1 Department of Physics and 2 Bioinformatics Center, Indian Institute of Science, Bangalore-560012, 3 Department of NMR, All India Institute of Medical Sciences, New Delhi-110029 and 4 International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: aromatic interactions/dehydrophenylalanine/de novo design/310 helix
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The peptide Boc-L-Ala-(Phe)4-L-Ala-(
Phe)3-Gly-OMe (Peptide I) was designed to understand the preferential helical screw sense observed in the consecutive
Phe-containing peptides studied in our laboratory and by others (Day et al., 1996
; Jain et al., 1997
; Ramagopal et al., 1998
). The results presented in this paper depict an unanticipated but interesting mutual recognition and association of ambidextrous helices mediated by a network of weak interactions (Desiraju and Steiner, 1999
) and highlight its possible exploitation in future work on 2de novo design. It is observed that the two shape-complement 310-helices in the asymmetric unit, a slightly distorted right-handed 310-helix (X) and a left-handed 310-helix (Y), are clamped together by a CH···O hydrogen-bonded
Phe zipper structure. They are further supported by
interactions (Malone et al., 1997
; Steiner, 1998
). The planar
Phe side chains from the adjacent helices stack one above the other, forming `extended phenyl embrace' arrangements at the helixhelix interface. Here we report how molecular recognition mediated by weak interactions can be exploited towards the construction and design of folded super secondary structural elements.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Circular dichroism (CD) measurements were carried out on a JASCO J720 spectropolarimeter with a data processor attached. A 1 mm pathlength was used. The spectrum was recorded in various solvents such as acetonitrile, dichloromethane, hexafluoro-2-propanol and trifluoroethanol. Chloroformmethanol titrations were also carried out by increasing the concentration of methanol from 0 to 80% and the data were expressed in terms of molar ellipticity.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A perspective view of the ambidextrous molecules in the asymmetric unit perpendicular to the two axes of the helices is given in Figure 1. The right-handed 310-helix (X) and left-handed 310-helix (Y) are anti-parallel to each other. The angle between the two helical axes is
15°.
|
|
|
|
In the crystal, the helices related by translational symmetry are observed as approximate helical rods and staggered by a small amount along the b-axis. A solvent molecule named DMF(S3) mediates the head to tail hydrogen bond (Karle, 1996) by making N2(Y)H···O1S3, C1S3H···O0'(Y) hydrogen bonds at its polar end and C3S3H···O8'(Y) hydrogen bond at its apolar end with a symmetry related molecule (Table I
, see figure in the supplementary material). Similarly, there is no direct NH···O=C head-to-tail hydrogen bond between the two right-handed helices. Rather the C
1(X) from the
Phe3 side chain forms a weak hydrogen bond with the free acceptor O9'(X) of the symmetry-related molecule (see figure in supplementary material for details).
A remarkable feature of the present structure is that the two shape-complement helices named X and Y are held together by symmetrically placed aromatic-backbone CH···O interactions distributed all along the helical axis (Figure 1, Table I
). Thus CH(phenyl)···O(carbonyl) hydrogen bonds (Burley and Petsko, 1988
) are observed between C
2(
Phe2), C
2(
Phe5), C
2(
Phe8) of molecule X to O8'(Y), O5'(Y), O2'(Y) of molecule Y, respectively, and similar hydrogen bonds are observed between C
2(
Phe2), C
2(
Phe5), C
2(
Phe8) of molecule Y to O8'(X), O5'(X), O2'(X) of molecule X, respectively (Table I
). The converse numbers associated with the atoms (donors and acceptors) involved in the hydrogen bonds represent the amazing regularity maintained to achieve the state of maximum possible hydrogen bonding (or weak interaction) between the two anti-parallel ambidextrous helices. Further,
Phe with its planar aromatic side chain stacks against another
Phe from the adjacent shape-complement helix leading to two `extended phenyl embrace' arrangements at the helixhelix interior (Figure 3
). It is interesting that the average of equivalent temperature factors of the six atoms of phenyl rings of the
Phe residues at the interface of the two antiparallel helices, and Y are lower than those of the
Phe residues not involved in the interface. These values further support our observation that the networks of weak interactions observed between the two helices are strong enough to bring molecular association.
|
CD spectroscopy has been used earlier to establish the screw sense of peptides containing dehydrophenylalanine residues (Pieroni et al., 1996). For the present decapeptide, CD studies were carried out in various solvents at room temperature. The CD spectra display couplets of bands which appear as a typical exciton splitting of the electronic transition of
Phe chromophore at 280 nm. This type of splitting pattern originates from a rigid mutual disposition of two or more
Phe residues, generally involved in a system of consecutive ß-turns, i.e. 310-helix (Pieroni et al., 1993
). The most striking feature of the CD spectra of the decapeptide is the sign of the couplet. A negative CD couplet ( +), typical of a right-handed 310-helix with a negative band at 285 nm, a positive band at 260 nm and a crossover point at
275 nm, is observed for the decapeptide in dichloromethane, hexafluoro-2-propanol and chloroform (Figures 4 and 5
). However, a positive CD couplet (+ ) with bands of opposite signs at
285 and
260 nm, characteristic of the left-handed screw sense of the helix (Pieroni et al., 1991
), is also observed for the peptide in dimethylformamide and 80% methanol (Figures 4 and 5
). The CD spectra of the decapeptide in chloroform with increasing concentration of methanol are indicative of the equilibrium shifting towards the left-handed conformer. Addition of methanol gradually appears to destabilize the right-handed form of the 310-helix. At a concentration of 60:40 (chloroform: methanol), almost equal proportions of the two conformers are present and, on further increase in methanol concentration, the left-handed helical conformation stabilizes in solution. Low molar ellipticity of the decapeptide in acetonitrile and trifluoroethanol may be attributed to the presence of both left-and right-handed conformers, simultaneously in solution, supporting the structure observed in the solid state. The equilibrium exhibited by the decapeptide may therefore shift towards the right-or left-handed conformer depending on the solvent conditions. Similar effects of solvent dependence on the handedness of the helix have also been reported by other workers (Pieroni et al., 1991
).
|
|
Solid-state and solution studies suggest that the energy barrier between the left-and right-handed conformers being less allows facile interconversion in solution and consequent association of the two conformers. Further, it appears that the 310-helical conformations and the shape complementarity of the conformers (X and Y) are the two key factors responsible for the stabilization of the CH···O hydrogen-bonded Phe zipper structure. In 310-helical peptides (ideal), the side chains stack one over the other along the helical axis creating a column of protuberant side chains at 120° to each other, hence forming a groove between them. This makes the space for side chains from adjacent helices to interdigitate in the groove. In the present case the planar
Phe side chains (hydrogen atoms in the same plane) with their favored disposition with respect to backbone are able to insert into the groove of the adjacent shape complement helix. Further, these interdigitated side-chains from shape complement helices are able to form good CH···O hydrogen bonds with the backbone carbonyl oxygens of the adjacent helix. This provides a good example of shape assisted cooperative recognition of helices through weak interactions. This type of interdigitation may be described as a `wedge into a groove' arrangement as opposed to a `knobs into holes' arrangement (Crick, 1953
) as proposed for
-helices forming coiled coils.
Conclusion
The structure reveals an unusual association of ambidextrous 310-helices through strong CH···O interactions. According to the `core hypothesis', the efficient packing of hydrophobic side chains in the interior primarily determines the native folded structure of globular proteins (Sander, 1994). Nature constructs this core mainly with the available hydrophobic residues from its toolbox of 20 amino acids. The hidden and plastic interactions involving hydrophobic groups play a substantial role in protein stability. In addition, the ability of a phenylalanine residue to lend extra stability to protein structures was pointed out by Burley and Petsko (Burly and Petsko, 1988). Moreover, almost all the de novo designed super secondary motifs containing only natural residues which fold/unfold cooperatively in water revealed the fact that aromatic residues play a substantial role in their stability (Baltzer, 1999
; De Grado et al., 1999). In a recent study, Ramagopal et al. showed that aromatic interactions can be cleverly exploited in the design of helical hairpin motif, without polar/apolar patterning or disulfide bridges (Ramagopal et al., 2001
). Similarly, it was shown that tryptophan zippers are capable of stabilizing small (12 residue) monomeric ß-hairpins (Cochran et al., 2001
). All these results highlight the importance of weak interactions in molecular recognition and in the de novo design of mini-proteins, where a smaller number of buried hydrophobic residues are expected to play a major role.
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baltzer,T. (1999) Top. Curr. Chem., 202, 3976.[ISI]
Burley,S.K. and Petsko,G.A. (1988) Adv. Protein Chem., 39, 125189.[ISI][Medline]
Cochran,A.G., Skelton,N.J. and Starovasnik,M.A. (2001) Proc. Natl Acad. Sci. USA, 98, 55785583.
Crick,F.H.C. (1953) Acta Crystallogr., 6, 689697.[CrossRef][ISI]
Day,S., Mitra,S.N. and Singh,T.P. (1996) Biopolymers, 39, 849857.[CrossRef][ISI][Medline]
DeGrado,W.F., Summa,C.M., Pavone,V., Nastri,F. and Lombardi,A. (1999) Annu. Rev. Biochem., 68, 779819.[CrossRef][ISI][Medline]
Desiraju,G.R. and Steiner,T. (1999). The Weak Hydrogen Bonds. IUCr Monographs on Crystallography. Oxford Science Publications, Oxford.
Jain,R.M and Chauhan,V.S. (1996) Biopolymers (Pept. Sci.), 40, 105119.[CrossRef][ISI][Medline]
Jain,R.M., Rajashankar,K.R., Ramakumar,S. and Chauhan,V.S. (1997) J. Am. Chem. Soc., 119, 32053211.[CrossRef][ISI]
Karle,I.L. (1996) Biopolymers (Pept. Sci.),40, 157180.[CrossRef][ISI][Medline]
Malone,J.F., Murray,C.M., Charlton,M.H., Docherty,R. and Lavery,A.J., (1997) J. Chem. Soc., Faraday Trans., 93, 34293436.[CrossRef][ISI]
Pieroni,O., Fissi,A., Pratesi,C., Temussi,P.A. and Ciardelli,F. (1991) J. Am. Chem. Soc., 113, 63386340.[ISI]
Pieroni,O., Fissi,A., Pratesi,C., Temussi,P.A. and Ciardelli,A. (1993) Biopolymers, 33, 110.[ISI][Medline]
Pieroni,O., Fissi,A., Jain,R.M. and Chauhan,V.S. (1996) Biopolymers, 38, 97108.[CrossRef][ISI][Medline]
Ramagopal,U.A., Ramakumar,S., Joshi,R.M. and Chauhan,V.S. (1998) J. Pept. Res., 52, 208255.[ISI][Medline]
Ramagopal,U.A., Ramakumar,S., Sahal,D. and Chauhan,V.S. (2001) Proc. Natl Acad. Sci. USA, 98, 870874.
Sander,C. (1994) TIBTEC, 12, 163167.
Sheldrick,G.M. (1997) The SHELX-97 Manual. University of Gottingen, Gottingen, Germany.
Steiner,T. (1998) Acta Crystallogr., D54, 2531, 584588.
Received September 11, 2001; revised January 10, 2002; accepted January 28, 2002.