Folding of a Zinc-Finger ββα-Motif Investigated Using Two-Dimensional and Time-Resolved Vibrational Spectroscopy

Abstract

Small proteins provide good model systems for studying the fundamental forces that control protein folding. Here, we investigate the folding dynamics of the 28-residue zinc-finger mutant FSD-1, which is designed to form a metal-independent folded ββα-motif, and which provides a testing ground for proteins containing a mixed α/β fold. Although the folding of FSD-1 has been actively studied, the folding mechanism remains largely unclear. In particular, it is unclear in what stage of folding the α-helix is formed. To address this issue we investigate the folding mechanism of FSD-1 using a combination of temperature-dependent UV circular dichroism (UV-CD), Fourier transform infrared (FTIR) spectroscopy, two-dimensional infrared (2D-IR) spectroscopy, and temperature-jump (T-jump) transient-IR spectroscopy. Our UV-CD and FTIR data show different thermal melting transitions, indicating multistate folding behavior. Temperature-dependent 2D-IR spectra indicate that the α-helix is the most stable structural element of FSD-1. To investigate the folding/unfolding re-equilibration dynamics of FSD-1, the conformational changes induced by a nanosecond T-jump are probed with transient-IR and transient dispersed-pump-probe (DPP) IR spectroscopy. We observe biexponential T-jump relaxation kinetics (with time constants of 80 ± 13 ns and 1300 ± 100 ns at 322 K), confirming that the folding involves an intermediate state. The IR and dispersed-pump-probe IR spectra associated with the two kinetic components suggest that the folding of FSD-1 involves early formation of the α-helix, followed by the formation of the β-hairpin and hydrophobic contacts.

Finding multiple reaction pathways via global optimization of action

“…The lowest OM action folding pathway is consistent with a recent experiment 57 published after the submission of this work, where the early formation of C-terminal α-helix is observed to be followed by the concurrent formation of the β-hairpin and hydrophobic contacts. A comparison of the root mean square deviation values indicates that the α-helix approaches to the native structure earlier than the β-hairpin.…”

“…After our manuscript was submitted, it came to our attention that Meuzelaar and co-workers reported that the folding of FSD-1 occurs via an intermediate state where only the α-helix is formed 57 . After this intermediate state, the β-hairpin and hydrophobic contacts form.…”

Efficient Vibrational Energy Transfer through Covalent Bond in Indigo Carmine Revealed by Nonlinear IR Spectroscopy

“…Time-resolved nonlinear infrared spectroscopy has the advantage of being able to monitor vibrational energy dissipation pathways, as well as structural dynamics of molecular systems in condensed phases. − Infrared pump–probe experiments under the so-called magic-angle polarization condition can provide vibrational lifetime information on a given vibrational mode. − How vibrational energy relaxes after excitation, whether it is through an intramolecular vibrational energy redistribution channel or through intermolecular energy transfer by interacting with the solvent environment, can be understood on the basis of time-resolved vibrational measurements. Time-resolved infrared spectroscopies, in particular IR pump–IR probe and two-dimensional infrared (2D IR) spectroscopies, − have the advantage of simultaneously monitoring vibrational energy dissipation channels and molecular structural dynamics.…”

Resolving Dynamics in the Ensemble: Finding Paths through Intermediate States and Disordered Protein Structures

“…Molecular dynamics (MD) simulations offer an atomic-level description but are unsuitable for slower dynamical processes as a result of the computational cost and complexity (Figure ). − ,,,− Structurally heterogeneous proteins like IDPs and proteins with IDRs are dynamic at the same time which can intrinsically sample a multitude of diverse structures, thus providing a robust data set for testing structural refinement methodologies and kinetic pathway sampling using time-resolved techniques with molecular models.…”

“…Time scales for protein dynamics grouped by technique. − ,,,− Relevant protein time scales are plotted vs the number of residues for the system of interest for studies done using MD (purple), TRXSS (green), IR (blue), and trp-fluorescence (orange). MD studies are primarily located around smaller proteins and shorter time scales.…”