Measurement of circular dichroism dynamics in a nanosecond temperature-jump experiment

Abstract

The use of a fast temperature jump (T-jump) is a very powerful experiment aiming at studying protein denaturation dynamics. However, probing the secondary structure is a difficult challenge and rarely yields quantitative values. We present the technical implementation of far-UV circular dichroism in a nanosecond T-jump experiment and show that this experiment allows us to follow quantitatively the change in the helical fraction of a poly(glutamic acid) peptide during its thermal denaturation with 12 ns time resolution.

Nanosecond T-Jump Experiment in Poly(glutamic acid): A Circular Dichroism Study

“…We observe a decrease in the absorption at this wavelength. This decrease in absorption is consistent with a temperature rise of 4 °C [19]. The nice feature about this signal is its excellent quality which allows us to carry out a fit and we see that fitting with a single exponential curve gives a very good result.…”

“…The most popular means to attain this wavelength are the Raman shifter and the dye laser. We have chosen another technique and implemented a nanosecond OPO pumped by the second harmonic of the Nd:YAG laser [19]. This device allows us to finely tune the pump wavelength to optimize the water absorption.…”

Spectroscopic studies of protein folding: Linear and nonlinear methods

“…Because the thermal energy initially concentrated in the laser excitation volume will gradually dissipate into the surroundings, the useful time window of this technique in which the temperature remains approximately constant ranges from a few to tens of milliseconds (ms), depending on the optical setup and sample holder. Because the T ‐jump technique does not require any specific protein modifications, it is, therefore, the most commonly used triggering method in the study of ultrafast protein folding events 13–23…”

Recent advances in ultrafast time-resolved chirality measurements: perspective and outlook

“…To achieve time‐resolved chirality measurements different experimental set‐ups have been implemented. To investigate protein folding: stopped flow CD achieves milliseconds time‐resolution ; microsecond time‐resolution has been employed to study cytochrome c , in which photoinduced electron transfer triggers protein folding ; this time‐resolution is reduced to the ns‐regime by using a nanosecond temperature‐jump to initiate unfolding . Whereas stopped flow CD and ns temperature‐jump are experimentally limited in their time resolution, processes like photolysis and photoinduced ring opening are limited only by the laser pulses used and the photoinduced artefacts.…”