Time course of chemical and structural events in protein crystals measured by microspectrophotometry

Abstract

The functional properties of proteins in the crystalline state have been investigated over the past 30 years by a variety of methods, including single crystal polarized absorption spectroscopy. This technique has provided information on the accumulation and equilibrium distribution of protein-ligand complexes in the crystal and, in a few cases, on the rates of interconversion of catalytic intermediates. It has been possible to detect synergistic effects in the binding of different ligands, cooperativity and half-site reactivity and even formation of active multiprotein complexes, obtained by diffusion of one small protein in the pre-formed crystals of the other. Lattice interactions restrain the conformational transitions of some proteins existing in multiple states in solution. The crystal offers the unique opportunity to analyse not only the structure but also the function of a single form of the protein. The relevance of these data to the planning and interpretation of structural studies, especially in the perspectives of time-resolved crystallography, will be discussed with reference to well-characterized systems.

T State Hemoglobin Binds Oxygen Noncooperatively with Allosteric Effects of Protons, Inositol Hexaphosphate, and Chloride

“…The gels were loaded into a Dvorak-Stotler flow cell (46), covered by a gas permeable silicon copolymer membrane. The flow cell was mounted on the thermostatted stage of a Zeiss MPM03 microspectrophotometer (47). The silica gels are optically isotropic and transparent (30,31).…”

Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches

“…Optical absorption spectroscopy was the first technique that was applied to single protein crystals in order to characterize the nature and population of a specific intermediate species trapped within crystals (Hadfield & Hajdu, 1993;Rossi et al, 1992). The importance of spectral characterization is that reactivity in the crystal can, and indeed should, be quantified by parallel optical studies.…”

A fast and portable microspectrophotometer for protein crystallography

“…© 1993 International Union of Crystallography Printed in Great Britain -all rights reserved catalysis (Eaton & Hochstrasser, 1967;Rossi & Bernhard, 1970;Makinen & Eaton, 1973;Vas, Berni, Mozzarelli, Tegoni & Rossi, 1979;Mozzarelli et al, 1982;Mozzarelli, Peracchi, Rossi, Ahmed & Miles 1989;Mozzarelli, Rivetti, Rossi, Henry & Eaton, 1991;Kuo, Lipscomb & Makinen, 1986;Makinen, Hill, Zeppezauer, Little & Burdett, 1987;Makinen, Zelano & Troyer, 1987;Metzler et al, 1988;Rossi, Mozzarelli, Peracchi & Rivetti, 1992;Rivetti, Mozzarelli, Rossi, Henry & Eaton, 1993). Commercial microspectrophotometers require the crystals to be specially mounted, restricting access to the sample for measurements from other directions.…”