Abstract
UV-visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2° off the X-ray beam, enabling spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection. The spectrometer was applied to NO reductase, a heme enzyme that catalyzes NO reduction to N2O. Radiation damage to the heme was monitored in real time during X-ray irradiation by evaluating the absorption spectral changes. Moreover, NO binding to the heme was probed via caged NO photolysis with UV light, demonstrating the extended capability of the spectrometer for intermediate analysis.
Highlights
X-ray crystallography is one of the most powerful tools for protein structural analysis at the atomic level and has been extensively used in structural biology
X-ray radiation damage has been a problem for accurate structural determination, for metalloproteins, because the metal centers are sensitive to hydrated electrons (Garman & Owen, 2006; Yano et al, 2005)
Because spectroscopy is useful for determining the physicochemical properties of proteins, spectroscopic analysis of a crystalline sample has been complementarily combined with X-ray crystallography to evaluate the sample status
Summary
X-ray crystallography is one of the most powerful tools for protein structural analysis at the atomic level and has been extensively used in structural biology. It is often difficult to monitor the X-ray-exposed volume of a crystal when the crystal is larger than the X-ray beam size To solve this problem, an on-axis geometry was recently adopted at SLS (Owen et al, 2009; Pompidor et al, 2013; Fuchs et al, 2014) and SPring-8 (Shimizu et al, 2013). A novel absorption spectrometer that adopts a nearly on-axis geometry but which does not interfere with X-ray diffraction data collection when operating is described In this spectrometer, a small prism mirror was placed near the X-ray beamstop that passes the white light to a crystal nearly collinear to (only 2 off) the X-ray beam and minimizes the interference with the X-rays diffracted from the crystal. The white light is focused using a 90 off-axis parabolic mirror PM1 ( f 101.6 mm) and directed to the sample by a small prism mirror Pr1 (5 mm  5 mm), which is mounted on a
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
