Helix formation in unsolvated peptides: side chain entropy is not the determining factor.

Understanding the factors that stabilize R-helices is critical to understanding protein folding.1 Structure and sequence information for naturally occurring proteins has revealed a preference for certain amino acids in R-helices.2 Thus, many studies have focused on the individual effects of the amino acids on R-helix stability,3,4 and algorithms have been developed that can predict helix content.5 Monte Carlo simulations have indicated that side chain entropy opposes helix formation and can account for most of the differences in the helix-stabilizing/destabilizing effects of the natural amino acids.6,7 Specifically, side chain entropy predicts a helix propensity scale with Ala > Leu > Val. The main complication in solution studies of R-helix stability is that the solvent environment can change the helix-stabilizing/destabilizing tendencies of the amino acids.8,9 Factors such as the solvation of the helix backbone may also play a role in the ability of a particular amino acid to stabilize an R-helix in solution.10 By studying unsolvated peptides, the intrinsic factors leading to R-helix stability can be elucidated.11 Here we report on the conformations of unsolvated leucine-based peptides and compare them to previous studies of the conformations of unsolvated glycine-, alanine-, and valine-based peptides.12-14 The results show that leucine forms helices more readily than alanine, but less readily than valine (Val > Leu > Ala). This indicates that side chain entropy is not the determining factor in helix formation in unsolvated peptides. The results of molecular dynamics simulations suggest that the controlling factor is the stability of the globular state (a compact three-dimensional geometry). Residues that make poor (high energy) globules are good at making helices. Information about the conformations of unsolvated peptides was obtained from high-resolution ion mobility measurements.15 The mobility of an ion in the gas phase depends on its average collision cross section with a buffer gas, which in turn depends on its structure.16-18 The experimental apparatus consists of an electrospray source followed by a 5 cm long ion gate and a 63 cm long drift tube containing helium buffer gas. The pressure in the drift tube is slightly above atmospheric pressure so that a steady flow of helium (∼2000 sccm) through the ion gate prevents neutral molecules from entering the drift tube. A drift voltage of 10 000 V is divided across 46 guard rings, providing a uniform electric field along the drift tube axis. After traveling through the drift tube, some of the ions exit through a small aperture (0.125 mm in diameter), where they are then focused into a quadrupole mass spectrometer and detected by an off-axis collision dynode and dual microchannel plates. The measured drift times are subsequently converted into collision cross sections for further analysis. All the peptides studied here were synthesized on an Applied Biosystems Model 433A peptide synthesizer using FastMoc (a variant of Fmoc) chemistry. Relative cross sections for Leun+H, Ac-Leun+H, Ac-LysLeun+H, and Ac-Leun-Lys+H are shown in Figure 1. The relative cross section scale used is Ωrel ) Ωmeas 21.03NL 22.08NK, where NL and NK are the number of leucine and lysine residues, respectively, 21.03 Å2 is the cross section per residue determined for an ideal polyleucine R-helix, and 22.08 Å2 is the average increment in the cross section for a C-terminus lysine. (1) Horovitz, A. H.; Matthews, J. M.; Fersht, A. R. J. Mol. Biol. 1992, 227, 560-568. (2) Chou, P. Y.; Fasman, G. D. Biochemistry 1974, 13, 211-222. (3) Pace, C. N.; Scholtz, J. M. Biophys. J. 1998, 75, 422-427. (4) Chakrabartty, A.; Baldwin, R. L. AdV. Protein Chem. 1995, 46, 141176. (5) Muñoz, V.; Serrano, L. Biopolymers 1997, 41, 495-509. (6) Creamer, T. P.; Rose, G. D. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5937-5941. (7) Piela, L.; Nemethy, G.; Sheraga, H. A. Biopolymers 1987, 26, 12731286. (8) Wang, C.; Liu, L.-P.; Deber, C. M. Phys. Chem. Chem. Phys. 1999, 1, 1539-1542. (9) Krittanai, C.; Johnson, W. C., Jr. Proteins: Struct., Funct., Genet. 2000, 39, 132-141. (10) Luo, P.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 49304935. (11) For some examples of studies of unsolvated peptides and proteins, see: Suckau, D.; Shi, Y.; Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M.; McLafferty, F. W. Proc. Natl. Acad. Sci. 1993, 90, 790-793. Campbell, S.; Rodgers, M. T.; Marzluff, E. M.; Beauchamp, J. L. J. Am. Chem. Soc. 1995, 117, 12840-12854. Schnier, P. D.; Price, W. D.; Jockusch, R. A.; Williams, E. R. J. Am. Chem. Soc. 1996, 118, 7178-7189. Kaltashov, I. A.; Fenselau, C. Proteins: Struct. Func. Genet. 1997, 27, 165-170. Valentine, S. J.; Clemmer, D. E. J. Am. Chem. Soc. 1997, 119, 3558-3566. Wyttenbach, T.; Bushnell, J. E.; Bowers, M. T. J. Am. Chem. Soc. 1998, 120, 5098-5103. Arteca, G. A.; Velázquez, I.; Reimann, C. T.; Tapia, O. Phys. ReV. E 1999, 59, 5981-5986. Schaaff, T. G.; Stephenson, J. L.; McLuckey, S. L. J. Am. Chem. Soc. 1999, 121, 8907-8919. (12) Hudgins, R. R.; Jarrold, M. F. J. Phys. Chem. B 2000, 104, 21542158. (13) Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1999, 121, 34943501. (14) Kinnear, B. S.; Kaleta, D. T.; Kohtani, M.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 2000, 122, 9243-9256. (15) Dugourd, P.; Hudgins, R. R.; Clemmer, D. E.; Jarrold, M. F. ReV. Sci Instrum. 1997, 68, 1122-1129. (16) Hagen, D. F. Anal. Chem. 1979, 51, 870-874. (17) Von Helden, G.; Hsu, M.-T.; Kemper, P. R.; Bowers, M. T. J. Chem. Phys. 1991, 95, 3835-3837. (18) Clemmer, D. E.; Jarrold, M. F. J. Mass Spectrom. 1997, 32, 577592. Figure 1. Relative cross-sections plotted against the total number of residues (excluding the acetyl group). The relative cross-section scale (in Å2) is given by Ωrel ) Ωmeas 21.03NL 22.08NK, where NL is the number of leucine residues and NK is the number of lysine residues. 7907 J. Am. Chem. Soc. 2001, 123, 7907-7908