Involvement of Local, Rapid Conformational Dynamics in Binding of Flexible Recognition Motifs

Abstract

Flexible protein sequences populate ensembles of rapidly interconverting states differentiated by small-scale fluctuations; however, elucidating whether and how the ensembles determine function experimentally is challenged by the combined high spatial and temporal resolution needed to capture the states. We used carbon–deuterium (C–D) bond vibrations incorporated as infrared probes to characterize with residue-specific detail the heterogeneity of states adopted by proline-rich (PR) sequences and assess their involvement in recognition of Src homology 3 domains. The C–D absorption envelopes provided evidence for two or three sub-populations at all proline residues. The changes in the subpopulations induced by binding generally reflected recognition by conformational selection but depended on the residue and the state of the ligand to illuminate distinct mechanisms among the PR ligands. Notably, the spectral data indicate that greater adaptability among the states is associated with reduced recognition specificity and that perturbation to the ensemble populations contributes to differences in binding entropy. Broadly, the study quantifies rapidly interconverting ensembles with residue-specific detail and implicates them in function.

Vibrational Spectroscopic Map, Vibrational Spectroscopy, and Intermolecular Interaction

“…Many experimental studies have taken advantage of the sensitivity to learn about protein or peptide conformational ensembles. 441,443,[448][449][450][451] An extensive QM/MM study reported by Corcelli and coworkers has aimed to account for the absorption lineshape of Cα-D backbone deuterated d1-alanine in aqueous solution; the power spectrum of the fluctuating electric dipole moment was determined from PM3 calculations of snapshots along a classical MD simulation. 452 In addition to investigating backbone structure, C-D probes have been introduced at side chains of amino acids to take advantage of their sensitivity to their local protein environment.…”

“…While less utilized than the nitrile or azido probes, a few research groups have been applying C–D bonds for the study of proteins and peptides. C–D probes have provided insight into protein folding, molecular recognition, and catalysis. − However, their widespread adoption likely has been hindered by their weak absorptions due to the small transition dipole strengths. High (mM) protein concentrations are typically required, and discerning the absorption bands requires careful matching of reference and sample transmission spectra to achieve flat background absorbance.…”

“…However, DFT calculations of vibrational frequencies have been reported for many C–D labeled amino acids, including glycine, alanine, proline, methionine, lysine, and histidine. ,− For backbone C α –D bonds and the γ-methylene CD 2 of proline, the calculated frequencies show dependence on amino acid conformation and therefore have been proposed as a way to measure local protein or peptide structure. Many experimental studies have taken advantage of the sensitivity to learn about protein or peptide conformational ensembles. ,,− An extensive QM/MM study reported by Corcelli and co-workers has aimed to account for the absorption line shape of C α –D backbone deuterated d 1 -alanine in aqueous solution; the power spectrum of the fluctuating electric dipole moment was determined from PM3 calculations of snapshots along a classical MD simulation …”

Altered coordination in a blue copper protein upon association with redox partner revealed by carbon–deuterium vibrational probes

“…21,22 First demonstrated as probes of redox state in cytochrome c, 22 a number of CD-labeled amino acids have since been applied to study protein molecular recognition, folding, and catalysis. [23][24][25][26][27] An important advantage for this study is that CD bonds are non-perturbative, enabling their incorporation at the delicate metal site. Additionally, unlike most electronic spectroscopy methods, IR spectroscopy can be applied to investigate the closed shell reduced state of Pc and thus analyze both the reactant and product of the redox reaction.…”