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1.  Protein energy landscapes determined by five-dimensional 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.
PMCID: PMC3852658  PMID: 24311594
five-dimensional crystallography; time-resolved crystallography; time-resolved microspectrophotometry; chemical kinetics; photoactive yellow protein
2.  Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography 
Nature chemistry  2013;5(3):212-220.
Trans-to-cis isomerization, the key reaction in photoactive proteins, cannot usually occur through the standard one-bond-flip mechanism. Due to spatial constraints imposed by a protein environment, isomerization is likely to proceed via a “volume-conserving” mechanism in which highly-choreographed atomic motions are expected, the details of which have not yet been directly observed. Here we employ time-resolved X-ray crystallography to structurally visualize isomerization of the p-coumaric acid chromophore in photoactive yellow protein with 100 picosecond time resolution and 1.6 Å spatial resolution. The structure of the earliest intermediate (IT) resembles a highly-strained transition state in which the torsion angle is located halfway between the trans and cis isomers. The reaction trajectory of IT bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue via E46Q mutation, which switches off the bicycle-pedal pathway.
PMCID: PMC3579544  PMID: 23422563
3.  Spectroscopic Studies of Model Photo-Receptors: Validation of a Nanosecond Time-Resolved Micro-Spectrophotometer Design Using Photoactive Yellow Protein and α-Phycoerythrocyanin 
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.
PMCID: PMC3794812  PMID: 24065094
nanosecond spectroscopy; time-resolved spectroscopy; photoreceptor proteins
4.  Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System 
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).
PMCID: PMC3412176  PMID: 22382353
Cytochrome c nitrite reductase; NrfA; Laue crystallography; UV/Vis spectropotentiometry; Protein film voltammetry
5.  Five-dimensional 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.
PMCID: PMC2824529  PMID: 20164643
time-resolved crystallography; chemical kinetics; protein structure; temperature dependence
6.  A Physical Picture of Protein Dynamics and Conformational Changes 
Journal of Biological Physics  2008;33(5-6):371-387.
A physical model is reviewed which explains different aspects of protein dynamics consistently. At low temperatures, the molecules are frozen in conformational substates. Their average energy is 3/2RT. Solid-state vibrations occur on a time scale of femtoseconds to nanoseconds. Above a characteristic temperature, often called the dynamical transition temperature, slow modes of motions can be observed occurring on a time scale between about 140 and 1 ns. These motions are overdamped, quasidiffusive, and involve collective motions of segments of the size of an α-helix. Molecules performing these types of motion are in the “flexible state”. This state is reached by thermal activation. It is shown that these motions are essential for conformational relaxation. Based on this picture, a new approach is proposed to understand conformational changes. It connects structural fluctuations and conformational transitions.
PMCID: PMC2565763  PMID: 19669525
Protein dynamics; Conformational relaxation; Mössbauer effect; Myoglobin
7.  Interaction of Nitric Oxide with Catalase: Structural and Kinetic Analysis 
Biochemistry  2011;50(21):4491-4503.
We present the structures of bovine catalase in its native form and complexed with ammonia and nitric oxide, obtained by X-ray crystallography. Using the NO generator 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, we were able to generate sufficiently high NO concentrations within the catalase crystals that substantial occupation was observed despite a high dissociation rate. Nitric oxide seems to be slightly bent from the heme normal that may indicate some iron(II) character in the formally ferric catalase. Microspectrophotometric investigations inline with the synchrotron X-ray beam reveal photoreduction of the central heme iron. In the cases of the native and ammonia-complexed catalase, reduction is accompanied by a relaxation phase. This is likely not the case for the catalase NO complex. The kinetics of binding of NO to catalase were investigated using NO photolyzed from N,N′-bis(carboxymethyl)-N,N′-dinitroso-p-phenylenediamine using an assay that combines catalase with myoglobin binding kinetics. The off rate is 1.5 s–1. Implications for catalase function are discussed.
PMCID: PMC3140772  PMID: 21524057

Results 1-7 (7)