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The nucleation event in α-helix formation is a fundamental process in protein folding. However, determining how quickly it takes place based on measurements of the relaxation dynamics of helical peptides is difficult because such relaxations invariably contain contributions from various structural transitions such as from helical to nonhelical states and helical to partial-helical conformations. Herein, we measure the temperature-jump (T-jump) relaxation kinetics of three model peptides that fold into a single-turn α-helix, using time-resolved infrared spectroscopy, aiming to provide a direct assessment of the helix nucleation rate. The α-helical structure of these peptides is stabilized by a covalent cross-linker formed between the side chains of two residues at the iand i 4 positions. If we assume that this cross-linker mimics the structural constraint arising from a strong side chain–side chain interaction (e.g., a salt bridge) in proteins, these peptides would represent good models for studying the nucleation process of an α-helix in a protein environment. Indeed, we find that the T-jump induced relaxation rate of these peptides is approximately (0.6 μs)−1at room temperature, which is slower than that of commonly studied alanine-based helical peptides but faster than that of a naturally occurring α-helix whose folded state is stabilized by a series of side chain–side chain interactions. Taken together, our results put an upper limit of about 1 μs for the helix nucleation time at 20 °C and suggest that the subsequent propagation steps occur with a time constant of about 240 ns. [ABSTRACT FROM AUTHOR]

Abstract: The non-natural amino acid p-cyanophenylalanine (PheCN) has recently emerged as a useful fluorescent probe of proteins; however, its photophysical properties have not been systematically examined. Herein, we measure the fluorescence quantum yield and the fluorescence lifetime of PheCN in a series of solvents. It is found that the fluorescence lifetime of PheCN shows a linear dependence on the Kamlet–Taft parameter α of the protic solvents used, indicating that the solute-solvent hydrogen bonding interactions mediate the non-radiative decay rate. Thus, results of this study provide a basis for quantitative application of PheCN fluorescence in protein conformational studies. [Copyright &y& Elsevier]

Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose four K+ binding sites. Here, we report on the two-dimensional infrared (2D IR) spectra of a semisynthetic KcsA channel with site-specific heavy (13C18O) isotope labels in the selectivity filter. The ultrafast time resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by molecular dynamics simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites. [ABSTRACT FROM AUTHOR]