Короткий зміст: | Introduction For their size, small molecules still present many challenges. In biophysics, much of the current research effort is focused on macromolecules, and the experimental direction defined by grand challenges such as the protein-folding problem. Small molecules play an important role in these larger efforts, as they provide a fertile testing ground for experimental techniques and computational models, and as such, their investigations are more relevant than ever. Small molecules also pose intrinsically interesting questions. In solvated environments, their surfacearea- to-volume ratio on a per-residue basis is disproportionately large, and thus, they provide an excellent vehicle for studying solvent interactions. For amino acids with small side chains, the reduced steric hindrance can allow multiple, thermally accessible conformations under ambient conditions. These features make for challenging spectroscopy. On the other hand, small molecules are amenable to high-level ab initio calculations that are computationally intractable for larger systems.1-5 The interplay between theory and experiment enables the careful isolation and study of different factors that affect the observed conformational distribution, such as the solvent environment. An oft-neglected, but relevant area of biophysics is molecular crowding.6-9 Many studies of biomolecular structure either ignore or fail to take into account the thermodynamically nonideal environment of the cell interior, in which the solvent is far scarcer than in an ideal aqueous solution. In a highly heterogeneous medium such as the cell cytoplasm, excluded volume effects can cause activities of solute species to deviate significantly from their idealized values in dilute solutions.10 These deviations have been shown to affect energetics, kinetics, equilibria, and transport.11 When available volumes are reduced, the influence of the solvent on structure may be amplified to the point where there may be dramatic changes in the potential energy landscape as well as spectroscopic observables. In one study, it was shown that, in the presence of crowding agents under physiological conditions, the amount of secondary structure is increased in R-helical VlsE, and R/- flavodoxin.12 Other studies have shown equally large effects on the structure and function of nucleic acids in crowded conditions.13 If investigations of small molecules are to be meaningful, the role of the solvent in helping to determine secondary structure should not only be better contextualized, but it should be embraced as (1) Havlin, R.; Laws, D. D.; Bitter, H.; Sanders, L.; Wemmer, E.; Pines, A.; Oldfield, E. J. Am. Chem. Soc. 2001, 123, 10362–10369. (2) Sun, H.; Oldfield, E. J. Am. Chem. Soc. 2004, 126, 4726–4734. (3) Cheng, F.; Sun, H.; Zhang, Y.; Mukkamala, D.; Oldfield, E. J. Am. Chem. Soc. 2005, 127, 12544–12554. (4) Wi, S.; Sun, H.; Oldfield, E.; Hong, M. J. Am. Chem. Soc. 2005, 127, 6451–6458. (5) Mukkamala, D.; Zhang, Y.; Oldfield, E. J. Am. Chem. Soc. 2007, 129, 7385–7392. (6) Zimmerman, S. B.; Minton, A. P. Annu. ReV. Biophys. Biomolec. Struct. 1993, 22, 27–65. (7) Ellis, R. J. Trends Biochem. Sci. 2001, 26, 597–604. (8) Ellis, R. J. Curr. Opin. Struct. Biol. 2001, 11, 114–119. (9) Minton, A. P. Curr. Biol. 2006, 16, R269–R271. (10) van den Berg, B.; Wain, R.; Dobson, C. M.; Ellis, R. J. EMBO J. 2000, 19, 3870–3875. (11) Minton, A. P. J. Biol. Chem. 2001, 276, 10577–10580. (12) Perham, M.; Stagg, L.; Wittung-Stafshede, P. FEBS Lett. 2007, 581, 5065–5069. (13) Zimmerman, S. B.; Trach, S. O. Nucleic Acids Res. 1988, 16, 6309– 6326. 10.1021/ja902917s CCC: $40.75 XXXX American Chemical Society J. AM. CHEM. SOC. XXXX, xxx, 000 9 A Downloaded by AUSTRIA CONSORTIA on July 6, 2009 Published on June 18, 2009 on http://pubs.acs.org | doi: 10.1021/ja902917s a primary determinant of spectroscopic observables. In the case of NMR, that observable is the chemical shift. Our current understanding of the connection between sequence identity, secondary structure, and chemical shift is now well-documented for globular proteins in aqueous solution.14 With the widespread use of higher-field NMR spectrometers and careful attention to experimental details, such as referencing, chemical shift measurements are being mined for more information than ever. The past year-and-half has seen dramatic developments in the ability to generate blind protein structures from chemical shifts alone. Combining chemical shifts and molecular dynamics, Cavalli et al. successfully determined the structures of 11 proteins, containing up to 123 residues, with a resolution of 2 Å or better.15 Vila et al. used a strategy focused entirely on 13CR chemical shifts to predict the structure of the 20-residue all- peptide BS2.16 Their method deploys a combination of experimentally observed and quantum chemically calculated 13CR shifts to derive torsional restraints for all the residues. Shen et al. have presented a yet new protocol for correlating six commonly observed chemical shifts with local structure.17 Their procedure uses an empirically optimized approach to select structurally similar fragments from the Protein Data Bank together with ROSETTA assembly methods. The efficacy of their method was successfully demonstrated, in a blind manner, on 9 protein targets, as large as 15.4 kDa. These exciting developments hold the promise of speedier structure determination, but for all their simplicity, they demand a more thorough understanding of dependences of the chemical shift on important factors, such as hydrogen bonding, solvation, and structure. In recent years, the term “microsolvation” has been much discussed in the literature.18-20 It refers to the chemical environment in which a solute molecule is surrounded by a defined number of water molecules in a very specific configuration, typically held together by transient hydrogen bonds. In essence, microsolvation is the site-specific and eVent-specific solvation at the atomic level. While the ideas of site-specific solvation have been around for a long time, the conjunction with event specificity makes microsolvation a particularly cogent and timely phenomenon for study using mode spectroscopic and computational tools. For example, water molecules have been found to play a key role in bacteriorhodopsin’s function.21 Chen et al. have demonstrated that the specific geometrical arrangement of hydrogen-bonded water molecule networks around the active site of heme oxygenase plays a role in determining both the enzyme’s rate of catalysis and the catalytic pathway.22 Individual water molecules have been implicated in the mechanisms of other enzymes, including carbonic anhydrase and horseradish peroxidase.23,24 Most recently, Bo et al. reported the ability to detect the onset of collective network motions in model peptides, as a function of solvation, using terahertz spectroscopy.25 Outside the biological realm, studies of the hydration environments around small organic molecules and ions show that hydration can alter molecular properties such as electron affinity and chemical reactivity.26 There is some evidence to suggest that solvent interactions can alter the sterics around a molecule and affect the reaction mechanisms and rates in some SN2 type reactions.27 The role of solvent in deciding conformational preferences of organic molecules is now wellknown and attributable to microsolvation.28 It has also been shown that optimally positioned water molecules can act as catalysts for radical reactions in the atmosphere.29 These experimental and computational realizations of microsolvation argue for deeper investigations of solvation mechanisms at the atomic level. The transient, dynamic nature of microsolvation makes its spectroscopic detection particularly challenging. Small molecules, and peptides in particular, provide an excellent point of entry. Alanine, for example, is known to induce helices, and yet, in short peptides, it exhibits a far richer geometrical palette.30-36 This intermediate regime, from 2-5 residues, poses many questions about the conformational preferences in different environments. Detailed studies of di- and tripeptides are beginning to add significant insights to structure, solvation, and spectroscopy, yet much remains unknown. Bour et al.’s combined computational and solution-state NMR study of L-alanyl-L-alanine presented evidence that the dipeptide occupies a single conformation in solution.37 Siegrist et al. have shown, using high-resolution terahertz spectroscopy, the ability to detect weak intramolecular hydrogen bonds in the three different crystalline polymorphs of trialanine, while others have linked T1-relaxation behavior with structural features.38 There has been an equal amount of attention devoted to the behavior of small peptides in the gas phase. We report here the results of a combined solution-state and solid-state NMR study, supported by ab initio calculations and X-ray diffraction, of four zwitterionic dipeptides, R-glycylglycine, glycyl-L-alanine, L-alanyl-glycine, and L-alanyl-Lalanine. We chose this system of dipeptides for three reasons. First, the crystalline form of each of the four dipeptides has (14) Wishart, D. J. Biomol. NMR 2003, 25, 173–195. (15) Cavalli, A.; Salvatella, X.; Dobson, C. M.; Vendruscolo, M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 9615–9620. (16) Vila, J. A.; Aramini, J. M.; Rossi, P.; Kuzin, A.; Su, M.; Seetharaman, J.; Xiao, R.; Tong, L.; Montelione, G. T.; Scheraga, H. A. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14389–14394. (17) Shen, Y.; et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 4685–4690. (18) Aikens, C.; Gordon, M. J. Am. Chem. Soc. 2006, 128, 12835–12850. (19) Blanco, S.; Lopez, J.; Alberto, L.; Alonso, J. J. Am. Chem. Soc. 2006, 128, 12111–12121. (20) Bachrach, S. M. J. Phys. Chem. A 2008, 112, 3722–3730. (21) Garczarek, F.; Gerwert, K. Nature 2006, 439, 109–111. (22) Chen, H.; Moreau, Y.; Derat, E.; Shaik, S. J. Am. Chem. Soc. 2008, 130, 1953–1965. (23) Isaev, A.; Scheiner, S. J. Phys. Chem. B 2001, 105, 6420–6426. (24) Derat, E.; Shaik, S.; Rovira, C.; Vidossich, P.; Alfonso-Prito, M. J. Am. Chem. Soc. 2007, 129, 6346–6347. (25) Bo , B.; Weingartner, H.; Brundermann, E.; Havenith, M. J. Am. Chem. Soc. 2009, 131, 3752–3755. (26) Yang, X.; Fu, Y. J.; Wang, X. B.; Slavicek, P.; Mucha, M.; Jungwirth, P.; Wang, L. S. J. Am. Chem. Soc. 2004, 126, 876–883. (27) Mohamed, A. A.; Jensen, F. J. Phys. Chem. A 2001, 105, 3259–3268. (28) Eliel, E. L.; Wilen, S. H.; Mander, L. N. Sterochemistry of Organic Compounds; Wiley-Interscience: New York, 1994. (29) Vohringer-Martinez, E.; Hansmann, B.; He andez, H.; Francisco, J. S.; Troe, J.; Abel, B. Science 2007, 315, 497–501. (30) Weir, a. F.; Lowrey, A. H.; Williams, R. W. Biopolymers 2001, 58, 577–591. (31) Mu, Y. G.; Stock, G. J. Phys. Chem. B 2002, 106, 5294–5301. (32) Wildman, K. A. H.; Lee, D.-K.; Ramamoorthy, A. Biopolymers 2002, 64, 246–254. (33) Kennedy, R. J.; Walker, S. M.; Kemp, D. S. J. Am. Chem. Soc. 2005, 127, 16961–16968. (34) Asakura, T.; Okonogi, M.; Nakazawa, Y.; Yamauchi, K. J. Am. Chem. Soc. 2006, 128, 6231–6238. (35) Yamauchi, K. J. Am. Chem. Soc. 2006, 128, 6231–6238. (36) Graf, J.; Nguyen, P. H.; Stock, G.; Schwalbe, H. J. Am. Chem. Soc. 2007, 129, 1179–1189. (37) Bour, P.; Budesinsky, M.; Spirko, V.; Kapitan, J.; Sebestik, J.; Sychrovsky, V. J. Am. Chem. Soc. 2005, 127, 17079–17089. (38) Siegrist, K.; Bucher, C. R.; Mandelbaum, I.; Walker, A. R. H.; Balu, R.; Gregurick, S. K.; Plusquellic, D. F. J. Am. Chem. Soc. 2006, 128, 5764–5775. B J. AM. CHEM. SOC. 9 VOL. xxx, NO. xx, XXXX A R T I C L E S Bhate et al. Downloaded by AUSTRIA CONSORTIA on July 6, 2009 Published on June 18, 2009 on http://pubs.acs.org | doi: 10.1021/ja902917s been characterized via single-crystal X-ray diffraction.39-42 The crystal structures provide independent structural data against which we may study hydrogen bonding in the crystalline state, and they provide a basis of calibration of the solid-state NMR results. Second, these four dipeptides are small enough to be amenable to high-level ab initio calculations, which allow us to control and isolate factors that are not otherwise accessible via experiment. Finally, the systematic appearance of a side chain methyl group enables a lateral comparison of structural and spectroscopic trends across the four molecules. Comparison of chemical shifts across phases allows us to correlate structural features with solvation. |