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

Abstract

Knowledge of dynamical structure of protein is an important clue to understand its biological function in vivo. Temperature-jump (T-jump) time-resolved transient mid-IR absorbance spectroscopy is a powerful tool in elucidating the protein dynamical structures and the folding/unfolding kinetics of proteins in solution. A home-built setup of T-jump time-resolved transient mid-IR absorbance spectroscopy with high sensitivity is developed, which is composed of a Q-switched Cr, Tm, Ho:YAG laser with an output wavelength at 2.09 μm as the T-jump heating source, and a continuous working CO laser tunable from 1580 to 1980 cm(-1) as the IR probe. The results demonstrate that this system has a sensitivity of 1 × 10(-4) ΔOD for a single wavelength detection, and 2 × 10(-4) ΔOD for spectral detection in amide I' region, as well as a temporal resolution of 20 ns. Moreover, the data quality coming from the CO laser is comparable to the one using the commercial quantum cascade laser.

Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics

“…Improved resolution of IR bands by time-resolved T-jump IR spectroscopy over equilibrium FTIR has been reported for a similar T-jump apparatus. 18 The likely explanation for this and for the slightly shifted maxima is that there is a slower phase (>0.5 ms) associated with the broad peak at 1660 cm −1 that is not captured by the transients at early time points. In particular the peak at 1680 cm −1 , arising from out-of-phase coupling of carbonyls in the β-sheets, is relatively weak in the FTIR data but strong in the kinetic IR data.…”

“…This is consistent with the S/N ratio of previous time-resolved T-jump IR spectrometers both in our lab and reported by others. 18,28 Equilibrium data can be used to determine the anticipated change in IR absorbance between two temperatures. Smaller signals can be detected by additional signal averaging, with a practical limit near 0.5×10 −5 ΔOD.…”

“…16 Recently, we and others have demonstrated that a Q-switched Ho:YAG laser, which has a fundamental output wavelength at 2.09 μm, can be used as an IR T-jump source. 17,18 The advantages of the Ho:YAG laser over the more widely used Nd:YAG laser/Raman shifter are its stability, beam quality and safety. There are non-linear processes in Raman shifting that result in shot-to-shot fluctuations of the pump pulse of ~15%.…”

The mechanism for thermal-enhanced chaperone-like activity of α-crystallin against UV irradiation-induced aggregation of γD-crystallin

“…The details of T-jump time-resolved IR absorbance difference spectrometer have been described previously ( 50 , 52 ). In the current apparatus, a 1.9- μ m pulsed laser generated from a home-built Q-switched Cr, Tm, Ho:YAG laser, was used as a laser heating source, which induced a rapid increase in temperature from 25 to 35°C for the solvent, as calibrated by the temperature-dependent FTIR spectra of D 2 O. IR absorbance changes were probed using a CO mid-IR laser at a spectral spacing of approximately 4 cm −1 in conjunction with an MCT detector (Kolmar, Newburyport, MA) and a digital oscilloscope (Tektronix, TDS520D, Santa Clara, CA).…”