Microscopic nucleation and propagation rates of an alanine-based α-helix

Abstract

An infrared temperature-jump (T-jump) study by Huang et al. (Proc. Natl. Acad. Sci. 2002 99, 2788–2793) showed that the conformational relaxation kinetics of an alanine-based α-helical peptide depend not only on the final temperature (Tf) but also on the initial temperature (Ti) when Tf is fixed. Their finding indicates that the folding free energy landscape of this peptide is non-two-state like, allowing for the population of conformational ensembles with different helical lengths and relaxation times in the temperature range of the experiment. Because α-helix folding involves two fundamental events, nucleation and propagation, the results of Huang et al. thus present a unique opportunity to determine their rate constants – a long-sought goal in the study of the helix-coil transition dynamics. Herein, we capitalize on this notion and develop a coarse-grained kinetic model to globally fit the thermal unfolding curve and T-jump kinetic traces of this peptide. Using this strategy, we are able to explicitly determine the microscopic rate constants of the kinetic steps encountered in the nucleation and propagation processes. Our results reveal that the time taken to form one α-helical turn (i.e., an α-helical segment with one helical hydrogen bond) is about 315 ns, whereas the time taken to elongate this nucleus by one residue (or backbone unit) is 5.9 ns, depending on the position of the residue.

Exposing the Nucleation Site in α-Helix Folding: A Joint Experimental and Simulation Study

“…This hierarchical structural assembly at the molecular level thus necessitates studying how and on what time scales protein secondary structures form in order to understand how proteins achieve their tertiary folds. In this regard, much effort has been devoted to elucidating the folding dynamics and mechanism of α-helix, − the most common structural motif found in proteins. The interest in studying the mechanism of α-helix formation is also motivated by the effort to design and develop individually folded, stable α-helices with novel biological functions and/or therapeutic utilities. ,− …”

“…Consequently, the reverse and unfolding process would run in a C-to-N manner. However, experimentally it has been difficult to directly validate this hypothesis due to the fact that there is not a distinct signal that only reports on the nucleation event . Herein, we aim to provide new insights into the α-helix folding mechanism by studying the folding kinetics of three cross-linked, alanine-based peptides using laser-induced temperature-jump ( T -jump) infrared (IR) spectroscopy and molecular dynamics (MD) simulations.…”

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

“…315 ns, whereas the time taken to elongate this nucleus by one residue is position-dependent (ca. 5.9 ns) …”

“…1600 cm −1 . The increased structural resolution via isotopic labeling of selected residues in the peptide sequence allowed Lin et al 86 to show that the T-jump-induced relaxation kinetics of this seemingly simple system not only are complex but also exhibit an unusual initial temperature dependence (when the final temperature is fixed), due to the coexistence of conformational ensembles that have different helicities (Figure 1) as well as the fact that the helical nucleation time constant is ca. 315 ns, whereas the time taken to elongate this nucleus by one residue is position-dependent (ca.…”

“…5.9 ns). 86 Hamm and co-worker 87 combined 2D IR spectroscopy and backbone 13 C� 18 O isotopic labeling to study the structural characteristics of a photoswitchable α-helix in its folded and unfolded states and concluded that the helix is not uniformly folded. Culik et al 88 further showed, using the min-protein Trp-cage as a model system, that it is possible to achieve secondary structural resolution in kinetic measurements of protein folding through the use of multiple conformational probes, including 13 C-labeled amide units.…”