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Biochemical and Biophysical Research Communications 316 (2004) 731737

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An application of experimental design using mutually orthogonal


Latin squares in conformational studies of peptides
K. Vengadesan,a T. Anbupalam,b and N. Gauthama,*
a
Department of Crystallography and Biophysics, University of Madras, Chennai 600 025, India
b
Department of Statistics, University of Madras, Chennai 600 005, India
Received 31 January 2004

Abstract

We address the questioncan we use experimental design methods to investigate peptide conformation and identify confor-
mational parameters that may contribute more signicantly to the potential energy than others? We used mutually orthogonal Latin
square design to sample the conformational space of peptides and analysed the samples using analysis of variance. We examined the
equality of the eect of the torsion angles on the conformational potential energy. The results showed that dierent torsion angles
contributed dierently to the conformational energy. We are able to identify those parameters that may have to be more carefully
considered in conformational studies of peptides.
2004 Elsevier Inc. All rights reserved.

Keywords: Experimental design; Mutually orthogonal Latin squares; Analysis of variance; Enkephalins; Conformational searching

Latin squares are widely used in the design of statis- This concept of orthogonal pairs of Latin squares can be
tical experiments in elds as diverse as agriculture, ed- extended to a set of N mutually orthogonal Latin
ucation, medicine, economics, psychology, marketing, squares (MOLS) which are superimposed on each other
health, and cryptography [15]. The Latin square design to study the eect of up to N factors [5]. Usually the
is used to simultaneously eliminate or block two Latin square and MOLS designs are analysed using
nuisance factors (one factor in the rows and one in ANOVA to estimate the contributions of each factor to
the columns of the square) that introduce variability the total variation in the whole set of measures, i.e., in
in the experiments, and then to compare the eects of the response.
the treatments on the response. However, Latin square Recently we used MOLS to sample the potential en-
design can also be useful in situations where the rows ergy hyper surface of peptides in an attempt to identify
and columns represent factors one actually wishes to the conformations corresponding to the minima on this
study. A single Latin square allows one to investigate surface [6,7]. In this we cast the peptide structure opti-
the eect of setting three factors each at M dierent mization problem as one in experimental design, where
values in only M2 trials, instead of M3 trials that would an experiment consists of setting all the conformational
be required for a complete and exhaustive sampling. The variables of the molecule to a specic set of values, and
design assumes that there is no interaction between the calculating the semi-empirical potential energy. The
factors, i.e., their eects on the response are independent variables are thus identied as the factors in the lan-
of each other. A mutually orthogonal Latin square de- guage of experimental design, and the values they are set
sign (also called a Graeco-Latin square design) is ob- to as the treatments. The response of the experiment
tained by superimposing two orthogonal Latin squares (or the eect of a particular treatment) refers to the
to simultaneously study the eect of four factors [5]. value of the potential energy calculated for that con-
formation. The nal aim of the study was to optimize
*
Corresponding author. Fax: +91-44-2235-2494.
the response. The use of MOLS allowed us to drastically
E-mail addresses: gautham@unom.ac.in, n_gautham@hotmail. reduce the size of the sampled conformational space,
com (N. Gautham). and still recover much of the information content of the

0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2004.02.114
732 K. Vengadesan et al. / Biochemical and Biophysical Research Communications 316 (2004) 731737

entire space. For a molecule with n conformational The use of MOLS in designing the experiments to sample peptide
variables, each sampled at m points, the size of the conformational space has been described in detail previously [6]. Here
conformational space is mn . Our method reduced the we briey reiterate some of the main points. We rst dene MOLS. A
Latin square of order N is dened as a set of N symbols, arranged in a
sample size to the order of m2 . The sampled measures N  N square, such that each symbol occurs exactly once in every row
were analysed using a procedure similar to the mean and once in every column. Two Latin squares are orthogonal if, when
eld technique [8] to locate minimum energy confor- they are superimposed, each symbol of the rst square occurs once,
mations. Since the basic assumption of the independence and only once, with each symbol of the second square. A set of MOLS
is a set of Latin squares, every pair of which is orthogonal [10]. It has
of the factors is true only to a limited extent, the above
been shown that if N is a prime power, one can construct N  1 MOLS
technique does not result in a single identiable global of order N [11,12]. Fig. 1 shows, as an example, a set of six mutually
optimum conformation for the molecule. Instead we orthogonal Latin squares of order 7. Now we may recast the statement
obtain one of the low energy conformations. If the cal- made earlier in this paper as follows. A set of N  1 MOLS of order N
culations are repeated, each time using a dierent set of may be used to study the eects of up to N  1 factors, each com-
MOLS to sample the space, each repetition results in a prising N treatments. In the model that we have considered, the three
dimensional structure of a peptide chain is specied by the n torsion
low energy conformation, either same as one obtained angles or factors, hr ; r 1; n. Each factor is capable of taking up m
before, or a new one. After a sucient number of rep- dierent values (i.e., treatments) in a range 0360, the step size thus
etitions we obtain no new structure, leading us to con- being 360/m. For example, each factor can have the treatments
clude that we have exhaustively searched the 0; 10; 20; . . . ; 350 for 10 step size, with m 36. If r is the index that
conformational space for minima. This method of labels the torsion angles and s is the index that labels the values taken
up by each torsion, i.e., the steps along each angle, then hr;s , r 1; n;
analysis diverges from the usual ANOVA techniques, s 1; m are the set of values that dene the complete conformational
and does not extract all the information present in a space of the molecule. The sampling has to be carried out among the
single set of experiments conducted using MOLS de-
sign. In this paper we report a more conventional
analysis of the variance in the data. This helps us to
identify the eects of the dierent conformational pa-
rameters in the molecule, and distinguish ones that
contribute signicantly to the nal optimal conforma-
tion from those that are less important.

Methods

The only hypothesis generally of interest in a Latin square design


analysis is the one concerning equality among the levels of the factors
[9]. In the present application to molecular structure, the factors are
the conformational variables and their levels or treatments are the
values they assume. The hypothesis of interest now concerns the eects
of dierent values for the variables. We model the molecule as com-
prising atoms connected by xed bonds. The only variables are the
torsion angles about the bonds. In this model, therefore, the factors are
the torsion angles, and the levels are the set of values from 0 to 360
that they assume. Note that since the levels are xed, and not chosen at
random, all the factors are so-called xed eects factors [9]. We are
interested in the questiondoes the energy dier signicantly over the
set of values assumed by each torsion angle? For each angle we set up
the null hypothesis (H0 ) that there is no eective dierence in setting it
to any particular value, and that the eect, measured here in terms of
the potential energy, is the same no matter which value is chosen for
that angle. The alternative hypothesis (H1 ) for each angle is that there Fig. 1. An example of a set of mutually orthogonal Latin squares,
is at least one value that shows a signicant dierence on the eect or showing six MOLS of order 7, i.e., N 7, n 6, and m 7. Symbols
the response, i.e., the energy. This may be stated formally as follows. If in the rst Latin square: A1, A2, A3, A4, A5, A6, and A7. Each of
h is the factor (torsion angle) and the subscript refers to its value, there these is repeated 7 times to give a total of 49 symbols, which have been
being m values, then arranged in a Latin square. Symbols in second Latin square: B1, B2,
B3, B4, B5, B6, and B7. The second Latin square is orthogonal to the
H0 : h1 h2 h3 h4       hm ; rst. Note that every pairing of a symbol from the rst square with one
H1 : h1 6 h2 6 h3 6 h4 6       6 hm ; from the second occurs exactly once. Similarly, the symbols in third
Latin square: C1; . . . ; C7; symbols in fourth Latin square: D1; . . . ; D7;
for at least one value of the angle:
symbols in fth Latin square: E1; . . . ; E7; and symbols in sixth square:
The acceptance of either H0 or H1 and the concomitant rejection of the F1; . . . ; F7. In the present application, each symbol within the sub
other are carried out on the basis of the ANOVA results. Commonly square represents a possible value for the corresponding torsion angle,
the decision to reject H0 is taken when the F ratio justies this at the and each sub square represents a possible conformation of the mole-
P 0:05 level of signicance, where F and P are statistics calculated cule. The MOLS method requires the potential function to be evalu-
from the data. ated at each of these N2 points in the conformation space.
K. Vengadesan et al. / Biochemical and Biophysical Research Communications 316 (2004) 731737 733

mn combinations of these values, each such combination specifying one


conformation of the molecule. We now use MOLS to pick up N2
combinations (i.e., N2 conformations) of these, where N is a prime
number greater than the larger of n or m. The potential energy at each
of the N2 locations is calculated. This is carried out by setting the
molecule to the specied conformation, and calculating the potential
energy, using the AMBER force eld parameters [13]. We included
only the inter-atomic electrostatic, van der Waals, hydrogen bond
energy terms, ignoring solvent eects, etc.
The computational procedure for the ANOVA of MOLS design is
a simple extension of the Latin square design ANOVA [5]. We trans-
formed the energy values to a logarithmic scale to accommodate the
exponential variation in them. We applied the analysis to peptide
molecules such as enkephalins and a few model homopolymer pep-
tides. The details of the computational experiments are given below
along with the results.

Results and discussion

Enkephalins

Enkephalins (HTyrGlyGlyPheMet/LeuOH)
are small, linear neuropeptides with morphine like ac-
tivity. They have been well studied both experimentally
and theoretically [1416]. The variable factors in our
model of Met-enkephalin comprised the 10 torsion an-
gles shown in Fig. 2; the side chain torsion angles were
set xed at the values given in the Insight II library [17].
The possible levels for each factor were chosen in the
range 0360. A step size of 10 would yield the 36 levels
(0; 10; 20; . . . ; 350). However, as explained in the
Methods section, the experimental design using MOLS
requires the number of levels m to be a prime number.
To accommodate this we assumed the value 0 to be
dierent from 360. This gave the 37 levels
(0; 10; 20; . . . ; 360) for each of the 10 factors. A set of 10
MOLS of order 37 was constructed and used to allocate
treatments to each factor. Thus, totally 372 computa-
tional experiments were performed. Each experiment
consisted of setting the 10 torsion angles to the values
decided by the MOLS experimental design and then
calculating the potential energy of the molecule. As an
illustration Table 1 gives the rst 10 conformations and
energies for Met-enkephalin. The 372 energy values that
resulted were subjected to ANOVA as described by
Montgomery [5]. The results of this analysis are given in
Table 2.
The rst column species the factors, in this case the
torsion angles, which are the source of variation in the Fig. 2. Schematic representation of extended conformation of Met-
potential energy. The signicance of each variable is enkephalin molecule with labelling of variables of 10 backbone vari-
ables (/1 , w1; , /2 , w2; , /3 , w3 , /4 , w4 , /5 , and w5 ) and the side chain
decided on the basis of the P value in column 6. Only variables were set xed at the values given in the Insight II library [17].
variables with P values less than 0.05 are considered Hydrogens attached to the carbon atoms are not shown for the clarity.
signicant. We may also use the P values as a guide to
the level of signicance of the variable, small values
indicating a greater eect of the corresponding variable bulky aromatic side chains of Tyr1 and Phe4 . Other
[5]. From the table it is clear that the variable /4 has the angles that strongly aect the conformation all occur in
greatest eect on the conformational energy. Variations the centre of the moleculeH0 is rejected for /2 , w2 , /3 ,
in this angle aect the interactions between the two w3 , /4 , w4 , /5 , and the hypothesis that the values of the
734 K. Vengadesan et al. / Biochemical and Biophysical Research Communications 316 (2004) 731737

Table 1
The torsion angle values (in degrees) with energy E (in kcal/mol) for rst 10 (of 372 ) Met-enkephalin conformations
/1 w1 /2 w2 /3 w3 /4 w4 /5 w5 E
1 280 110 270 40 10 0 110 70 260 320 2.28
2 270 210 340 150 350 30 0 270 320 340 10.61
3 60 240 30 130 70 270 10 120 280 220 2.45
4 30 200 150 350 260 260 270 360 340 260 10.12
5 300 270 90 260 320 40 230 0 140 330 )2.08
6 130 90 60 270 120 170 340 10 250 140 8.39
7 350 290 230 70 80 300 320 260 300 250 12.06
8 260 310 40 0 160 210 40 190 60 230 17.15
9 230 250 170 300 110 250 130 40 0 100 4.96
10 210 130 140 10 90 320 70 50 220 170 4.95

Table 2
Analysis of variance for the Met-enkephalin experiment
Source of variation Degrees of freedom Sum of squares Mean square F0 P value Signicance codes
/1 36 832.4 23.1 1.0657 3.66  1001 NS
w1 36 579.6 16.1 0.7420 8.67  1001 NS
/2 36 2956.9 82.1 3.7854 1.51  1012 ***
w2 36 1206.7 33.5 1.5448 2.20  1002 *
/3 36 3094.0 85.9 3.9608 1.83  1013 ***
w3 36 1795.1 49.9 2.2980 2.71  1005 ***
/4 36 8860.4 246.1 11.3427 0.00  1000 ***
w4 36 1153.7 32.0 1.4769 3.59  1002 *
/5 36 3087.9 85.8 3.9530 2.02  1013 ***
w5 36 726.8 20.2 0.9304 5.88  1001 NS
Error 1008 21872.2 21.7
Total 1368 46165.6
Signicance codes: ***P < 0:001, **P < 0:01, *P < 0:05, and NS P P 0:1.

angles matter in arriving at the nal conformation is set of 10 MOLS of order 37 may be carried out in (37!)10
accepted at various levels of signicance. All these aect dierent ways. However, according to the theory of
the relative orientation of the two bulky aromatic experimental design using Latin squares, all of these
groups. Of these /2 , /3 , /4 , and /5 show the greatest would lead to the same results. We conrmed this by
eect. This is probably related to the fact that in the repeating the calculations 10 times, each time choosing a
Ramachandran map [18], / has a more restricted range dierent set of MOLS. The results were consistent, and
of values than w. In particular for glycine, which are all angles show the same eects on the structure, though
residue numbers 2 and 3 in the pentapeptide, the the P values uctuate somewhat.
Ramachandran map shows that every value of w can Table 3 shows the results obtained for Leu-enkeph-
lead to an allowed conformation, while this is so for alin. They are very similar to the ones for Met-
only about 60% of the / values. The angles at the two enkephalin. Once again w angles are much less signicant
ends of the molecule do not contribute to the confor- than / angles, and /4 has the greatest eect on the
mational energy even at the P 0:05 level. This is not conformational energy. Again /5 has a strong eect,
surprising, considering that they only aect interactions presumably because the side chain of Leu5 is about as
between the end atoms and their immediate neighbours, bulky as Met5 . Repeating the experiments for Leu-en-
and do not contribute in any major way to the folding of kephalin showed the same consistent behaviour as for
the peptide. It is interesting to note that, on the one Met-enkephalin, though the angles /1 and w4 show
hand, w1 is not signicant, while on the other hand, /5 uctuating P values.
which occupies a similar position at the other end of the
chain has a strong eect on the conformational energy. Homopolymers
One probable reason for this has been discussed above
w angles are much less signicant than / angles. The The experiments and the analyses have also been
other reason could be the presence of two bulky side carried out for the homopolymer pentapeptides (Gly)5 ,
chains Phe4 and Met5 on either side of /5 , whereas w1 is (Ala)5 , and (Aib)5 . Table 4 shows the results for (Gly)5
anked by a glycine residue on one side. Changes in /5 , As for the enkephalins, the torsion angles at the two
therefore, may lead to stronger variations in the inter- ends of the molecule do not contribute strongly to the
actions between the side chains. The construction of the conformational energy. Again, as for the enkephalins, /
K. Vengadesan et al. / Biochemical and Biophysical Research Communications 316 (2004) 731737 735

Table 3
Analysis of variance for the Leu-enkephalin experiment
Source of variation Degrees of freedom Sum of squares Mean square F0 P value Signicance codes
/1 36 995.1 27.6 1.318 1.01  1001 NS
w1 36 819.6 22.8 1.0856 3.37  1001 NS
/2 36 2998.8 83.3 3.972 1.60  1013 ***
w2 36 1361.4 37.8 1.8032 2.80  1003 **
/3 36 1636.4 45.5 2.1675 9.87  1005 ***
w3 36 2124.1 59 2.8134 1.16  1007 ***
/4 36 7848.2 218 10.3951 2.22  1016 ***
w4 36 1179.8 32.8 1.5626 1.93  1002 *
/5 36 5398.1 149.9 7.1499 2.22  1016 ***
w5 36 769.6 21.4 1.0193 4.39  1001 NS
Error 1008 21139.6 21
Total 1368 46270.4
Signicance codes: ***P < 0:001, **P < 0:01, *P < 0:05, and NS P P 0:1.

Table 4
Analysis of variance for the (Gly)5 experiment
Source of variation Degrees of freedom Sum of squares Mean square F0 P value Signicance codes
01
/1 36 508.6 14.1 0.9462 5.61  10 NS
w1 36 376.9 10.5 0.7012 9.07  1001 NS
/2 36 3726.3 103.5 6.9321 7.77  1016 ***
w2 36 1191.4 33.1 2.2165 6.10  1005 ***
/3 36 4311.7 119.8 8.0212 2.22  1016 ***
w3 36 1826.1 50.7 3.3972 1.50  1010 ***
/4 36 3446.1 95.7 6.4108 3.33  1016 ***
w4 36 1627.8 45.2 3.0282 1.05  1008 ***
/5 36 1491.3 41.4 2.7743 1.78  1007 ***
w5 36 792.1 22 1.4737 3.67  1002 *
Error 1008 15051.1 14.9
Total 1368 34349.4
Signicance codes: ***P < 0:001, **P < 0:01, *P < 0:05, and NS P P 0:1.

angles have a greater eect than w angles. However, (Aib)5 is dierent from the other pentapeptides. The
neither of these observations has as much support as constituent amino acid, a amino isobutyric acid, is not a
previous. In particular w5 at the carboxy end of the standard one and its Ramachandran map is highly re-
pentapeptide contributes to the conformational behav- stricted in / [19]. This feature, however, is not reected
iour to a much greater amount than for the neuropep- in our results (Table 6). All the angles in the middle of
tides above. Repetitions of the calculations using the molecule are signicant with P < 0:05, and w3 and
dierent sets of MOLS show that these results are /2 have the greatest eect on the conformation. This is
consistent. consistently so even when the calculations are repeated
The results for (Ala)5 are given in Table 5. Again the several times. Thus this pentapeptide does not follow the
rst two torsion angles /1 and w1 are not signicant. pattern we have observed in the other cases above.
However, unlike the previous cases, the angles at the
carboxy terminus also contribute to the conformational Decapeptides
energy. Repetitions of the experiment show that in some
cases the F value corresponding to w5 is good enough to We have also carried out the analysis on a pair of
classify it as having a strong eect with P > 0:05. The decapeptides. The sequences were originally chosen
other angles in the middle of the chain all have signi- from the CSD [20], to evaluate the ecacy of our
cant eects on the energy. The eect of /2 is the stron- structure prediction methodology, since our predictions
gest, though its dierence with the other angles is not as could then be compared with the experimental structure.
large as in the case of the enkephalins. This is probably a Here we discuss the analysis of the results as a study in
consequence of all side chains being the same, as well as experimental design. The rst decapeptide has the se-
small. Once again / angles have a greater eect than w quence LLLLBLLLLB [21]. Only the backbone / and w
angles, but now the dierences in the P values are not as angles were varied and the side chain angles were kept
striking as in the previous cases. xed as in the case of the pentapeptides. The rst set of
736 K. Vengadesan et al. / Biochemical and Biophysical Research Communications 316 (2004) 731737

Table 5
Analysis of variance for the (Ala)5 experiment
Source of variation Degrees of freedom Sum of squares Mean square F0 P value Signicance codes
/1 36 392.2 10.9 0.8702 6.89  1001 NS
w1 36 409.5 11.4 0.9085 6.25  1001 NS
/2 36 2049.7 56.9 4.5477 8.88  1016 ***
w2 36 742.9 20.6 1.6483 9.99  1003 **
/3 36 1574.3 43.7 3.4929 4.86  1011 ***
w3 36 1311.6 36.4 2.91 3.95  1008 ***
/4 36 1551.4 43.1 3.4422 8.82  1011 ***
w4 36 987.4 27.4 2.1908 7.85  1005 ***
/5 36 1090.6 30.3 2.4197 7.82  1006 ***
w5 36 726.4 20.2 1.6116 1.33  1002 *
Error 1008 12619.9 12.5
Total 1368 23456
Signicance codes: ***P < 0:001, **P < 0:01, *P < 0:05, and NS P P 0:1.

Table 6
Analysis of variance for the (Aib)5 experiment
Source of variation Degrees of freedom Sum of squares Mean square F0 P value Signicance codes
/1 36 226.7 6.3 0.6484 9.47  1001 NS
w1 36 321.4 8.9 0.9194 6.07  1001 NS
/2 36 1306.5 36.3 3.7375 2.67  1012 ***
w2 36 511.8 14.2 1.4642 3.92  1002 *
/3 36 955.1 26.5 2.7322 2.82  1007 ***
w3 36 1058.9 29.4 3.0291 1.04  1008 ***
/4 36 783.3 21.8 2.2407 4.80  1005 ***
w4 36 940.5 26.1 2.6906 4.43  1007 ***
/5 36 571.7 15.9 1.6354 1.11  1002 *
w5 36 277.5 7.7 0.7939 8.03  1001 NS
Error 1008 9787.9 9.7
Total 1368 16741.2
Signicance codes: ***P < 0:001, **P < 0:01, *P < 0:05, and NS P P 0:1.

calculations indicated that w5 and w6 were the only smaller peptides, and to a much lesser extent for larger
signicantly eective angles. When the calculations were ones, torsions that control interactions between bulky
repeated using other sets of MOLS, the results were not amino acid side chains exert a greater inuence on the
consistent. The second decapeptide has the sequence energy. A feature common to all peptides studied was
WIABIVBLBP [22]. Once again the results were equiv- the greater signicance of / angles as compared to w
ocal, with one important dierencethe angle /2 con- angles.
sistently has an F value greater than 2.0, which indicates These studies were carried out on the assumption
that it is signicant at P < 0:001. This may be due to the that the variables are independent. This assumption
bulky tryptophan and isoleucine residues on either side clearly has only very limited validity. Nevertheless, the
of it. analysis leads to a preliminary appreciation of the role
of each parameter in determining the minimum energy
conformation of a biomolecule. Most protein and
Conclusion peptide structure prediction methods have to over-
come the very dicult obstacles relating to multiple
We used MOLS design to study the eects of each minima [23] and combinatorial explosion [24]. Our
torsion angle on the conformational energy of a few studies may be useful in tacking these problems, at
oligopeptides. The aim of the study was to explore the least for small peptides. The information obtained
possibility of identifying crucial conformational pa- may be used to design optimization algorithms that
rameters that could then be concentrated on in any concentrate on the most important parameters. It may
conformational study. The results showed that there are also be possible to extend the analyses by the use of
dierences in torsion angle eects. In general torsion other statistical tests to compare and group the most
angles at the ends of the peptide chain do not play a signicant values of each conformational parameter.
signicant role in determining the molecular potential One may also try experimental designs other than
energy corresponding to a particular conformation, MOLS, such as fractional factorial design or response
while angles in the middle do. In particular for the surface design [5].
K. Vengadesan et al. / Biochemical and Biophysical Research Communications 316 (2004) 731737 737

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