Abstract
Time-resolved spectroscopic experiments have been performed with protein in solution and in crystalline form using a newly designed microspectrophotometer. The time-resolution of these experiments can be as good as two nanoseconds (ns), which is the minimal response time of the image intensifier used. With the current setup, the effective time-resolution is about seven ns, determined mainly by the pulse duration of the nanosecond laser. The amount of protein required is small, on the order of 100 nanograms. Bleaching, which is an undesirable effect common to photoreceptor proteins, is minimized by using a millisecond shutter to avoid extensive exposure to the probing light. We investigate two model photoreceptors, photoactive yellow protein (PYP), and α-phycoerythrocyanin (α-PEC), on different time scales and at different temperatures. Relaxation times obtained from kinetic time-series of difference absorption spectra collected from PYP are consistent with previous results. The comparison with these results validates the capability of this spectrophotometer to deliver high quality time-resolved absorption spectra.
Highlights
Proteins catalyze the chemical reactions that make life possible
Light intensity is controlled by an internal neutral density (ND) filter and can be adjusted from ~18 μW to ~500 μW, as measured at the sample
The light is coupled by large diameter (50 mm) focusing optics located within the instrument
Summary
Proteins catalyze the chemical reactions that make life possible. The biological and pharmaceutical relevance of these molecules is the major driving force for their characterization in terms of structure, function and dynamics [1,2,3]. We test the capabilities of this TR-MSP with photoactive yellow protein (PYP), of which the structure is known to sub-Ångström resolution [35], and which is structurally and kinetically very well characterized in crystal and solution [36,37,38]. These forms have very distinct spectra that can be switched into each other with lights of different wavelengths (see below and Figure 1) Unlike a photocycle, this reaction cannot be re-initiated conveniently to allow for the collection of multiple spectra. The design of the microspectrophotometer must be augmented by another light source in addition to the pulsed laser This additional light source utilizes a second wavelength, which is used after the switching reaction, to push the spectrum of the formed species back to its original state for subsequent iterations of the time-resolved experiment. Spectra that emerge during the course of the transition between either form can be recorded with nanosecond time resolution
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