Equilibrium versus Nonequilibrium Peptide Dynamics: Insights into Transient 2D IR Spectroscopy

Abstract

Over the past two decades, two-dimensional infrared (2D IR) spectroscopy has evolved from the theoretical underpinnings of nonlinear spectroscopy as a means of investigating detailed molecular structure on an ultrafast time scale. The combined time and spectral resolution over which spectra can be collected on complex molecular systems has led to the precise structural resolution of dynamic species that have previously been impossible to directly observe through traditional methods. The adoption of 2D IR spectroscopy for the study of protein folding and peptide interactions has provided key details of how small changes in conformations can exert major influences on the activities of these complex molecular systems. Traditional 2D IR experiments are limited to molecules under equilibrium conditions, where small motions and fluctuations of these larger molecules often still lead to functionality. Utilizing techniques that allow the rapid initiation of chemical or structural changes in conjunction with 2D IR spectroscopy, i.e., transient 2D IR, a vast dynamic range becomes available to the spectroscopist uncovering structural content far from equilibrium. Furthermore, this allows the observation of reaction pathways of these macromolecules under quasi- and nonequilibrium conditions.

Figure 1

Various effective time scales of techniques for observing conformational changes in biomolecules.

Figure 2

Typical transient 2D IR optical design with pulse shaping module.

Figure 3

(top) A photolyzable linker constrains a peptide in an energetically unfavorable configuration until released via photodissociation, after which the peptide relaxes to an equilibrium position. (bottom) Simulated 2D IR spectra before and after release of the photolinker. A 2D IR difference spectrum is also shown.

Figure 4

A disulfide linker constrains the peptide until photodissociation via a 270 nm pulse, resulting in peptide relaxation back to an equilibrium conformation. Adapted with permission from Refs. and . Copyright 1997 and 2007 American Chemical Society.

Figure 5

a) Transient linear IR and b) Two-Dimensional infrared spectra of a β-turn peptide. c) horizontal slices of the 2D spectrum as indicated by the lines in part b. Reprinted with permission by Macmillan Publishers Ltd: Nature. Ref. , copyright 2006.

Figure 6

Photodecomposition of s-tetrazine into nitrogen and hydrogen cyanide.

Figure 7

Helical reorganization after photorelease of tetrazine linker indicates rotation of the backbone around the psi dihedral angle. Reproduced with permission from Ref. . Copyright 2013 American Chemical Society.

Figure 8

Normalized diagonal traces of 2D IR spectra modeled as a two exciton system. Reproduced with permission from Ref. . Copyright 2013 American Chemical Society.

Figure 9

(left) Azobenzene undergoes isomerization from trans to cis causing the α-helix to become unfolded. Upon irradiating with 450 nm, the cis-azobenzene reverses to the trans isomer, allowing the helix to refold. (right) 2D IR spectra taken of various isotopic labels within the helix before and after photoisomerization. Adapted with permission from Ref . Copyright 2010 American Chemical Society.

Figure 10

T-Jump transient 2D IR spectra of insulin a) sample chart of spectral changes in t-2D IR. Difference spectra of insulin at b) T_f = 45 °C and c) T_f = 50 °C following a time jump with different polarization and concentrations. Reproduced with permission from Ref. . Copyright 2016 American Chemical Society.