Although ethanol has been reported to affect cholesterol homeostasis in biological membranes, the molecular mechanism of action is unknown. Here, nuclear magnetic resonance (NMR) spectroscopic ...techniques have been used to investigate possible direct interactions between ethanol and cholesterol in various low dielectric solvents (acetone, methanol, isopropanol, DMF, DMSO, chloroform, and CCl
4). Measurement of
13C chemical shifts, spin-lattice and multiplet relaxation times, as well as self-diffusion coefficients, indicates that ethanol interacts weakly, yet specifically, with the HC-OH moiety and the two flanking methylenes in the cyclohexanol ring of cholesterol. This interaction is most strong in the least polar-solvent carbon tetrachloride where the ethanol–cholesterol equilibrium dissociation constant is estimated to be 2
×
10
−3 M.
13C-NMR spin-lattice relaxation studies allow insight into the geometry of this complex, which is best modeled with the methyl group of ethanol sandwiched between the two methylenes in the cyclohexanol ring and the hydroxyl group of ethanol hydrogen bonded to the hydroxyl group of cholesterol.
13C-NMR relaxation experiments (T 1, T 2, T 1 ρ, and NOE) were performed on selectively enriched residues in two peptides, one hydrophobic staple α-helix-forming peptide GFSKAELAKARAAKRGGY and one ...β-hairpin-forming peptide RGITVNGKTYGR, in water and in water/trifluoroethanol (TFE). Exchange contributions, R ex, to spin−spin relaxation rates for 13Cα and 13Cβ groups were derived and were ascribed to be mainly due to peptide folding−unfolding. To evaluate the exchange time, τex, from R ex, the chemical shift difference between folded and unfolded states, Δδ, and the populations of these states, p i, were determined from the temperature dependence of 13C chemical shifts. For both peptides, values for τex fell in the 1 μs to 10 μs range. Under conditions where the peptides are most folded (water/TFE, 5 °C), τex values for all residues in each respective peptide were essentially the same, supporting the presence of a global folding−unfolding exchange process. Rounded-up average τex values were 4 μs for the helix peptide and 9 μs for the hairpin peptide. This 2−3-fold difference in exchange times between helix and hairpin peptides is consistent with that observed for folding−unfolding of other small peptides.
The study of backbone and side-chain internal motions in proteins and peptides is crucial to having a better understanding of protein/peptide "structure" and to characterizing unfolded and partially ...folded states of proteins and peptides. To achieve this, however, requires establishing a baseline for internal motions and motional restrictions for all residues in the fully, solvent-exposed "unfolded state." GXG-based tripeptides are the simpliest peptides where residue X is fully solvent exposed in the context of an actual peptide. In this study, a series of GXG-based tripeptides has been synthesized with X being varied to include all twenty common amino acid residues. Proton-coupled and -decoupled (13)C-nmr relaxation measurements have been performed on these twenty tripeptides and various motional models (Lipari-Szabo model free approach, rotational anisotropic diffusion, rotational fluctuations within a potential well, rotational jump model) have been used to analyze relaxation data for derivation of angular variances and motional correlation times for backbone and side-chain chi(1) and chi(2) bonds and methyl group rotations. At 298 K, backbone motional correlation times range from about 50 to 85 ps, whereas side-chain motional correlation times show a much broader spread from about 18 to 80 ps. Angular variances for backbone phi,psi bond rotations range from 11 degrees to 23 degrees and those for side chains vary from 5 degrees to 24 degrees for chi(1) bond rotations and from 5 degrees to 27 degrees for chi(2) bond rotations. Even in these peptide models of the "unfolded state," side-chain angular variances can be as restricted as those for backbone and beta-branched (valine, threonine, and isoleucine) and aromatic side chains display the most restricted motions probably due to steric hinderence with backbone atoms. Comparison with motional data on residues in partially folded, beta-sheet-forming peptides indicates that side-chain motions of at least hydrophobic residues are less restricted in the partially folded state, suggesting that an increase in side-chain conformational entropy may help drive early-stage protein folding. Copyright 1999 John Wiley & Sons, Inc.
A simple method is presented to accurately determine (15)N-(1)H NOEs in biomolecules in the presence of H(N)-water proton chemical exchange. Three measurements are required: one with nonselective ...proton saturation and two with different water saturation conditions to determine the equilibrium value of the (15)N signal. This approach is exemplified with data on two peptides, one helix-forming 17-mer and one compactly folded 56-mer. Results indicate that (15)N-(1)H NOEs determined using the standard approach with short recycle times (3 to 4 s) can be significantly in error when H(N)-water proton chemical exchange is relatively rapid, water proton relaxation is relatively slow, and (15)N-(1)H NOEs are away from the value of -1. This new method avoids such inaccuracies resulting from the use of short recycle times.
A new approach to visualizing spectral densities and analyzing NMR relaxation data has been developed. By plotting the spectral density function, J(ω), as F(ω)=2ωJ(ω) on the log–log scale, the ...distribution of motional correlation times can be easily visualized. F(ω) is calculated from experimental data using a multi-Lorentzian expansion that is insensitive to the number of Lorentzians used and allows contributions from overall tumbling and internal motions to be separated without explicitly determining values for correlation times and their weighting coefficients. To demonstrate the approach, 15N and 13C NMR relaxation data have been analyzed for backbone NH and CαH groups in an α-helix-forming peptide 17mer and in a well-folded 138-residue protein, and the functions F(ω) have been calculated and deconvoluted for contributions from overall tumbling and internal motions. Overall tumbling correlation time distribution maxima yield essentially the same overall correlation times obtained using the Lipari–Szabo model and other standard NMR relaxation data analyses. Internal motional correlational times for NH and CαH bond motions fall in the range from 100 ps to about 1 ns. Slower overall molecular tumbling leads to better separation of internal motional correlation time distributions from those of overall tumbling. The usefulness of the approach rests in its ability to visualize spectral densities and to define and separate frequency distributions for molecular motions.
This study presents a site‐resolved experimental view of backbone CαH and NH internal motions in the 56‐residue immunoglobulin‐binding domain of streptococcal protein G, GB1. Using 13CαH and 15NH NMR ...relaxation data T1, T2, and NOE acquired at three resonance frequencies (1H frequencies of 500, 600, and 800 MHz), spectral density functions were calculated as F(ω) = 2ωJ(ω) to provide a model‐independent way to visualize and analyze internal motional correlation time distributions for backbone groups in GB1. Line broadening in F(ω) curves indicates the presence of nanosecond time scale internal motions (0.8 to 5 nsec) for all CαH and NH groups. Deconvolution of F(ω) curves effectively separates overall tumbling and internal motional correlation time distributions to yield more accurate order parameters than determined by using standard model free approaches. Compared to NH groups, CαH internal motions are more broadly distributed on the nanosecond time scale, and larger CαH order parameters are related to correlated bond rotations for CαH fluctuations. Motional parameters for NH groups are more structurally correlated, with NH order parameters, for example, being larger for residues in more structured regions of β‐sheet and helix and generally smaller for residues in the loop and turns. This is most likely related to the observation that NH order parameters are correlated to hydrogen bonding. This study contributes to the general understanding of protein dynamics and exemplifies an alternative and easier way to analyze NMR relaxation data.
Abstract
This study presents a site‐resolved experimental view of backbone C
α
H and NH internal motions in the 56‐residue immunoglobulin‐binding domain of streptococcal protein G, GB1. Using
13
C
α
...H and
15
NH NMR relaxation data
T
1
,
T
2
, and NOE acquired at three resonance frequencies (
1
H frequencies of 500, 600, and 800 MHz), spectral density functions were calculated as
F
(ω) = 2ω
J
(ω) to provide a model‐independent way to visualize and analyze internal motional correlation time distributions for backbone groups in GB1. Line broadening in
F
(ω) curves indicates the presence of nanosecond time scale internal motions (0.8 to 5 nsec) for all C
α
H and NH groups. Deconvolution of
F
(ω) curves effectively separates overall tumbling and internal motional correlation time distributions to yield more accurate order parameters than determined by using standard model free approaches. Compared to NH groups, C
α
H internal motions are more broadly distributed on the nanosecond time scale, and larger C
α
H order parameters are related to correlated bond rotations for C
α
H fluctuations. Motional parameters for NH groups are more structurally correlated, with NH order parameters, for example, being larger for residues in more structured regions of β‐sheet and helix and generally smaller for residues in the loop and turns. This is most likely related to the observation that NH order parameters are correlated to hydrogen bonding. This study contributes to the general understanding of protein dynamics and exemplifies an alternative and easier way to analyze NMR relaxation data.
Here, we report a method to simultaneously determine CH
2 cross-correlation spectral densities and
T
1 relaxation times in the laboratory and rotating frames. To accomplish this, we have employed an ...indirect approach that is based on measurement of differences in relaxation rates acquired with and without cross-correlation terms. The new method, which can be employed using multidimensional NMR and standard relaxation pulse sequences, is validated experimentally by investigation of a selectively
13C-enriched hexadecapeptide and the uniformly
13C-enriched immunoglobulin-binding domain of streptococcal protein G (GB1). Use of this approach makes determination of CH
2 cross-correlation spectral densities in uniformly
13C-enriched proteins now routine and provides novel information concerning their internal motions.
NMR relaxation-derived spectral densities provide information on molecular and internal motions occurring on the picosecond to nanosecond time scales. Using (13)C and (15)N NMR relaxation parameters ...T(1), T(2), and NOE acquired at four Larmor frequencies (for (13)C: 62.5, 125, 150, and 200 MHz), spectral densities J(0), J(omega(C)), J(omega(H)), J(omega(H) + omega(C)), J(omega(H) - omega(C)), J(omega(N)), J(omega(H) + omega(N)), and J(omega(H) - omega(N)) were derived as a function of frequency for (15)NH, (13)C(alpha)H, and (13)C(beta)H(3) groups of an alanine residue in an alpha-helix-forming peptide. This extensive relaxation data set has allowed derivation of highly defined (13)C and (15)N spectral density maps. Using Monte Carlo minimization, these maps were fit to a spectral density function of three Lorentzian terms having six motional parameters: tau(0), tau(1), tau(2), c(0), c(1), and c(2), where tau(0), tau(1) and tau(2) are correlation times for overall tumbling and for slower and faster internal motions, and c(0), c(1), and c(2) are their weighting coefficients. Analysis of the high-frequency portion of these maps was particularly informative, especially when deriving motional parameters of the side-chain methyl group for which the order parameter is very small and overall tumbling motions do not dominate the spectral density function. Overall correlation times, tau(0), are found to be in nanosecond range, consistent with values determined using the Lipari-Szabo model-free approach. Internal motional correlation times range from picoseconds for methyl group rotation to nanoseconds for backbone N-H, C(alpha)-H, and C(alpha)-C(beta) bond motions. General application of this approach will allow greater insight into the internal motions in peptides and proteins.
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