Infrared Spectroscopic Discrimination between the Loop and α-Helices and Determination of the Loop Diffusion Kinetics by Temperature-Jump Time-Resolved Infrared Spectroscopy for Cytochrome c

Abstract

The infrared (IR) absorption of the amide I band for the loop structure may overlap with that of the alpha-helices, which can lead to the misassignment of the protein secondary structures. A resolution-enhanced Fourier transform infrared (FTIR) spectroscopic method and temperature-jump (T-jump) time-resolved IR absorbance difference spectra were used to identify one specific loop absorption from the helical IR absorption bands of horse heart cytochrome c in D2O at a pD around 7.0. This small loop consists of residues 70-85 with Met-80 binding to the heme Fe(III). The FTIR spectra in amide I' region indicate that the loop and the helical absorption bands overlap at 1653 cm(-1) at room temperature. Thermal titration of the amide I' intensity at 1653 cm(-1) reveals that a transition in loop structural change occurs at lower temperature (Tm=45 degrees C), well before the global unfolding of the secondary structure (Tm approximately 82 degrees C). This loop structural change is assigned as being triggered by the Met-80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25 degrees C to 35 degrees C breaks the Fe-S bond between the Met-80 and the iron reversibly, which leads to a loop (1653 cm(-1), overlap with the helical absorption) to random coil (1645 cm(-1)) transition. The observed unfolding rate constant interpreted as the intrachain diffusion rate for this 16 residue loop was approximately 3.6x10(6) s(-1).

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

“…2,55,57,58 In other words, the α -helix folding kinetics can be described by a series of discrete steps where the nucleation event is the slowest and occurs first, followed by a faster elongation process. In this regard, many experimental studies 4,5,7,8,12–14,16,19,20,23,25–28,31,34,35,38,41,42,59–61 have focused on the kinetics of α -helix formation, attempting to resolve those key conformational events. While taken together, these studies provided many microscopic details into the folding dynamics of α -helix, we still lack a complete understanding of the folding mechanism of this simple structural motif.…”

Terahertz-infrared spectroscopy of Shewanella oneidensis MR-1 extracellular matrix

“…1, 2, and 3) with the spectra, derived by least-square fits of the IR transmission coefficients. We associate the rich sets of absorption features above ≈ 1000 cm −1 with intramolecular modes and vibrations of molecular groups, as discussed for BSA in [28,29] and for CytC in [30][31][32]. Tables 1, 2, and 3 summarize parameters of absorption Fig.…”

A Q-switched Ho:YAG laser assisted nanosecond time-resolved T-jump transient mid-IR absorbance spectroscopy with high sensitivity

“…To examine the spectral sensitivity of the current apparatus, a previous studied sample cyt c was chosen as the reference. 35 The T-jump-induced difference absorbance spectrum of cyt c was acquired at room temperature (25 • C) with a ∆T = 10 • C. The corresponding T-jump induced transient IR absorbance spectrum of cyt c at 2 µs delay after the laser pulse (labeled as triangles) is shown in Fig. 7.…”

“…8,11 Although the transient difference IR spectra covering the whole amide I ′ region have scarcely reported, it has been realized in our group with the development of a CO gas laser. 13,15,35,36 Recently, the advent of quantum cascade laser (QCL) in mid IR region with a broader tuning range and higher power provides a better IR probe source to replace lead salt infrared diode lasers. 37,38 Besides, other timeresolved broad band mid IR spectroscopic methods have also been reported including step-scan FTIR, 39 broad band fs IR pulse probe, 40 and 2D IR spectrum 41 coupled to T-jump.…”