J. Am. Chem. SOC.

1993,115, 4849-4858 4849

Peptide Models 6. New @-Turn Conformations from ab Initio
Calculations Confirmed by X-ray Data s f ProteinsL
Andrhs Perczel,t** Michael A. McAllister,t Phl Cshszhr,t*g and Imre G. Csizmadia'*+
Contribution from the Department of Chemistry, University of Toronto, Ontario,
Canada M5S 1 AI , Department of Organic Chemistry, Eat& University, Budapest 11 2
POB 32 H-1518. Hungary, and Apotex Inc., 150 Signet Drive, Weston, Ontario,
Canada M9L 1 T9
Received May 20, 1992
Abstract: In an attempt to determine intrinsically stable hairpin geometries, a number of triamide conformations of
For-Ala-Ala-NH2 were investigated using ab initio calculations (HF/3-21 G). Previous ab initio calculations of selected
diamides of single amino acid residues (e.g., For-Ala-NH2) suggested that the a,-type backbone conformation (6 =
-54', +=-45') is not a minimal energy structure, although in globular proteins the (aL),, units (referred to as a-helices)
are the most frequently found conformations. The lack of the aL conformation made the application of ab initio
calculations in peptide geometry analyses questionable. In contrast, for triamides (e.g., For-Ala-Ala-NH2) the appearance
of the aL backbone subconformation is confirmed in the a,6, conformation (usually referred to as type I &turn). This
intrinsically stable conformation is the most frequently found hairpin structure in proteins. The existence of the eL
conformation (4 =d o o , +=120') in chiral diamides (such as For-Ala-NH2, For-Ser-NH2, or For-Val-NH2) has
never been confirmed by ab initio studies, although X-ray analyses of proteins revealed the existence of the polyproline
I1 conformation [(eL),,] a long time ago. The herein presented stable Y ~ E ~ and bDeL hairpin conformations, calculated
by abinitiomethods, legitimize the"missing" e, backbonegeometry. The fact that somelegitimate backboneconformations
(a, and e,) appear only in triamides and not in diamide systems assigns a specific role to triamide models in understanding
protein conformations. The importance of some triamide conformations, especially type I and type I1 &turns, is
emphasized. This study summarizes all the possible [18 (30) conformations depending on the d or T "selection rule"]
hairpin geometries determined for For-Ala-Ala-NH2 using ab initio computations. We were able to identify all 30
ab initio yielded conformations as backbone substructures of globular proteins, determined by X-ray crystallography.
The 30 optimized triamide structures present a unique opportunity to understand the conformational behavior of
@-turns @bends or hairpins). This may have far-reaching consequences in understanding the j3-turn-mediated protein
folding.
Introduction
Besides the two major secondary structural elements'-) [the
a-helix (& =-60 f 30, qi =-60 f 30),, and the 8-pleated
sheet (& =-150' f 30, +i =150' f 30),], the third most
frequently found4J structural unit in globular proteins is the @-turn
conformation.G8 Smith and Pease* reviewed in detail the reverse
turns in peptides and proteins. Reverse turns, hairpins, 8-bends,
or &turns are structural elements consisting of four successive
amino acid residues (labeled 1,2,3, and 4) at positions i, i +1,
i +2, and i +3 in proteins. The variety of definitions suggested
in the past quarter of a century clearly illustrates the evolution
of the &turn concept. Adhering to the original definitions of
Venkatachalam: &turns are classified into conformational types
by their values of cj i +, , +i +l , &+I, and +i+2 torsional angles. On
the basis of the four backbone torsional angle values of the second
* Address correspondence to this author. Current address: Laboratoire
de Chime Thorique, UniversitC Nancy 1 C.N.R.S., 54506 Vandoeuore-les-
Nancy, France.
+University of Toronto.
5 Apotex Inc.
1 Dedicated to the memory of Professor M. KajtBr, the late virtuoso of
stereochemistry.
(1) Pauling, L.; Corey, R. Proc. Natl. Acad. Sci. U.S.A. 1951, 37, 729-
740.
(2) Pauling, L.; Corey, R.; Branson, H. Proc. Natl. Acad. Sci. U.S.A.
(3) Levitt, M.; Chothia, C. Nature (London) 1976, 261, 552-558.
(4) Crawford, J . L.; Lipscomb, W. N.;Schellman,C. G. Proc. Natl. Acud.
(5) Zimmerman, S . S.; Scheraga, H. A. Proc. Narl. Acad. Sci. U.S.A.
(6) Venkatachalam, C. Biopolymers 1968, 6, 1425-1436.
(7) Sibanda, B. L.; Thorton, J . M. Nurure. (London) 1985,316,170-174.
(8) Smith, J .; Pease, L. Crit. Reu. Biochem. 1980, 8, 315-399.
Edtvijs University.
1951, 37, 205-21 1.
Sci. U.S.A. 1973, 70, 538-542.
1977, 74, 41264129.
and third residues, there are three major types of folded
conformations: I, 11, and I11 &turns (Chart I). About a decade
later, in the analysis of the @turn content of globular proteins,
a distance criteria was also i ntrod~ced.~~.~ Accordingly, the
Cy - Ci distance must be shorter than 7 A. Often an
intramolecular H-bond can befound in @-turns, where the NH
of the i +3 residue points toward the carbonyl oxygen of the ith
residue (1 - 4-type H-bond) as shown in Figure 1. Although
this 1 - 4 hydrogen bond has never been proved to be a necessary
condition for @-turns, it is frequently found in peptides and proteins
on the basis of X-ray9aqc and NMR structure determinations,"J
and therefore a misconception has developed over the years that
such a hydrogen bond is an essential structural featureof 0-turns.
This H-bond pattern was also used as a criterion for @-turn
assignment in proteins.9a~c
The Vankatachalam6-predicted +i +2 =0' value for several
types of &turns was not satisfied in several structure assignments
in globular proteins. Consequently, Chou and Fa~man~bsuggested
a larger tolerance for the +i+2 torsional angle (-50' I +1+2 I
50'). More recently, Wilmot and Thorton" demonstrated that
the 1c;+z =0' criterion is one of the reasons that numberous turns
are identified as "distorted." The experimental value of $i +2 is
often around 45' or -45'. This finding is in perfect agreement
with our previous analysisl2J3 of ab initio Ramachandran maps,
(9) (a) Levitt, M.J . Mol. Biol. 1976,104,59-107. (b) Chou, P. Y.; Fasman,
G. D. J. Mol. Biol. 1977, 115, 135-175. (c) Kabsch, W.; Sander, C.
Biopolymers 1983, 22, 2577-2637.
(IO) Dyson, H. J .; Rance, M.; Houghten, R. A.; Lerner, R. A.; Wright,
P. A. J. Mol. Biol. 1988, 201, 161-200.
(11) Wilmot, C. M.; Thorton, J . M. Protein Eng. 1990, 3, 479493.
(12) Perczel, A,; Angyan, J . G.; Kajtar, M.; Viviani, W.; Rivail, J .-L.;
Marcoccia, J .-F.; Csizmadia, I. G. J. Am. Chem. SOC. 1991,113,62564265.
OOO2-7863/93/1515-4849$04.00/0 0 1993 American Chemical Society
4850 J. Am. Chem. Soc., Vol. 115, No. 11, 1993 Perczel et al.
0
dMODEL g dCRl l l CAL < A
Figure 1. (a) Sequenceof four successiveamino acid residues forming
a &turn backboneconformation. (b) A schematic representation of the
1 - 4 hydrogen bond in a &turn structure and the 7-A upper limit of
the critical distance (&, =d) whichassigns the secondary structural
elements according to the "classical" definition. Inmodel compounds
(e.g., For-Ala-Ala-NH2) the model distance (&del) isalways shorter
than the critical distance for a peptide with real Ci' and Cy atoms (c).
chart I
+i,l *i+2 topological code
TYPE
I -60 -30 -90 0 a a
0 c a
L L aL'L aL6L
L D ' L I D ' LdD
X I -60 120 80
1 x 1 -60 -30 -60 -30 aLaL
chart n
H2 02 H CH3 H4
I II \ / I
N2 N4
/ \ I II
H3 03
H CH3
ll
01
where none of the nine conformational minima had +values
around 0" while values around - 4 5 O (aL, tSD, or r,,) and around
45" (ao, tSL, or T~ conformations) were found. Therefore, we
propose to distinguishI3 the three different forms of type I and
type I1 &turns on the basis of +i +z. These conformations are
identified by the topological codes given in Chart I. The type I11
&turn is determined as a single turn of a 3 helix and has therefore
a single topological code only.
The smallest N- and C-terminal protected @-turn model,
incorporating two chiral amino acids, has the structure shown in
Chart 11.
In an N-formyl dipeptidamide model, the two amino acids
represent the second and the third residues of a &turn (cf. Figure
1). In this model the Hl-C'l bond of the N-terminal formyl
group is shorter by -0.5 A and the N4-H4* bond length of the
C-terminal amide group is shorter by 0.6 A, as compared to the
corresponding Cp- C'1 and the N4 - CP,4 bonds. Due to this
shortening, the distances dmdcl and may differ from each
other by no more than 0.6 A +0.5 A =1.1 A, depending on the
orientation of the two bonds (Figure 1).
Although the classification of 8-turns is traditionally made on
the basis of the backbone torsional anglevalues (4i , +i ), the degree
(13) Perczel, A.; Kajtar, M.; Marcoccia, J.-F.; Csizmadia, I. G. J. Mol.
Srrucr. 1991, 232, 291-319.
Figure 2. (Left) Hairpin conformation of a polypeptidechain. A
schematic illustration of anuntwisted &turn and a backbonetwisted by
T degrees is shownonthe right.
b"L b"L
Figure 3. yLyL conformation of For-L-Ala-L-Ala-NHz corresponding to
a rather twisted backbonegeometry ( T ~ ~ + : - ~ ~ + =167.8').
Table I. Different Types of &Turnsa in Peptides Identified on the
Basis of Experimental Studies6,8J I
type 41 $1 $2 $2 new code(s)b
I -60 -30 -90 0 a L a L , ~ L Y L ~
I' 60 30 90 0 ~ D ~ D , ~ D Y D , 4,
I1 -60 120 80 0 c L ~ D , ~ L Y D .
11' 60 -120 -80 0 W Y L ~ cDYL,
I11 -60 -30 -60 -30 QLCYL
111' 60 30 60 30 aDaD
IV ambiguously defined
V ambiguously defined
VIa -60 120 -90 0 ( L aL , ~ L Y L ,
VIb -120 120 -60 0 B L ~ L ~ B ~ Y L , ~ L ~ ~
VI11 -60 -30 -120 120 C&
VI1 ambiguously defined
"Only types I, 11, and I11havemore than sporadic Occurrencein
globular proteins according to X-ray studies. Indegrees.
of folding or unfolding of a @-turn can be defined in a simpler
way, on the basis of the twisting of the hairpin conformation. In
agreement with the pioneering work of Levitt? wehave recently
introducedI4 the CY - CP,, - C: , - CK3 torsional angle labeled
as T (see Figure 2) that describes the overall angularity of the
backbone conformation with values -180" I T I 180". The
global minimum (the yLr L conformation), for example, has T?L?L
= 168.4', which can quantitatively describe the degree of
unfolding (Figure 3). Considering the criterion that the Cy -
C: distance must be shorter than 7 A, only a fraction of the
@-turn conformations (-90" I T I 90") can be assigned as such.
For the first threeoftheeight different types (I-VIII) ofb-turns
(Table I), their mirror image conformations (types 1', 11', and
111' &turns) have also been suggested previously.6 Although
both the type I @-turn and the type I' @-turn conformations
incorporate only L-amino acids, they have conformationally
(14) McAllister, M. A.; Perczel, A.; Csaszar, P.; Csizmadia, I. G. J. Mol.
Szrucr., in press.
8- Turn Conformations Confirmed by Protein X-ray Data
chart m
01 H R H3
-300 40 8~ TL
J. Am. Chem. SOC., Vol. 11.5, No. 11, 1993 4851
do 8L YL
I I I I I
enantiomeric peptide backbones.i2 Therefore, the conformational
enantiomer of the aLuL conformation (type I [or 1111&turn) is
the a,u, conformation labeled traditionally as the type I [or
1111&turn. (Duplications reported in Table I originate from
the historic evolution of the concept; both the type I and the type
I11 &turns have an aLaL-like backbone geometry, but these were
derived from different sources and therefore are labeled differ-
ently.) The extraction of the remaining &turn conformations,
fulfilling the angularity (-90 5 7 I 90) or distance (d I 7 A)
criteria, is hardly possible on the basis of earlier approaches.
Even if more &turn conformations exist than can be detected by
analyzing a 4D-RamachandranI3-type potential energy hyper-
surface (PEHS), these structures, due to the rate Occurrence of
these conformations in globular proteins, cannot be extracted
from the X-ray data analyses of these proteins.
Multidimensional conformational analysis (MDCA)I5 pre-
dicted2 nine minima on the full Ramachandran map,16 E =
E(&#), where the torsional angles are defined according to
IUPAC-IUB17 convention as shown in Chart 111. (Let us assume
thatw, andw2areconstants;usuallywl andw2=180 or sometimes
O O ) . Each of these nine minima represents the only energy
minimum in a given catchment region.I8
Figure 4 shows the Occurrence of the nine minima (aL, aD, BL,
yu yo, 6L, 6,, eL, e,) in an idealized fashion. It should benoted
that according to the IUPAC-IUB conventioni7 the 4 and the #
values vary between -180 and 180O. This domain is indicated
by the dashed square in the center of Figure 4. For various
reas0ns,12.~3 during conformational analysis it is convenient to
use a different cut of the same potential energy surface (PES),
namely any of the four identical quadrants encircled by the solid
lines in Figure 4. With this choice of representation, the spatial
arrangement of the nine minima may be specified as shown in
Chart IV. Torsional angles and #TOP span the range from
Oo to 360 in accordance with the earlier suggestion,I2 while in
the IUPAC-IUB ~nventi on ~ these 4 and $ variables vary from
During the conformational analysis of PCONH-CHK-CON-
HQ using ab initio cal c~l ati ons,~~-~~ the absence of the aL and
(15) (a) Csizmadia, 1. G. Generaland theoreticalaspectsof the thiolgroup.
In The Chemistry of functional groups. The chemistry of the thiol group;
Patai. S., Ed.; Wiley and Sons: New York, 1974; pp 1-109 (particularly
pages 36-41 including Figures 23 and 24 as well as Table 20). (b) Bertran,
J. Multidimensional Theoretical Stereochemistry and Conformational Potential
Energy Surface Topology. In New Theoretical Concept for Understanding
Organic Reactiorw;Csizmadia, I. G. Ed.; D. Reidel Publishing Co.: Dordrecht,
1989: BD 1-31.
-180 to 180.
r r -
.. _ _ ,
(16) Ramachandran, G. N.; Ramakrishnan, C.; Sasisekharan, V. J. Mol.
(17) IUPAC-IUB Commission on Biochemical Nomenclature. Biochem-
Bi d. 1963, 7, 95-98.
istry 1970, 9, 3471-3479.
(18) Potential Energy Hypersurfaces; Mezey, P. G. Ed.; Elsevier Science
.- -
Publishers: Essex, 198-j.
(19) (a) Sellers, H. L.; Schafer, L. J . Am. Chem. SOC. 1978,100, 7728-
7729. (b) Schafer, L.; Sellers, H. L.; Lovas, F. J.; Suenram, R. D. J. Am.
Chem. Soc. 1980,102,6566-6568. (c) Schafer, L.; Van Alsenoy, C.; Scarsdale,
J. N. J . Chem. Phys. 1982,76,1439-1444. (d) Klimkowski, V. J.; Schafer,
L.; Momanay, F. A.; Van Alsenoy, C. J. Mol. Srruct. 1985, 124, 143-165.
(e)Scarsdale, J. N.; Van Alsenoy,C.; Klimkowski, V. J .; Schafer.L.; Momany,
F. A. J. Am. Chem. SOC. 1983,105,3438-3445. (f) Schafer, L.; Klimkowski,
V. J.; Momany, F. A.; Chuman. H.; Van Alsenoy. C. Biopolymers 1984,23,
(20) Viviani, W.; Rivail, J.-L.; Perczel, A.; Csizmadia, I. G. J. Am. Chem.
SOC., submited for publication.
2335-2347.
- 240t
eL peptide conformations was noted. By the calculation of several
dozens of relaxed grid points, the shape of the PES for For-
Ala-NH2 was recently analyzed in the vicinity of the uL
conformation, but no minimum was f0und.2~Nevertheless, the
shapeof thePES, in thearea where thea,conformationisexpected
to be, suggests that even a minor stabilizing force (such as a
hydrogen bond) could result in a local minimum, giving legitimacy
to the aL backbone structure. To the best of our knowledge,
various experimental methods, including X-ray crystallography,
have never identified the uL backbone conformation for any single
amino acid diamides, which agrees perfectly with the ab initio
results. Nevertheless, each of the nine minima specified in Chart
IV are legitimate both in terms of multidimensional conforma-
tional analysis and on the basis of X-ray-analyzed structures in
larger peptides and pr0teins.2~9~~ (The conformational oligomers
(u& and (e,),,, the so-called a-helix and poly-L-proline I1
secondary structural units, Occur frequently in globular proteins.)
However, the absence of the mentioned minima (aL and e,) in the
(21) (a) Head-Gordon, T.; Head-Gordon, M.; Frish, M. J.; Brooks, C., 11;
Pople, J. A.; In?. J. Quantum Chem. Quantum Biol. Symp. 1989, 16, 311-
319. (b) Head-Gordon, T.; Head-Gordon, M.; Frish, M. J.; Brooks, C., 11;
Pople, J. A.; J. Am. Chem. Soc. 1991,113, 5989-5997.
(22) Bohm, H.-J.; Brode, S. J. Am. Chem. SOC. 1991, 113, 7129-7135.
(23) Bertran, J. Peptide conformational Potential Energy Surfaces and
their relevance to protein folding. In Molecular aspect of biotechnology:
computational models and theories; Perczel, A., Viviani, W.. Csizmadia, I.
G., Eds.; Kluwer Academic Publishers Co.: Dordrecht, 1992; pp 39-82.
(24) McAllister, M. A,; Perczel, A.; Csaszar, P.; Vladia, W.; Rivail, J.-L.;
Csizmadia, I. G. J. Mol. Srruct., in press.
(25) (a) Aubry, A.; Marraud, M.; Protas, J .; Nccl, J. C. R. Acad. Sci.
Paris 1974, 287c, 163-166. (b) Aubry, A.; Marraud, M.; Protas, J.; Neel,
J. C. R. Acad. Sci. Paris 1973,276~, 1089-1092. (c) Aubry, A. These pour
Docteur de Sciences Physiques, Universite de Nancy, France, 1976,102-107.
(d) Aubry, A.; Marraud, M.; Protas, J.; Neel, J. C. R. Acad. Sci. Paris 1971,
273c, 959-961. (e) Aubry, A.; Marraud, M.; Protas, J.; Neel, J. C. R. Acad.
Sci. Paris l974,287c, 163-166. (f) Aubry, A.; Cung, M. T.; Marraud, M.
Cryst. Srruct. Commun. 1982,II, 129-133. (g) Aubry, A.; Marraud, M.;
Protas, J.; Neel, J. C. R. Acad. Sci. Paris 1974,287~ 697-700. (b) Aubry,
A.; Marraud, M.; Cung, M. T.; Protas, J. C. R. Acad. Sci. Paris 1985,280~.
861-863. (i) Aubry, A,; Protas, J.; Marraud, M. Acra Crysfallogr. 1977,
833, 2534-2539. (j) Aubry, A.; Protas, J .; Marraud, M.; Neel, J. Acta
Crystallogr. 1976, 832, 2749-2759.
4852 J. Am. Chem. SOC., Vol. 115, No. 11, 1993 Perczel et a/.
Table 11. Experimental Conformation of Selected Single Amino
Acid Diamides Obtained from X-ray Crystalloaraphy
amino confor-
acid N-terminal C-terminal QP + mation ref
MeCO-
PrCO-
MeCO-
MeCO-
MeCO-
MeCO-
MeCO-
BuCO-
BuCO-
BuCO-
-NHiPr
-NHiPr
-NHiPr
-NMe2
-NMe2
-NMe2
-NHEt
-NHEt
-NHMe
-NHMe
-78 160 CL
-1 12 142 tL
-169 175 B L
-1 26 162 B L
-92(-90) 123(122) CL
-132 77 6,
-9 1 144 tL
-9 1 144 CL
-72 165 CL
-92 151 EL
25a
25b
25c
25d
25e
25f
2%
25h
25i
25j
In degrees. There were two molecules of different geometries in the
unit cell. Side chain torsional angles are xI =70, x2 =88.
I
+
Figure 5. Illustration of the Grand Canyon region of a 2D-
Ramachandran map that includes the BL, yL, et, and 6, conformations.
The idealized conformations are denoted by open stars, while the arrows
indicate the approximate shifts of the ideal conformation to the actual
ones.
simplest peptide model (PCONH-CHR-CONHQ) could have
far-reaching consequences, resulting in the conclusion that
polypeptide backbone conformations cannot be modeled using
PCONH-CHR-CONHQ-type models. This would mean that,
from a conformational point of view, proteins cannot besimply
regarded as the polymers of -CONH-CHR-CONH-systems but
must beconsidered as built from larger substructures. According
to selected crystallographic data, amino acid diamides adopt
minimal energy conformations (Table 11) close to cL, Y ~ , and 6,
on the E =E(4, $) PES. These three minima are also close to
each other geometrically. These crystallographically determined
minima are located in a common region of a Grand Canyon
(as shown in Figure 5) that has been constructedz3 using Poples
ab initio energy contour diagram.2 Type I and I1 @-turns
incorporate the aL and tL conformations at the second [or ( i +
l)th] position of the hairpin conformation (see Table I). These
(26) (a) Aubry, A.; Marraud. M.; Protas, J . C. R. Acad. Sci. Paris 1975,
ZSOC, 509-512. (b) Aubry, A.; Protas, J .; Boussard, G.; Marraud, M. Acta
Crystallogr. 1977,833,2399-2406. (c) McLarfi, M.; Aubry, A.; Marraud,
M. Eur. Biophys. J. 1986,14,43-51. (d) Boussard, G.; Marraud, M.; Aubry,
A. Int. J. Pept. Protein Res. 1986, 28, 508-517. (e) Aubry, A.; Protas, J .;
Boussard, G.; Marraud, M. Acta Crystallogr. 1979,835,694-699. ( f ) Aubry,
A.; Protas, J .; Boussard, G.; Marraud, M. Acta Crystallogr. 1980,836, 321-
326. (g) Aubry, A.; Boussard, G.; Marraud, M. Acta Crystallogr. 1981,837,
1474-1477. (h) Aubry, A.; Protas, J .; Boussard, G.; Marraud, M. Acta
Crystallogr. 1980,836,2822-2824. (i) Aubry, A.; Protas, J .; Boussard, G.;
Marraud, M. Acta Crystallogr. 1980,836,2825-2827. (j) Aubry, A,; Lecomte,
C.; Boussard, G.; Marraud, M. J. Chim. Phys. Phys. Chim. Biol. 1983.80,
609-614. (k) Aubry, A.; Marraud, M. Acta Crystallogr. 1985, C41,65-67.
(I ) Aubry, A.; Vitoux, B.; Marraud, M. Biopolymers 1985, 24, 1089-1 100.
(m) Aubry, A.; Cungt, M. T.; Marraud, M. J. Am. Chem. SOC. 1985, 107,
7640-7647. (n) Aubry, A.; Ghermani, N.; Marraud, M. Int. J. Pept. Protein
Rb. 1984, 23, 113-122. ( 0) Milner-White, E. J .; Ross, B. M.; Ismail, R.;
Belhadj-Mostefa, K.; Poet, R. J. Mol. Biol. 1988,204,777-782. (p) Boussard,
G. Marraud, M.; Aubry, A. Biopolymers 1979,18,1297-1331. (9) Boussard,
G.;Marraud,M. J. Am. Chem.Soc. 1985,107,1825-1828. (r)Liang,G.-B.;
Rito, C. J .; Gellman, S. H. J. Am. Chem. SOC. 1992, 114, 44404442.
backbone geometries were not obtained from ab initio studies of
chiral diamide model ~I ~- ~~ (e.g., For-Ala-NHz or For-Val-NH2).
The conformational enantiomers of these @-turns, the type I and
the type 11 structures, incorporate not the unstable CY, and t L
subconformations but the stable aD and tD backbone orientations.
Several papers have been published9- I detailing results obtained
by using theoretical predictions and statistical analyses of @-turn
distribution frequencies in globular proteins. However, the
question of whether &turns may be considered not only as a
recognizable secondary structural element in proteins9 but also
as intrinsically stable conformational elements of simple triamides
has still not been answered. Although experimental evidence
accumulated from studies of model compounds (e.g., -Pro-Xxx-)
in apolar such as CC4, suggests that @-turn
conformations are the most enthalpically favored patterns for
several small peptides, the lack of gas-phase evidence may suggest
the following alternatives. It is quite possible that @-turn
conformations exist only in an environment created by solvation,
long-range interactions, intermolecular H-bonds, etc. On the
other hand, 8-turns may be intrinsically stable structures and
they may exist even in a vacuumwithout the stabilizing interaction
of any side chain or environmental effect.
TheX-ray crystallography of dipeptidederivatives (e.g., PCO-
Xxx-Yyy-NHQ) having a hairpingeometry (seeTable 111) shows
that the folded conformation is always stabilized by intermolecular
and/or environmental In solution the stabilizing
effect of solvents can never be excluded. Since gas-phase data
on tripeptide structures are currently not available, there are no
experimental data which prove or disprove the intrinsic stability
of @-turns. In the 1970s, Scheraga and others investigated @-turn
conformations using molecular mechanics cal cul ati ~ns.~~ The
structural analysis of Ac-Xxx-Yyy-NHMe-type peptides resulted
in useful geometrical data, although these force-field calculations
cannot answer the question of existence or nonexistence due
to the experimental origin of the parameters used. For example,
force-field calculations (e.g., ECEPP/2) for Ac-L-Ala-NHMe
resulted in a stable aL structure that must be regarded as an
artifact in view of recent ab initio cal c~l ati ons.~~J ~-Z~ Reliable
answers can only beexpected from high-level ab initio calculations.
Although some attempts have been made28 to include correlation
energy in the case of diamides of single amino aeids, the triamide
systems presently remain much too large for such sophisticated
calculations. Even at a lower level of theory, only a limited number
of computations have been published13,i423.29,30 on selected
triamide conformations. The more than two dozen optimized
dipeptide diamide geometries reported here should provide a
unique opportunity for analysis of the conformational behavior
of @-turns, which hopefully will lead to a more accurate
understanding of protein 3D-structures.
Computational Methods
The fully relaxed minimal energy conformations were calculated using
full geometry optimizations by gradient methods with the GAUSSIAN
90 program3 using the 3-21G basis seP2 on a Cray X-MP/28
supercomputer. This work is exploratory in its nature, and calculations
using a larger basis set at the HF or MP level of theory may beperformed
(27) (a) Zimmerman, S. S.; Scheraga, H. A. Biopolymers G77,16,81L
843. (b) Zimmerman, S. S.; Scheraga, H. A. Biopolymers 1978, 17, 1849-
1869. (c) Zimmerman, S. S.;Scheraga, H. A. Biopolymers 1978,17,1871-
1884. (d) Zimmerman, S. S.; Scheraga, H. A. Biopolymers 1978,17,1885-
1890.
(28) Frey, R. F.; Coffin, J .; Newton,S. Q.; Ramek, M.; Cheng, V. K. W.;
Momany, F. A.; Schafer, J. Am. Chem. Soc. 1992, 114, 5369-5376.
(29) Chesnut, D. B.; Phung, C. G. Chem. Phys. Lett. 1991,183,505-509.
(30) Sapsa, A.-M.; Daniel, S. B.; Erickson, B. W. Tetrahedron 1988,44,
(31) Frisch, M.; Head-Gordon, M.; Trucks, G. W.; Foresman, J . B.;
Schelegel, H. B.; Raghavachari, K.; Robb, M. A.; Brinkley, J . S.; Gonzalez,
C.; Defrees, D. J .; Foz, D. J .; Stewart, J . J . P.; Topiol, S.; Pople, J . A,;
GAUSSIAN 90(Revision F Version); Gaussian Inc.: Pittsburgh, PA, 1990.
(32) Binkley, J . S. ; Pople, J . A.; Hehre, W. J . J. Am. Chem. SOC. 1980,
102,939-947.
999-1006.
8- Turn Conformations Confirmed by Protein X-ray Data
Table 111. Selected Experimental Conformations of Dipeptide Derivatives Determined by X-ray Crystallography
J. Am. Chem. SOC., Vol. 115, No. 11, 1993 4853
amino acids N-terminal C-terminal @ l a *I 42 *2 &turn type conformation ref 26
Pro-Gly BuCO- -NHMe -7 1 157 -76 175 ~ L L a
Pro-Ala PrCO- -NHlPr -59 136 66 14 I1 CLQD b
Pro+ Ala IPrCO- -NHPr -62 137 96 3 I1 C L ~ D b
Pro- Asp BuCO- -NHMe -57 134 59 26 I1 C L ~ D C
Pro- Asn BuCO- -NHMe -59 138 66 11 I1 C L ~ D C
Pro-Asp(0Me) BuCO- -NHiPr -66 -20 -9 1 6 I C
Pro-His BuCO- -NHMe - 63 -22 -7 0 -20 I a L h d
D- Ala-Pro BuCO- -NHI Pr 60 -140 -89 9 11 ~ D Y L e
D- Ala-D-Pro BuCO- -NHIPr 64 -152 83 -156 WD f
Gly-Pro BuCO- -NHMe -79 174 -8 5 -22 VIa C L ~ L g
Pro-Gly BuCO- -NHIPr -64 137 84 -3 I1 CLYD h
Ala-Pro PrCO- -NHPr -129 76 -67 -22 VIb ha, i
Gly-Gly BuCO- -NHPr -69 -25 -8 9 3 I a L a 1 j
Ala-Gly BuCO- -NHI Pr -68 132 -8 3 2 VIa C L ~ L j
Pro-Thr BuCO- -NHMe -66 -22 -103 7 I ~ L Y L k
Pro-Pro BuCO- -NHMe -60 138 -9 5 -7 VIa C L ~ L 1
Pro-D-Pro BuCO- -NHMe -58 134 83 -7 I1 CLYD I
Pro-Pro BuCO- -NHMe -60 138 -9 5 -7 VIa C L ~ L m
Pro-Cys(Me) BuCO- -NHMe -6 1 132 62 17 I1 C L ~ D m
Pro-Phe BuCO- -NHMe -64 139 62 23 I1 f LaD m
Pro-Tyr BuCO- -NHMe -59 137 73 9 I1 ~ L Q D m
Pro-D-Tyr BuCO- -NHMe -64 137 73 9 I1 C L ~ D m
Pro-Ser BuCO- -NHMe -60 -30 -7 5 -1 1 I ( Y L ~L n
Pro-D-Ser BuCO- -0Me -64 -29 76 17 a~% n
Pro-D-Ser BuCO- -NHMe -59 133 76 8 I1 1 f f D n
In degrees.
Table IV. Comparison of the Q and $ Torsional Angles Obtained in
ab Initio Calculations under Normal and Tight Optimizations on
Selected Conformations of For-L-Ala-NHz and For-L-Ala-L-Ala-NH2
torsional anglea
compd conformation type normalb tightC
For-Ala-NH2 6 Q 63.8 63.8
* 32.7 32.7
+ 170.9 170.5
* 67.7 67.3
* 67.0 67.0
* 66.4 66.4
BI 4 -168.4 -168.3
71 Q -84.4 -84.5
For-Ala-Ala-NH2 y, yI Q -84.2 -84.2
Cp -84.9 -85.0
change
0.0
0.0
0.1
0.4
0.1
0.4
0.0
0.0
0.1
0.0
a In degrees. Max force <3 X au. Max force 51 X au.
in the future on selected minima. The authors are fully aware of the
limitations of the 3-21G basis set, although work by Pople and
co-workers21albindicates that in the case of peptides even the 3-21G basis
set is sufficient. The initial geometries of the selected triamides were
generated on the basis of the previously optimized For-~-Ala-NHz
structures.12 The For-~-Al a-~-Al a-NHz geometries were then subse-
quently optimized. The final forces along the internal coordinates in the
relaxed structures ranged from 2.2 X to 2.1 X 10-4 au, while the
value of the root mean square of the forces (rms) was between 5.4 X lo-
and 5.7 X au. One may be concerned about the convergence of
geometry optimization under these normal conditions. The ultimate test
of accuracy, in this particular case, is not so much the magnitude of the
residual force but the self-consistency of the 4 and +values as these
torsional angles characterize peptide conformations. In order to check
this point, normal and tight optimizations have been carried out on
selected conformations. As may beseen from Table IV, the absolute
value of torsional angle changes, Le., IAq51 and lA+I. are very small. Since
the geometry-optimized q5 and * values are needed in the present paper
mainly to determine which catchment region a computed conformation
belongs to (these characteristic conformations are separated from each
other by several tens of degrees), the achieved accuracy is more than
adequate (Table IV).
The y, yl conformation was found as the global minimum (E[RHF]
=-656.963 681 hartrees) and was used as reference point for AE
calculations.
scope
Several different types of criteria (such as distance, torsional
angle, or the 1 - 4 hydrogen bonding) have been used to identify
Table V. Optimized ab Initio SCF (3-21G) Geometries for Type I
&Turn Conformations of For-L-Ala-L-Ala-NH2
initb
Wnvc
01
41
*I
42
$2
w3
&del d
Tmcdel
&rite
Tcrit
0 1-HN4
01-*N4
01-NH4-N4
01-HN3
01***N3
01-HN3-N3
02-HN4
02--N4
02-NH4-N4
w2
max force
rms force
E
AE
SDV
LDBg
Q D ~ D
( Y D ~ D
171.2
60.4
28.3
179.0
62.3
24.9
179.1
5.33
-66.9
5.97
-70.8
2.03
3.03
172.4
3.03
3.10
3.06
3.23
91.1
6.5 X
0.956 139
4.73
28
77
-84.5
1.8 x 10-5
~ D Y D
67,
173.5
62.3
37.0
74.1
174.9
6.06
-79.3
6.64
-80.4
3.76
4.00
97.1
3.20
3.27
1.93
2.83
147.1
1.1 x 10-4
0.950 869
8.04
3
10
-172.9
-58.0
-85.3
3.6 x 10-5
4,
171.0
64.1
16.8
-175.6
150.8
-40.0
-174.2
4.30
-11.0
4.64
-12.7
2.16
3.11
160.3
2.87
3.05
90.7
4.10
4.61
114.9
8.4 x 10-5
2.7 x 10-5
0.949 9 15
8.64
1
4
d l
%Bl
176.2
60.2
33.0
-177.5
-173.6
169.8
177.5
7.03
61.6
7.42
57.8
6.42
6.06
-64.5
3.18
3.19
5.1 1
5.16
-81.5
-87.1
3.6 x 10-5
1.1 x 10-5
0.954 609
5.69
26
72
Torsion angles ( w. 6, $) in degrees, distances in angstroms, forces
in au, energy (E) in hartrees, and energy differences (AE) in kcal/mol
relative to E(yLyL) [-656.963 681 hartree)]. Initial backbone confor-
mation (calculated by ECEPP/2). Converged backbone conformation.
Cp and CP,, in accordance with classical &turn definition must be
shorter than 7 A. In For-Ala-Ala-NH2 the two Ca atoms are replaced
by hydrogens (H1 and H4*) (cf. Chart 11); therefore, the model distance
(dmdeJ is shorter than CP - CP,, (Figure 1) by no more than 1.1 A. In
such a case, T is H1-C2a-C3a-H4. e Critical distances for j3-turn
assignment (Cp - CP,,) were extrapolated using ab initio calculated bond
lengths and bond angles on the basis of the determined N-H and C-H
distances. In such a case, T is Cla-C2*-C3*-C4. /Small Data Base
(for the list of nonidentical proteins, see ref 13). g Large Data Base (for
the list of proteins, see ref 13).
twisted hairpin conformations since Vankatachalam.6 We now
use an objective measure, recently defined14 and based on the
angularity (or twisting) parameter 7, in agreement with the work
4854 J. Am. Chem. SOC., Vol. 115, No. 11, I993
Table VI. Optimized ab Initio SCF (3-21G) Geometries for Type I and Type. 11' ,!?-Turn Conformations of For-L-Ala-L-Ala-NHf
Perczel et al.
init
conv
$1
$1
42
$2
a3
dmodei
Tmodel
&it
Tcrit
01-*HN4
01 --N4
01-HNbN4
01*.*HN3
0 1 *-N3
0 1 -HN 3-N 3
02-*HN4
02-*N4
02- HNbN4
WI
W2
max force
rms force
E
AE
SDB
LDB
&&, a L a L ,
a L h
-171.7
-68.6
-17.5
177.8
21.3
176.0
4.83
41.1
5.32
43.7
2.1 1
3.07
162.5
3.01
3.15
3.50
3.98
111.9
5.8 X 10"
0.958 677
3.13
129
435
-113.1
-89.0
1.5 x 10-5
&& ~ L c L ,
-175.9
-121.3
17.6
176.4
169.6
178.1
7.25
19.5
7.56
20.8
7.55
6.98
-51.9
3.63
4.12
112.7
5.10
5.15
-169.4
-87.31
5.6 x 10-5
1.6 x 10-5
0.956 57
4.46
14
37
&&,
6 L 6 D
-172.8
-126.8
23.8
171.1
-45.6
-176.5
5.66
-9.9
5.95
-5.6
5.49
5.74
99.4
3.55
4.15
121.2
4.55
4.85
101.8
-173.7
8.7 x 10-5
2.7 x 10-5
0.945 712
11.28
4
9
c D L c D ~ L
cD&
-177.4
56.1
177.2
26.6
176.5
5.29
5.82
-36.9
1.99
2.98
169.9
3.33
3.21
-74.6
3.34
3.89
115.9
1.4 X 10-4
0.955 958
4.85
1
1
-129.7
-1 12.4
-35.0
4.9 x 10-5
CDYL
CDYL
-171.9
64.0
-172.8
-175.3
-86.0
66.2
-176.2
5.99
-36.7
6.52
-41.5
3.58
4.19
121.6
4.46
4.09
-62.1
2.05
2.90
141.9
1.3 X lo4
0.952 062
7.29
1
8
4.6 x 10-5
~ D Y D
CDYD
-171.3
-178.1
66.9
174.9
75.8
-86.0
-177.7
6.97
71.3
7.52
69.5
5.04
5.91
148.6
4.64
4.23
-59.9
1.91
2.80
146.5
1.8 X lo4
0.947 257
10.31
1
8
3.9 x 10-5
W D
C D ~ D
-169.6
-178.2
-166.1
-170.6
-179.0
67.7
63.7
8.00
77.8
8.56
75.4
7.79
7.14
-71.7
4.72
4.28
4.61
4.02
-62.1
8.0 X 10-6
2.8 X 10-6
0.940 468
14.57
1
1
-58.2
Units, abbreviations, and parameters are the same as those used in Table V.
Scheme I
type I ' 8-turns
'DID ' D ' D 'D~D " D ' D
INITIAL CEOIIFIRIES FINAL CEOM!ZTFlIES
--------> FI type XI' 8-turns
%' L
INITIAL GEOM!ZTFlIES FINAL GEOMETRIES
of Levitt,g* such that for a reverse &turn the value of this T
(torsional angle involving Cq, C>l, C>2, C$3 peptide backbone
atoms) must be in the -90" 5 T 5 90" range. The following
problems were studied: (1) How many of the 81 legitimateI3
backbone conformations of a triamide (e.g., For-Ala-Ala-NH2)
would qualify as 8-turns? (2) Can ab initio molecular orbital
(MO) computations indicate whether a @-turn has intrinsic
stability? If yes, which of the total of 8 1 legitimate minima have
such an "intrinsic" stability? (3) Is a 1 - 4-type intramolecular
H-bond a necessary structural condition for 8-turns? If not,
which types of &turns have a 1 .- 4 hydrogen bond and which
do not have such a bond? (4) Can any of the "intrinsically
unstable" cy, and/or eL diamide conformations be stabilized and
therefore included in a &turn?
Results and Discussions
In the first part of this study type I' and type 11' @-turn
conformations were analyzed ((YDcYD, (YDYD,
These conformations were expected to be minimal energy
geometries on the basis of previous conformational analyses of
ab initio surfaces.12 The computed results are given in Tables
V and VI (cf. Scheme I).
All three relaxed conformations containing an cyD-typegeometry
(LYDCYD, LYDYD, a&) at the first position are typical type I' 8-turns,
althoughonly thecyDcyD structureincorporatesa 1 -4-typeH-bond
(Table V and Figures 6 and 7). The conformational parameters
of eDy, did not change qualitatively during the optimization, and
the structure remained a @-turn. Only the optimization of the
eDa,-type initial geometry resulted in a qualitatively new backbone
(DaL, cDYL,
Scheme II
pzq --------> 'L 'L ~ ' L ~L
' L' L
INITIAL GEOMETRIES FINAL GEOIIFIRIES
conformation (eDSL) which is a more "open" conformation (Scheme
I). The original t D a L conformation with ~ , D . L =6.47 A and T,D@L
=-55.3" values fulfills both the distance (d I 7 A) and the
angularity (-90" I T I 90") criteria of a &turn, as does the eDbL
geometry ( d r ~ d ~ =5.82 A and T, D~ L =-36.9"). On the basis of
an earlier molecular mechanics (MM) study on dipeptide
diamides,13 two more conformations (cDyD and cDcD) among the
type I'and type 11'8-turns were also incorporated in the analysis.
Although the d values of the e D y D and eDeD conformations are
longer than expected for a conventional @-turn ( d t D7 D =7.52 A
and d,D,D =8.56 A), on the basis of the angularity of the backbone
conformation (T value) both of these are hairpin conformations,
while the T r ~ + =69.5" and T,D& =75.4" values are smaller than
90" but larger than -90", respectively.
In contrast to the above @-turns, the type I @-turns (CYLOLL~ OLLYL,
CYL ~L ) as well as the type I1@-turn conformations (CLCYD, BLYD, e L b D )
have an intrinsically unstable aL or eL conformation as their first
residue. Starting with the ab initio geometry optimization for
the type I 8-turn conformation, using geometries obtained from
molecular mechanics, neither the aLaL nor the aLy, conformations
were found to beminimum energy structures (Scheme I1 and
Table VI).
The aLSL structure incorporates a stabilizing intramolecular
H-bond, which may suggest that all aL conformations must be
stabilized with a hydrogen bond as found in the aLbL conformation.
On the basis of the topological analysis of an idealized PES2J 3
or a MM calculation associated with a diamide system, three
different type I @-turn structures ((YLcYL, CYLYL, L Y L ~ L ) are expected.
These three different backbone conformations were also confirmed
by the backbone analyses of X-ray determined protein structures
(Table VII); 11 13 Occurrences for the aLcyL, 14 occurrences for
the cyL?,, and 129 occurrences for the ( ~ ~ 6 ~ substructures were
found in the Small Data Base." The present finding that only
the aLbL conformation is a minimum energy structure is congruent
with previous ab initio calculations, as this is the only (YLxL or CYLXD
J. Am. Chem. SOC.. Vol. 115, No. 11, 1993 4855
-IZOJ I
-180 I ,
, I ,
4 5 6 ; I -9- 10 l l
C R I T I C AL DIST. ( A)
-180 ~ , I , I 1 180
4 ' 4 5 6 7 8 Ib Ill
Figure 6. (a) Critical distance (d,,,,) vs T (the angularity of the
conformation) of the 75(81) Ac-Ala-Ala-NHCH, conformations, cal-
culated by the ECEPP/2 method. (The six conformations marked by
*, shown among the 81 symbols in this figure caption below, are the
annihilated conformations [for details, see refs 13 and 141) represented
by 0 on the top portion of this figure. Conformations incorporating 1
- 4-type H-bonds are plotted as A. The numbers and the conformations
they represent are as follows: 1, 5, a D6 L ;
C R I T I C AL DI S T ( A )
2, a D a L ; 3, adB,; 4,
6, ai ~i ; 7, 8, ~ D Y D ; 9, ~ D Y L ; 10, a L a D ; 11, a L a L ; 12, aL @L ; 13, aL 6D;
14, ai6i; 15, ai~i); 16, ~ I Y , ; 17, ~ L c L * ; 18, ~ L Y D ; 19, B L ~ D; 20, B L ~ L ; 21,
6oaI; 30, 6di ; 31,6&; 3276dL; 33,6D~D; 34, 35, ~ D Y D ; 36, ~ Y L ; 37,
61~~1,; 38, 6iai; 39, 6i h; 40, 616,; 41, 42, hie,, 43, 6ifu 44, ~ L Y D ; 45,
CI,YII; 54, %YI; 55, Cia,; 56, CL ~L * ; 57, Y L ~ L ; 58, CL@L; 599 e~6D.i 60, L~L*;
61, 1 CD*; 62 ~l ~l ; 63, c I YD; 64, cLYL; 65, Y D ~ D ; 66, YDG 67, YDBL; 689 Y D ~ ;
69, Y I J ; 709 Y n G 71, ~ ~ 6 1 ; 72, YDYD; 73, YDYL; 74, Y L ~ D ; 75, YLBL; 76, Y L ~ D ;
77, ~ ~ 6 , ; 78, yIt D, 79, yIeL; 80, ~ ~ 7 ~ ; 81, -yLyL. (b) d,,,, vs T distribution
of the 30 hairpin conformations of For-Ala-Ala-NH2, calculated by the
ab initio method. Note that according to the original definition, based
on the critical distance9 only 18 of the 30 conformations are @-turns and
only 5 (aI,aI, [type HI'@-turn], a D 6 D [type 1'0-turn], aL 6L [type I &turn],
c1,6, [type 11' @-turn], and ~ ~ 6 , ) have the 1 - 4-type H-bond (tus).
&@I; 22, @I&); 23, 8161; 24, BIG 25, B L t L ; 26, BLYD; 27, B L Y G 28, 29,
6171; 46, Cl)a,; 47, Co al ; 48, CD@L; 49, C D ~ D ; 50, C D ~ L ; 51, CDCD; 52, CDt L*; 53,
conformation of For-Ala-Ala-NHz which has an intramolecular
H-bond (XL stands for a ~ , &, y ~ , b ~ , and EL; XD stands for CYD,
y ~ , 6 ~ , and e ~ ) . The present ab initio calculation resulted in the
aLbL (type I &turn) conformation =-69', $,+I =-18', 4 +2
=-113,$1+~=2l0),whichisremarkab1ycloseto thatpredicted
by Vankatachalam =-60, $ i + ~ =-30, &+z =-go', tJ1+2
=0'): On the basis of molecular mechanics cal c~l ati ons,~~J ~
three additional 8-turn conformations (adL, aL 6D, and OILEL), each
containing the aL subconformation, may also be minimal energy
~tructures.~3,~~ The aLBL conformation was recently assigned to
be a &turn by Wilmot and Thortonll on the basis of protein
X-ray data analyses, where the torsional angles are close to #,+I
=-60, =-30, q$+2 =-120, $,+2 =120'. The present
ab initio calculations do not confirm the existence of such an a$,
or aLeL conformation for For-Ala-Ala-NH2. These two initial
conformers were shifted to 6dL, (Table VI). Although this is a
qualitatively new backbone conformation (&fL =-121 O,
$:ifL =18', &fL =-169O, #f)tL =170), it has never been
Table VII. Frequency of Type I and I' as Well as Type I1 and 11'
@-Turn Structures in Selected Proteins"
Type I
a L a L b ~ L Y L a L &
1113 (8103) 14 (62) 129 (435)
Type I'
Type 11'
15 (43) 1(8) 1(1)
"Frequency values are for the Small Data Base (SDB) and in
parentheses for the Large Data Base (LDB) (see ref 13). aLaL triamides
may participate in the a-helix, the 3,o helix, or the type I @-turn.
Scheme III
Fi --------'
INIIIAI. GEOMETRIES FINAL GEOMETRIES
assigned previously as a 8-turn. It fulfills the angularity criteria
of the hairpin conformation with T =20.8'. A similar shift of
the aL6, was also observed to bea 6,6, &turn conformation. This
second structure seems to be a "perfect" @-turn on the basis both
criteria, with a critical distance significantly shorter than 7 A (d
=5.95 A) and a backbone angularity rather close to 0 (T =-So).
Due to its relatively high energy compared to the L y L confor-
mation (AI3 =11.28 kcal/mol), only sporadic Occurrence is
expected, as confirmed by X-ray analyses (Table VI). The
appearance of 4 (9) representatives of 6,6, in the Small Data
Base (SDB) and Large Data Base (LDB)l3confirms the instability
of the calculated conformation. Since it is unreasonable to expect
that any type of statistical analysis based on protein X-ray data
will reveal the existence of such a rare &turn, the present
theoretical results are of unique value.
According to Table I, all three forms of the type I1 @-turn
backbone conformation incorporate an eL substructures in their
i+l positions. In all optimizations the eL conformation of the
first residue has been shifted to the more stable 6, conformation
(Scheme 111). In contrast to preliminary molecular mechanics
investigations,13J4 none of these three eLxD conformations were
found to be minima for For-Ala-Ala-NH2 according to our ab
initio calculations.
All three type I1 ,%turn (t ,~,) structures were shifted to the
corresponding Sg, minima on the 4D-Ramachandran type map
(Scheme 111). Two of the three new backbone conformations
and 6DyD) aredistorted and becomeextended conformations.
Such a backbone angularity change is authentically monitored14
by the shift AT(~,cY, - aDaD) =-152' - -18' =134" and AT-
(cLYD + 6 D y D ) =-163' + -66' =+97'. Although the third
conformational switch (~~6, --c 6,6,) resulted in a new backbone
category, it still may be regarded as a 8-turn conformation (daD6D
=6.87 A and T6D6D =-51.4'). Of the three most important
B-turns, type I ( a L a L , ~ L Y L , aL U type 11(eLaD, ~ L Y D ~ and
type I11(aLaL), only one (aLaL) was found to be a minimal energy
conformation in the ab initio calculations. Using only the X-ray
determined 8-turn geometries as input conformations for ab initio
studies, even with their mirror images [type I'(aDaO, ~ D Y D , CY&,
type 11' ( c D~ L , cDYL, ED&), and type 111' (aDaD) @-turns], no more
than seven 8-turn-like backbone conformations can becalculated
for For-Ala-Ala-NHz (aDaD, ct,~,, aDbD, eDyL, t D y D , eDe,, and (~~6,).
T U Wr. Ab Initio SCF (3-21G) FTum Conformations of For-L-Ala-L-Ala-NH2 Predicted by Multidimensional conformntional Annlysce.
init BLW
WOV BLUD
0 1 179.2
41 -167.6
$1 168.4
01 172.1
h 62.1
h 35.3
0 3 179.1
d d 7.13
T r y 49.2
dail 7.56
Tai l 49.0
01-HN4 5.96
01-N4 6.69
01-HNCN4 134.8
01-NH3 5.06
01-N3 5.15
OI-NH3-N3 -89.7
02-NH4 3.20
02-N4 3.24
02-NH4-N4 -83.0
BL~D
BL-
178.0
-169.0
172.4
-171.5
65.7
-175.5
-179.2
8.43
40.2
8.83
42.8-
8.81
8.17
-47.3
5.05
5.18
91.6
4.55
4.14
-59.8
BLrO
BLrO
179.2
-167.2
168.4
172.1
75.6
-57.2
-178.2
7.27
28.0
7.73
31.1
6.29
6.%
128.8
4.99
5.13
92.7
1.93
2.82
146.7
BLTL
BLTL
178.9
-167.7
169.3
-177.5
-85.1
68.2
-178.8
7.67
-83.0
8.22
-83.0
6.53
7.16
125.5
5.08
5.15
-88.8
2.04
2.89
140.7
WL
WL
176.6
-176.6
-43.6
-178.5
-167.4
170.2
177.7
7.74
-16.2
8.00
-18.4
8.72
8.20
-55.7
4.57
4.87
102.1
5.09
5.14
-87.2
ab
aobD
174.2
178.0
-45.6
178.7
-172.9
-49.7
-173.0
6 d D
6.54
-51.5
6.87
-51.4
6.23
6.65
110.7
4.41
4.84
109.8
4.61
4.87
99.7
b L '
b L
175.2
-174.2
154.1
171.7
179.7
6.36
45.2
6.42
43.3
8.1 1
7.37
-39.0
4.46
4.85
107.7
4.53
4.31
-7 1 .O
-55.0
-79.0
aDa,
awL
b 7 L
174.7
178.9
-44.3
-173.2
-85.5
68.8
-179.3
6.89
85.2
7.23
82.2
6.63
7.01
108.8
4.45
4.85
108.0
2.04
2.88
140.3
bD'
OLcD
-177.0
-161.8
55.7
-152.1
62.6
-173.8
-179.1
6.47
-62.3
6.60
-59.8
8.05
7.33
-40.7
4.01
4.57
I 18.8
4.56
4.18
-61.7
d L
d L
175.0
76.1
-5 1.2
-178.7
-161.6
169.2
177.6
7.06
-0.5
7.51
-4.7
6.23
5.54
42. 5
2.01
2.88
144.3
4.95
5.04
-89.2
r D b
WD
174.1
73.8
-57.6
178.9
-170.5
4 5 . 4
-179.3
5.81
46. 7
6.34
-47.3
3.19
3.62
107.7
1 .88
2.81
151.8
4.62
4.89
99.7
roak
rDh
roaL
172.0
75.5
-52.7
-171.9
-122.1
22.9
176.9
5.34
40.4
5.77
38.3
3.61
4.20
120.1
1.91
2.83
149.5
3.54
4.06
114.5
W L
YDfL
-177.2
72.6
46. 7
160.6
-73.7
168.8
179.0
5.81
57.0
6.09
53.2
5.99
5.34
-45.9
1.90
2.80
147.8
4.35
4.12
-70.1
YWL
*/DyL
174.3
73.8
-58.3
-173.7
-83.6
67.1
-179.4
6.33
84.4
6.80
80.6
5.26
5.28
-85.3
1.90
2.81
149.1
1.99
2.85
142.3
YLao
-172.4
-85.8
64.0
176.6
62.4
33.0
178.9
5.50
-65.5
5.97
-64.9
3.76
4.64
148.5
2.01
2.88
143.6
3.21
3.27
-84.7
nb
-177.5
-79.3
75.8
-173.6
176.7
-35.2
-178.2
5.28
43.7
5.84
47.0
2.20
3.14
-157.4
2.07
2.82
130.4
4.58
4.89
102.0
' WD
n-
176.5
-80.9
-1 56.8
64.3
-176.5
-178.9
5.54
-51.2
5.70
-47.9
5.90
5.27
-47.1
1.99
142.8
4.62
4.22
-60.2
75.8
2.85
r&
- d L
-176.1
-86.8
71.4
-179.7
-164.0
168.6
177.8
7.66
77.7
8.25
78.4
6.40
5.74
-45.0
2.16
2.96
135.4
4.99
5.07
-88.9
nm nn
-174.3 -174.4
-84.5 -84.2
68.6 67.0
72.7 -84.9
176.6 -174.1
-51.3 66.4
-177.6 -1789
6.27 7.92
413.5 167.8
6.74 8.76
-79.9 168.4
5.22 5.57
5.33 5.71
90.7 93.1
1.98 2.02
146.6 142.4
1.90 2.00
146.9 142.6
2.87 2.88
2.79 2.86
maxfora
ma, force
E
AE 6.91 8.87 4.34 1.65 7.47 14.33 5.88 7.00 6.11 6.87 8.73 6.11 6.57 2.11 5.09 4.53 5.40 4.17 2.34 0.00
1.5 X 104 2.1 X 104 1.7 X 104 LOX IW 4.7 X IW5 3.1 X IO-' 1.3 X I@' 1.2X 104 1.1 X 1 0 4 1.1 X lW 1.6X 104 6.7 X le5 1.6X 104 6.0X IW5 1.2X 104 1.2X 104 1.7X lW 3.3X 10-5 1.6X l(r 6AX l(r
3.6 X 10-5 4.5 X 10-5 4.4 X 10-5 2.7 X 10-5 1.5 X 10-5 9.7 X I&' 3.8 X 10-5 3.3 X 10-5 2.9 X 1W5 3.0 X IPS 3.6 X 1 V 2.6 X 4.0 X les 3.1 X IO-' 5.1 X 10-5 1.0 X 10-5 4.0 X l(r 2.1 X 105
0.952 663 0.949 539 0.956 752 0.%1 052 0.951 769 0.940 844 0.954 315 0.952 531 0.954 077 0.952 732 0.949 771 0.953 949 0.953 218 0.960 326 0.955 569 0.956 465 0.955 072 0.957 037 0.959 946 0.963 681
4.7 X IC5 1.9 X
SDB 12 9 1 20 3 4 9 2 1 1 1 1 2 0 9 2 1 38 9 a
41 113 19 LDB 35 31 3 83 I 1 29 12 5 3 5 3 6 13 5 19 16 5
=163.1O. CThe &*D conformation contaihpan 8-membrrad intramolecular H-bond, where 03-H2 =2.07 A and 03-N2 =2.99 A withan H-bond angle(03-H2-N2) =151.2'.
Units, abbreviations. and parameters arc the same as those used inTableV. ' The ~ DCL conformation contains an 8-membered intramolecular H-bond, whcre 03-H2 =2.07 h, 03-N2 =3.05 A with an H-boad angle (03-H2-N2)
fl-Turn Conformations Confirmed by Protein X-ray Data J. Am. Chem. Soc., Vol. 115, No. 11, 1993 4851
b ead b r e d
b l g d
b l g t
d l b e d l d d
Figure 7. Continued
4858 J. Am. Chem. Soc.. Vol. 115, No. 11, 1993 Perczel et al.
Figure7. All 30 &turn conformations resulting from ab initio calculations.
By contrast, MDCA considerations predict a significantly larger
number of @-turn geometries. Using molecular mechanics
(ECEPP/Z), 26 @-turn conformations are expected if the stricter
d =7 A criterion is accepted, and a total of 36 different backbone
orientations are predicted if the -90' I T 5 90' threshold value
is applied. These preliminary considerations were extremely
useful, while abinitiocalculations resulted in 18 @-turns according
to the stronger distance criteria and an additional 12 (total of 30)
(Figure 6b) according to the backbone angularity criteria (-90'
<7 <90'). Selected conformational parameters for the 30 @-turn
backbone conformations are listed in Tables V, VI, and VIII.
The yLaD conformation has a folding pattern similar to that
of aL6,, but the middle amide plane is twisted by - 150' (Figure
7). The critical distance is somewhat larger in y L a D (d =5.97
A) than that found in aLgL (d =5.32 A), but the similar absolute
value of 7 in both conformations ( T ~L ~L =44', T-,L~D =-65' )
reflects a highly similar degree of folding. This conformation is
relatively close to a type 11@-turn. If @-turns are evaluated on
the basis of their degree of refolding (T =0), then the y D@L
conformation will be the most perfect one ( T ~D ~L =-4.7') (Figure
6b). Such a folding pattern (+,+I 80, J/i+l =d o o , 4i +2 =
-160, J/i+2 =160), assigned and labeled here as a @-turn for
the first time, was also found in globular proteins (see Table
VIII), despite its small probability.
conformations (Table VIII) are at least 1 A shorter than the
previously defined 7 A as an upper limit for @-turns. On the basis
of the backbone angularity value ( 7) , these conformations are
perfect hairpin geometries (Figure 7). By contrast, the 1 - 4
intramolecular H-bond is missing in all of these conformations,
strongly suggesting that for 8-turn-like geometries such an
interaction (1 - 4 H-bond) is not a necessary condition. It seems
that such an intramolecular H-bond is necessary where the 8-turn
structure incorporates the a, conformational subunit. It is also
interesting to note that two different @-turn conformations, 6,y,
and yDyL (Table VIII), result in very similar dcrit values as well
as almost identical T values, daD,L =7.23 A, dyDtL =6.80 A and
76DyL 82', TyDyL =81, respectively. This suggests that more
than a single combination of the subconformations may result in
the same degree of hairpin twisting.
Up to the present, ab initio calculations have shown that, due
to the unfavorable eclipsed interaction of the amide proton and
the@ carbon atoms (H-N-Cu-CB torsional angle is approximately
-20), the cL backbone conformation is unstable. (As published
The critical distances in the a, &, , ?,eD, yLaD, yD6,, and
previously,lzJ9-24 the cL backbone conformation was annihilated
in For-Gly-NHz, For-Ala-NHZ, and For-Val-NHz.) The &EL
and yDc, relaxed conformations (rms forces 3.8 X 10-5 and 4.7
X au, respectively) reported herein are unique exceptions
(see Table VI11 and Figure 7). There is no direct interaction
(like an intramolecular hydrogen bond) between the third and
the second amide groups of the molecule oriented in the t L
conformation. However, it is presumable that indirect effects
may influence the 6- polarity of the carbonyl oxygen in the central
amide. The increased 6- charge on the oxygen can stabihe the
eL conformation of the "second half" of the moiecule. This
speculation, however, must beinvestigated by ab initiocalculations
using larger basis sets and taking correlation effects into account.
Conclusion
For the first time, ab initio-type calculations on the multidi-
mensional conformational problem of dipeptide diamides resulted
in a complete set of relaxed @-turn conformations of For-Ala-
Ala-NH2. The geometries are intrinsically stable hairpin con-
formations. It was shown that if an a,substructureis incorporated
in a @-turn conformation (e.g., a,gL in type I @-turn), a favorable
H-bond interaction is required to stabilize such a @-turn. By
contrast, for @-turns not containing an a, conformational subunit,
the existence of such a 1 - 4 H-bond is not required. Thus, while
the aDaD conformation contains a 1 - 4-type H-bond, the 6L&
folding pattern contains no H-bond3 at all. Therefore, ab initio
calculations confirmed that, while the 1 - 4 H-bond may be
present in @-turns, it is not a necessary condition for the
stabilization of such structures. These calculations lead to the
conclusion that the 1 - 4-type intramolecular H-bond is more
related to the a,-type substructure than to the true nature of a
&turn backboneconformation. The ab initiocalculations reported
in this paper resulted in 18 @-turns according to the stronger
distance criteria (d I 7 A) and an additional 12 structures (a
total of 30 @-turns) according to the backbone angularity criteria
Acknowledgment. The authors wish to express their gratitude
to the Ontario Center for Large Scale Computing (OCLSC) for
the generous allocation of Cray X-MP/28 Supercomputer time.
The continued financial support of the NSERC of Canada is
gratefully acknowledged. This research was also supported in
part by a grant from the Hungarian Scientific Research
Foundation (OTKA No. 111-2245).
(-90' <7 <90').