various cross correlations. 
While the three peptides had comparable degrees of order about each 
C.sup..alpha. H in TFE, order on SDS micelles about each C.sup..alpha. H 
followed predictions precisely. Increased helical stability was 
demonstrated in helical loops surrounding the LL structure and decreased 
order was found in the region of the 
.diamond-solid..diamond..diamond-solid..diamond..diamond-solid. structure. 
EQUIVALENTS 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, many equivalents to the embodiments of 
the invention described specifically herein. Such equivalents are intended 
to be encompassed in the scope of the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
LeuTyrGlnGluLeuGlnLysLeuThrGlnThrLeuLys 
1510 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
LeuTyrGlnGluLeuGlnLysLeuLeuGlnThrLeuLys 
1510 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LeuTyrGlnGluLeuGlnLysLeuThrLeuThrLeuLys 
1510 
__________________________________________________________________________