| Краткое содержание: | 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
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10.1021/ja902917s CCC: $40.75 XXXX American Chemical Society J. AM. CHEM. SOC. XXXX, xxx, 000 9 A
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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.
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andez, H.; Francisco, J. S.;
Troe, J.; Abel, B. Science 2007, 315, 497–501.
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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.
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2007, 129, 1179–1189.
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Sychrovsky, V. J. Am. Chem. Soc. 2005, 127, 17079–17089.
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5764–5775.
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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. |
