Nanosecond laser temperature-jump optical rotatory dispersion: Application to early events in protein folding/unfolding

Abstract

Pumping picosecond optical parametric oscillators by a pulsed Nd:YAG laser mode locked using a nonlinear mirror Appl.Nanosecond time-resolved optical rotatory dispersion ͑TRORD͒ techniques are coupled with laser temperature-jump ͑T-jump͒ triggering in an instrument that measures ultrafast protein folding-unfolding dynamics with high specificity to secondary structure. Far-ultraviolet ͑UV͒ ORD can be measured with this instrument over a wide wavelength range at times as early as 35 ns after a 3 ns laser T-jump pulse. The fundamental of a Nd:YAG laser is passed through a D 2 -filled Raman shifter to generate a pulse of 1.5 m infrared ͑IR͒ light that is efficiently absorbed by water. The resulting T-jump is stable for at least 1 ms before decaying back to the starting temperature with a time constant of ϳ30 ms. The ability to measure entire TRORD band shapes during this temporal window makes it possible to distinguish between changes in the signal due to a genuine unfolding or refolding process and changes due to artifacts. The technique, applicable to a wide variety of proteins, is demonstrated here in submillisecond unfolding studies of RNAse A and cytochrome c.

Time-resolved temperature-jump infrared spectroscopy of peptides with well-defined secondary structure: A Trpzip β-hairpin variant as an example

“…Additionally one of the beam lines is delayed by extending its path, so that it reaches the sample after the first pulse has passed. The use of two counterpropagating pulses of approximately equal intensity on the sample results in a temperature gradient of less than 5% through the cell [28][29][30]. Furthermore, the focus of each beam was adjusted to lie beyond the sample windows for both front and back excitation to avoid high power density within the sample.…”

Vibrational circular dichroism signal enhancement using self-heterodyning with elliptically polarized laser pulses

“…In the VCD experiment, a smaller modulation amplitude of the PEM also leads to larger dissymmetry between left and right handed polarization states due to residual static birefringence ͑of modulator or sample cell windows͒, which can only be corrected partially. 12 The crossed polarizer or "quasinull" technique was introduced and is currently used by Kliger and co-workers 11,19,20 for time-resolved UV-visible CD and ORD spectroscopy. The most important practical modification of this method in our mid-IR setup is the use of the PEM, which replaces the mechanical rotation of a polarizer or a strain plate.…”

“…Most promising, however, is the possibility to record transient vibrational ORD signals, which have been found to be less sensitive to pump-induced polarization artifacts in electronic spectroscopy. 19 Although it has only been little explored in the past for vibrational transitions, transient ORD has the potential to provide the same local structural information as VCD.…”

Probing Early Events in Ferrous Cytochrome c Folding with Time- Resolved Natural and Magnetic Circular Dichroism Spectroscopies.

“…Consequently, the most common way to generate a T-jump takes advantage of the absorption of laser light from 1.4 µm to 2 µm by an aqueous sample, whereby a 10–30K T-jump can be induced within the ∼10 ns pulse width of a laser. The T-jump initiation method has been coupled with time-resolved absorption, fluorescence, resonance Raman, infrared and, more recently, ORD detection methods [28]. …”