Advances in Grating‐Based Photoacoustic Spectroscopy for the Study of Protein Dynamics

Abstract

Recent advances in transient grating spectroscopy are described in relation to extracting photoacoustic parameters. The time resolution for measurement of dynamically driven acoustics has been extended to the picosecond level. This improvement was achieved by adopting counter-propagating beam geometries and resolving the acoustic phase shift in analogy to phase modulation spectroscopy. At the other extreme, the dynamic range of this technique has been extended to milliseconds in order to follow dynamical processes central to biological functions. In addition, diffractive optics were used for generating the necessary excitation and probe beam geometries. A novel optical setup was developed which permits both the rapid exploration of fringe spacing dependencies in separating thermal from nonthermal contributions to the observed signal as well as heterodyne detection without active feedback. The latter capability significantly increases the signal-tonoise ratio and permits separation of the real and imaginary components from the nonlinear four-wave-mixing signal. Applications of these new methods are demonstrated by following the functionally relevant structural relaxation processes of heme proteins over 10 decades in time.

Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: From “protein quakes” to ligand escape for myoglobin

“…2. The Re part of the grating signal arises from changes in the index of refraction, from processes such as absorption, heat deposition, and volume changes of the protein itself (22). DeoxyMb has an extremely short excited state lifetime (Ͻ Ͻ1 ps).…”

“…1 and has previously been discussed in detail (21)(22)(23). Briefly, the excitation pulses (527 nm, 25 ns full width at half maximum) and probe (1.064 m continuous wave) beams were coincident and impinged normally on the diffractive optic through one of three gratings to give grating fringe spacings of ⌳ ϭ 30 m, 15 m, and 7.5 m at the sample.…”

“…A differential detection method was used to further improve the signal-to-noise ratio: detector 2 measured only the reference laser noise for subtraction from the heterodyned signal at detector 1. Unambiguous separation of the Re (Re) and Imaginary (Im) parts of the signal was accomplished by using the two cover slips as described previously (22). The signals were recorded with a 500-MHz digitizer (Textronix 520D; Beaverton, OR).…”

“…The preparation of the horse heart MbCO and deoxyMb is standard and has been described elsewhere (22). MbCO (deoxyMb) samples were kept under a CO (N 2 ) environment while the experiment was in progress.…”

Observation of the cascaded atomic-to-global length scales driving protein motion

“…Horse-heart myoglobin and cytochrome c (cyt c) were purchased from Sigma-Aldrich, dissolved in 0.2 M Tris buffer (pH 7.0), and used as described (18,19). The final sample concentrations gave an OD 530 of 1.…”