Defining redox state of X-ray crystal structures by single-crystal ultraviolet visible microspectrophotometry

• Exciting results have been emerging from the field of single-crystal X-ray crystallography, giving unprecedented detail of freeze-trapped reaction intermediates from important classes of macromolecules that contain chromophores. These structures have been coupled with single-crystal UV-visible microspectrophotometry. This has defined the distinct catalytic intermediates present in the crystal structures, allowing the correlation of electronic transitions with the observed structural transitions. Of particular note is that many of these structures have been generated “on the fly” during kinetic turnover in the crystal. Most enzymatic reactions proceed through distinct catalytic intermediates that, under favorable conditions, may accumulate transiently in the crystal during turnover. In some cases, the physical constraints of the contacts within crystals may also lead to a significant slowing of the reaction at certain points along the pathway where conformational changes are required. This can lead to a transient build-up of spectrally distinct intermediates in the crystal that can be trapped by flash freezing in liquid nitrogen, allowing a complete single-crystal data set to be collected to the highest possible resolution at a later time. Similar build-up of intermediates may be achieved by altering the pH, temperature, or the solvent environment around the protein in the crystal, or by producing engineered variants that build up an intermediate of interest. The chapter focuses on the technical considerations required to carry out UV-visible microspectroscopy of single crystals.

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NATURVETENSKAP  -- Biologi -- Cellbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Cell Biology (hsv//eng)

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