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Acta Crystallographica Section A: Foundations of Crystallography (1)
Acta Crystallographica Section D: Biological Crystallography (1)
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry (1)
Schmidt, Marius (3)
Srajer, Vukica (3)
Henning, Robert (2)
Elliott, Sean J. (1)
Goelzer, Tyler (1)
Graber, Tim (1)
Ihee, Hyotcherl (1)
Judd, Evan T. (1)
Pacheco, A. Andrew (1)
Purwar, Namrta (1)
Sayyed, Bilal (1)
Tenboer, Jason (1)
Tripathi, Shailesh (1)
Youngblut, Matthew (1)
Year of Publication
Protein energy landscapes determined by five-dimensional crystallography
Acta Crystallographica Section D: Biological Crystallography
Barriers of activation within the photocycle of a photoactive protein were extracted from comprehensive time courses of time resolved crystallographic data collected at multiple temperature settings.
Free-energy landscapes decisively determine the progress of enzymatically catalyzed reactions [Cornish-Bowden (2012 ▶), Fundamentals of Enzyme Kinetics, 4th ed.]. Time-resolved macromolecular crystallography unifies transient-state kinetics with structure determination [Moffat (2001 ▶), Chem. Rev. 101, 1569–1581; Schmidt et al. (2005 ▶), Methods Mol. Biol. 305, 115–154; Schmidt (2008 ▶), Ultrashort Laser Pulses in Medicine and Biology] because both can be determined from the same set of X-ray data. Here, it is demonstrated how barriers of activation can be determined solely from five-dimensional crystallography, where in addition to space and time, temperature is a variable as well [Schmidt et al. (2010 ▶), Acta Cryst. A66, 198–206]. Directly linking molecular structures with barriers of activation between them allows insight into the structural nature of the barrier to be gained. Comprehensive time series of crystallographic data at 14 different temperature settings were analyzed and the entropy and enthalpy contributions to the barriers of activation were determined. One hundred years after the discovery of X-ray scattering, these results advance X-ray structure determination to a new frontier: the determination of energy landscapes.
five-dimensional crystallography; time-resolved crystallography; time-resolved microspectrophotometry; chemical kinetics; photoactive yellow protein
Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System
Judd, Evan T.
Elliott, Sean J.
Pacheco, A. Andrew
Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “Small Tetra-heme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated ~20 mg crude ccNiR/L culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for E. coli ccNiR, and is stable for over two weeks in pH 7 solution at 4° C. UV/Vis spectropotentiometric titrations and protein film voltammetry identified 5 independent 1-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the 5 reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed amongst the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good quality crystals, with which the 2.59 Å resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).
Cytochrome c nitrite reductase; NrfA; Laue crystallography; UV/Vis spectropotentiometry; Protein film voltammetry
Acta Crystallographica Section A: Foundations of Crystallography
Here it is demonstrated how five-dimensional crystallography can be used to determine a comprehensive chemical kinetic mechanism in concert with the atomic structures of transient intermediates that form and decay during the course of the reaction.
A method for determining a comprehensive chemical kinetic mechanism in macromolecular reactions is presented. The method is based on five-dimensional crystallography, where, in addition to space and time, temperature is also taken into consideration and an analysis based on singular value decomposition is applied. First results of such a time-resolved crystallographic study are presented. Temperature-dependent time-resolved X-ray diffraction measurements were conducted on the newly upgraded BioCARS 14-ID-B beamline at the Advanced Photon Source and aimed at elucidating a comprehensive kinetic mechanism of the photoactive yellow protein photocycle. Extensive time series of crystallographic data were collected at two temperatures, 293 K and 303 K. Relaxation times of the reaction extracted from these time series exhibit measurable differences for the two temperatures, hence demonstrating that five-dimensional crystallography is feasible.
time-resolved crystallography; chemical kinetics; protein structure; temperature dependence
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