High-Resolution Measurements of Peptide Torsion Angles
A R T I C L E S
determine through-space and through-bond interactions, much
like high-resolution NMR studies of soluble proteins. The
success of this approach relies on obtaining hundreds or
thousands of constraints for even small proteins, and these
studies have been particularly successful for nanocrystalline
samples. When a protein exists in an array of structures or in
an extended-type structure, these measurements are not able to
determine structure as accurately. Samples created by fibrillar-
ization, freezing, or lyophilization have also been studied,8,9,22
but quite often their resolution is compromised due to line
broadening from side-chain conformational variability as well
as other inhomogeneous broadening mechanisms, making
spectral assignments untenable.
When samples have unresolved resonances or higher-resolu-
tion structural information is needed to answer mechanistic
questions, coherent measurements can be used to obtain
quantitative distances between specific atoms in order to refine
an extended structure or to determine conformational hetero-
geneity. A variety of MAS ssNMR methods have been
developed to extract information about local atomic structure
in noncrystalline, macroscopically disordered proteins.13,23,24
Given the large proton homonuclear dipolar couplings, these
methods typically rely on the spin characteristics of rare spin-
1/2 nuclei, such as 13C and 15N, either in concert with each other
or with their directly bonded protons, invariably requiring the
incorporation of isotopically enriched amino acids. Extracting
backbone secondary structure information can be accomplished
by measuring internuclear spin interactions. NMR experiments
that focus on internuclear distances25-27 or the relative orienta-
tions of the dipolar interactions28-32 and/or relative chemical
shift anisotropies21,33-41 (CSAs) of particular spin networks have
been developed and demonstrated to measure backbone second-
ary structure in peptides and proteins. However, many of these
experiments have only been tested on a small subset of model
compounds with known secondary structures and then applied
to unoriented or noncrystalline compounds which are, through
independent experiments, known to fall into secondary structure
regimes particularly relevant to the experiment and/or isotopic
labeling scheme selected. Each method has its advantages and
drawbacks, yet what is desirable is an experiment that can be
used to discern the full range of secondary geometries and which
retains a consistent accuracy over that range. This approach
should evaluate interactions which are relatively free of
experimental parameters requiring time-intensive independent
measurements (such as relaxation, chemical shift anisotropy
orientations, and radio frequency (rf) field homogeneity) or
assumptions about the generality of a particular parameter.
Additionally, for systems in which no prior structural informa-
tion is available or which have a mixture of conformations,
consideration of the labeling schemes and pulse sequences
applicable to a wide array of secondary structures is required.
To our knowledge, no sequence has demonstrated the ability
to retain its accuracy over a broad range of secondary structures;
in fact, many are known to be relevant for extended or â-sheet-
type secondary structures and are of limited accuracy for turns
and helices.21,28-31,40,41
This work addresses the experimental accuracy and relevance
of using 13C′ (i) f 13C′ (i + 1) interactions to determine
backbone torsion angles. This particular pair of spins was chosen
due to their ease of isotopic enrichment, consistent relaxation
and CSA characteristics, the sufficiently large size of their
interactions, and the lack of redundancy in their relative CSA
orientations over the allowed φ, ψ torsion angle space.5,13,33-36
Thus, determining structures within both helices and more
extended conformations should be possible. The DQ-DRAWS
experiment (chosen to select 13C′ (i) f 13C′ (i + 1) interactions)
offers a robust technique for examining these interactions
without detrimental effects to biological NMR samples even at
high fields and with currently achievable rf fields and spinning
speeds.27 Since these are homonuclear interactions, two-spin
coherences can be DQ filtered, an advantage which obviates
corrections for single quantum interactions from naturally
abundant 13C nuclei with overlapping resonances.5,42,43
(17) Igumenova, T. I.; McDermott, A. E.; Zilm, K. W.; Martin, R. W.; Paulson,
E. K.; Wand, A. J. J. Am. Chem. Soc. 2004, 126, 6720-6727.
(18) Franks, W. T.; Wylie, B. J.; Stellfox, S. A.; Rienstra, C. M. J. Am. Chem.
Soc. 2006, 128, 3154-3155.
(19) Franks, W. T.; Zhou, D. H.; Wylie, B. J.; Money, B. G.; Graesser, D. T.;
Frericks, H. L.; Sahota, G.; Rienstra, C. M. J. Am. Chem. Soc. 2005, 127,
12291-12305.
(20) Wylie, B. J.; Sperling, L. J.; Frericks, H. L.; Shah, G. J.; Franks, W. T.;
Rienstra, C. M. J. Am. Chem. Soc. 2007, 129, 5318-5319.
(21) Rienstra, C. M.; Hohwy, M.; Mueller, L. J.; Jaroniec, C. P.; Reif, B.; Griffin,
R. G. J. Am. Chem. Soc. 2002, 124, 11908-11922.
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(23) Luca, S.; Heise, H.; Baldus, M. Acc. Chem. Res. 2003, 36, 858-865.
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654-663.
The overriding goal of this work is to demonstrate that with
only three types of well-established parameters, 13C′ isotropic
chemical shifts, peptide bond lengths, and peptide bond angles,
one can utilize DQ-DRAWS experiments to determine backbone
torsion angles in proteins with high accuracy-regardless of the
underlying secondary structure of the system or protein being
studied. To this end, a series of tripeptides with published crystal
structures spanning a variety of torsion angles and with varying
hydrogen-bonding patterns was selected for study. A total of
ten compounds were chosen, three with torsion angles typical
for helices, two with torsion angles typical for extended
conformations, one with torsion angles similar to a â-sheet, one
with torsion angles commonly seen in a turn conformation, and
three with torsion angles intermediate between classical R-helix
(26) Ishii, Y. J. Chem. Phys. 2001, 114, 8473-8483.
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