Enhanced Sensitivity to Local Dynamics in Peptides by use of Temperature-Jump IR-Spectroscopy and Isotope Labeling

David Scheerer,Heng Chi,Dan Mcelheny,Tim A Keiderling,Karin Hauser

doi:10.1016/j.bpj.2019.11.1188

Abstract

Site-specific isotopic labeling of molecules is widely used in IR-spectroscopy to resolve local contributions to vibrational modes. The frequency shift of the corresponding IR band depends on the substituted masses, but also on hydrogen bonding and on vibrational coupling both within the molecular structure and to the solvent molecules. The impact of these different factors was analyzed by use of equilibrium and dynamic IR spectra for a designed three-stranded β-sheet peptide with selected 13C isotope substitutions at multiple positions in the peptide backbone. NMR based structures provided a basis for molecular dynamics simulations of equilibrium conformational fluctuations. DFT-based simulations of the vibrational spectra were used to analyze specific site interactions. Experimentally, single strand labels give rise to isotopically shifted bands at different frequencies depending on the specific sites, demonstrating sensitivity to the local environment. Cross-strand double labeled peptides exhibited two resolved bands, which could be uniquely assigned to specific residues, and indicated weak local-mode coupling. Temperature-jump IR-laser spectroscopy was applied to monitor structural dynamics and revealed an enhanced sensitivity to coupling interactions as compared to equilibrium FTIR. Site-specific relaxation rates were altered upon 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. Our results demonstrate that isotope labels do not just provide local probes, but they also sense the complexity of the molecular environment.

Highlights

961-Pos Computational Analysis of Missense Mutations in Creatine Transporter Protein Associated with Creatine Deficiency Syndrome Mahesh Koirala, Emil Alexov
Using a combination of biophysical approaches, including SEC-SAXS, HDX-MS and SR-CD, we show that calcium-induced disorder-to-order transitions and acylation are involved in CyaA secretion and folding into a compact and functional state
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Summary

Introduction

961-Pos Computational Analysis of Missense Mutations in Creatine Transporter Protein Associated with Creatine Deficiency Syndrome Mahesh Koirala, Emil Alexov. We recently detected interactions between foldable and intrinsically disordered nascent proteins (not bearing an N-terminal signal sequence) and ribosomal proteins by dynamic fluorescence depolarization in the frequency domain and by chemical cross-linking. These results suggest that the ribosome facilitates in vivo protein folding by directly interacting with nascent chains.

Results

Conclusion