Abstract
Site‐specific isotopic labeling of molecules is a widely used approach in IR spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, as well as on hydrogen bonding and vibrational coupling. The impact of these different factors was analyzed with a designed three‐stranded β‐sheet peptide and by use of selected 13C isotope substitutions at multiple positions in the peptide backbone. Single‐strand labels give rise to isotopically shifted bands at different frequencies, depending on the specific sites; this demonstrates sensitivity to the local environment. Cross‐strand double‐ and triple‐labeled peptides exhibited two resolved bands that could be uniquely assigned to specific residues, the equilibrium IR spectra of which indicated only weak local‐mode coupling. Temperature‐jump IR laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them, relative to that of equilibrium FTIR spectroscopy. Site‐specific relaxation rates were altered upon the introduction of additional cross‐strand isotopes. Likewise, the rates for the global β‐sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. This study reveals that isotope labels provide not only local structural probes, but rather sense the dynamic complexity of the molecular environment.
Highlights
Protein activity is intimately linked to local structure and dynamics, for example, by positioning functional groups or substrates in enzymes, thereby allowing the structure to execute biochemical processes
The predicted IR spectra for labeled variants of such a near-ideal structure are illustrated in Figure S2 in the Supporting Information. This design goal was approached in our previous study of the pG2 peptide, but the DProÀGly turn sequence, in that case, led to spectral interferences that inhibited interpretation of the impact of isotopic labeling on strand dynamics.[12]
Spectral effects of isotopic labeling are highly sensitive to molecular structure and dynamics
Summary
Fold, and its dynamics requires methods that sense the coupling of specific peptide units, which form the fundamental polymer chain and that are capable of a relatively fast response upon structural changes. Isotopic substitution provides a means of studying site-specific, fast dynamics of b-sheets that can be accessed using laser-induced temperature-jump (T-jump) spectroscopy with tunable single-wavelength IR detection.[8] we used T-jump IR techniques to gain new insights into the spectroscopic and folding properties of isotopically labeled bsheet model peptides. To provide some measure of spectral sensitivity to structure variations, spectral simulations for a set of 23-residue all-Ala peptides, each constrained to selected conformations, as determined by NMR spectroscopy (see above), were carried out at the DFT level (BPW91/6-31G**/PCM) using Gaussian 16.[23] The methods used closely followed our previous study,[12] and are detailed in the Supporting Information. To account for the differences in central and outer-strand hydration effects, DFT force fields (FF) were empirically adjusted to better reflect experimental frequency shifts of single-labeled variants
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