Allen Orville

Despite their immense value, the atomic coordinates derived from ground-state x-ray crystal structures reveal only a portion of the functional story for many important and complex systems in Biology. Consequently, a frontier challenge for many structural biologists is to determine time-resolved crystal structures directly from systems engaged in catalysis. In structural biology this leads to a desire to collect structural, spectroscopic, and if possible, kinetic data too, from the same crystalline samples. To these ends, single-crystal electronic absorption and Raman spectroscopies can be correlated with x-ray crystal structures at several synchrotron facilities, including Diamond Light Source and the Central Laser Facility. However, a set of general tools to fully correlate enzyme kinetics with time-resolved crystallography are not yet readily available. To address this grand challenge, we are working to extended enzyme kinetics strategies to crystalline samples, and to couple these methods to room-temperature, time-resolved, serial crystallographic methods with correlations to single-crystal spectroscopy and other structural methods. Achieving our goals will be transformative and impact nearly all aspects of structural biology.

Serial crystallography is a rapidly developing field with the potential to produce time-resolved, atomic resolution movies of macromolecules engaged in catalysis. It uses a large number of several µm³ or smaller crystals, from each of which only a small fraction of diffraction data is collected, which are then merged together to form the whole dataset. Our guiding hypothesis is that most enzyme microcrystals (on the order 2 μm³ and smaller) will equilibrate with substrate(s) many times faster than their overall reaction cycle. If this technique is applied to µm³-sized crystals at synchrotrons, then time domains in the µs and longer can be probed. X-ray free electron lasers (XFEL) sources enable us to collect diffraction and spectroscopic data from the same sample on the fs and longer time-scale and with smaller crystals.

We are developing tools that will exploit a variety of triggering strategies for use at i) the LCLS at SLAC National Accelerator Laboratory in the USA, ii) SACLA in Japan, iii) on the Harwell campus at Diamond Light Source, the Central Laser Facility, and for cryo-EM at the electron Bio-Imaging Centre, as well as iv) at the European XFEL in Hamburg, Germany. For example, our novel LCLS acoustic injection systems use focused sound waves to eject on demand picoliter to nanoliter crystal-containing droplets directly into the LCLS x-ray pulse. Our acoustic conveyor belt moves droplets through a series of visible-light laser beams to support time-resolved, pump-probe studies. At the LCLS we are studying several light-triggered reactions, including phytochromes (photon receptors that impact optogentics) and photosystem II (spectroscopy and structure of the water oxidizing complex). Rapid mixing jets and microfluidics systems will support enzyme turnover in time-resolved serial crystallography, which will focus initially on metalloenzyme catalysis.

Feifei Li, E. Sethe Burgie, Tao Yu, Annie Héroux, George C. Schatz, Richard D. Vierstra, Allen M. Orville, "X‑ray Radiation Induces Deprotonation of the Bilin Chromophore in Crystalline D. radiodurans Phytochrome", J. Am. Chem. Soc. (2015) 137, 2792-2795; DOI: 10.1021/ja510923m; PMID: 25650486

Lu Huo, Ian Davis, Fange Liu, Babak Andi, Shingo Esaki, Hiroaki Iwaki, Yoshie Hasegawa, Allen M. Orville Nature Communications (2015) Jan 7; 6:5935; DOI: 10.1038/ncomms6935; PMID: 25565451

Héroux, A., Allaire, M., Buono, R., Cowan, M. L., Dvorak, J. Flaks, L. LaMarra, S., Myers, S. F., Orville, A. M., Robinson, H. H., Roessler, C. G., Schneider, D. K., Shea-McCarthy, G., Skinner, J. M., Skinner, M., Soares, A. S., Sweet, R. M. and Berman, L. E. "Macromolecular Crystallography beamline X25 at the NSLS" J. Synchrotron Rad. (2014). 21, 627-632; PMID: 24763654

Roessler, C. G., Kuczewski, A., Orville, A. M., Allaire, M., Soares, A. S., Héroux, A., "Acoustic Methods for High-Throughput Protein Crystal Mounting at Next-Generation Macromolecular Crystallographic Beamlines" J. Synchrotron Radiation 20, 805 - 808 (2013) PMID: 23955046; please see the associated video at: http://www.youtube.com/watch?v=2xIs3eba8RI

Daughtry, K.D., Xiao, Y., Stoner-Ma, D., Cho, E., Orville, A.M.*, Liu, P.*, and Allen, K.N.*, "Quaternary Ammonium Oxidative Demethylation: X-ray Crystallographic, Resonance Raman and UV-visible Spectroscopic Analysis of a Rieske-type Demethylase," J. Am. Chem. Soc., 134 (5), 2823 – 2834 (2012) PMID: 22224443

Orville, A.M."Single-Crystal Spectroscopy Correlated with X-Ray Crystallography Provides Complementary Perspectives on Macromolecular Function" Chapter 9 in Methods in Protein Biochemistry, edited by Harald Tschesche, ISBN 978-3-11-218934-4, DE GRUYTER, Berlin, Germany, pages 143-164 (2012)

Soares, A.S., Engel, M.A., Stearns, R., Datwani, S., Olechno, J., Ellson, R., Skinner, J.M., Allaire, M., and Orville, A.M. "Acoustically Mounted Microcrystals Yield High Resolution X-ray Structures" a Rapid Report in Biochemistry 50, 4399-4401 (2011); PMID: 21542590

Orville, A.M., Buono, R., Cowan, M., Héroux, A., Schneider, D.K., Shea-McCarthy, G., Skinner, J.M., Skinner, M.J., Stoner-Ma, D., Sweet, R.M. "Correlated Single-Crystal Electron Absorption Spectroscopy and X-ray Crystallography at NSLS Beamline X26-C" J. Synchrotron Radiation 18, 358-366 (2011); PMID: 21525643

Stoner-Ma, D., Skinner, J.M., Héroux, A., Schneider, D.K., Sweet, R.M. Orville, A.M. "Single-Crystal Raman Spectroscopy and X-ray Crystallography at Beamline X26-C of the NSLS" J. Synchrotron Rad. 18, 37-40 (2011); PMID: 21169688

Finnegan, S., Yuan, H., Wang, Y-F., Orville, A.M., Weber, I.T., Gadda, G. "Structural and Kinetic Studies on the Ser101Ala Variant of Choline Oxidase" Archives of Biochemistry and Biophysics 501, 207-213 (2010); PMID: 20561507

Yi, J., Orville, A.M., Skinner, J.M., Skinner, M.J., Richter-Addo, G.B. "Synchrotron X-ray-Induced Photoreduction of Ferric Myoglobin Nitrite Crystals Gives the Ferrous Derivative with Retention of the O-bonded Nitrite Ligand" Rapid Report in Biochemistry, 49, 5969-5971 (2010). PMID: 20568729

Major, D.T., Héroux, A., Orville, A.M., Valley, M.P., Fitzpatrick P.F., Gao, J. "Differential quantum tunneling contributions in nitroalkane oxidase catalyzed and the uncatalyzed proton transfer reaction" Proc. Nat. Acad. Sci. USA, 106, 20734-20739 (2009); PMID: 19926855

Héroux, A., Bozinovski, D.M., Valley, M.P., Fitzpatrick, P.F. and Orville, A.M. "Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction", Biochemistry 48, 3407-3416 (2009); PMID: 19265437

Orville, A.M., Lountos, G.T., Finne­gan, S., Gadda, G., Prabhakar R. "Crystallographic, Spectroscopic, and Computational Analysis of a Flavin-C4a-Oxygen Adduct in Choline Oxidase" Biochemistry 48, 720-728 (2009); PMID: 19133805

Quaye, O., Lountos, G.T., Fan, F., Orville, A.M., Gadda, G. "Role of Glu312 in Binding and Positioning of the Substrate for the Hydride Transfer Reaction in Choline Oxidase" Biochemistry 47, 243-256 (2008); PMID: 18072756

Fitzpatrick, P.F., Bozinovski, D.M., Héroux, A., Shaw, P.G., Valley, M.P., Orville, A.M., "Mechanistic and Structural Analyses of the Roles of Arg409 and Asp402 in the Reaction of the Flavoprotein Nitroalkane Oxidase", Biochemistry 46, 13800-13808 (2007)

Pau, M.Y.M., Davis, M.I., Orville, A.M., Lipscomb, J.D. and Solomon, E.I.; "Spectroscopic and Electronic Structure Study of the Enzyme-Substrate Complex of Intradiol Dioxygenases: Substrate Activation by a High-Spin Ferric Non-heme Iron Site" J. Am. Chem. Soc. 129, 1944-1958 (2007)

Lountos, G.T., Jiang, R., Wellborn, W.B., Thaler, T.L., Bommarius, A.S. and Orville, A.M., "The Crystal Structure of NAD(P)H Oxidase from Lactobacillus sanfranciscensis: Insights into the Conversion of O₂ into Two Water Molecules by the Flavoenzyme", Biochemistry 45, 9648-9659 (2006)

Nagpal, A., Valley, M.P., Fitzpatrick, P.F., and Orville, A.M., "Crystal Structures of Nitroalkane Oxidase: Insights into the Reaction Mechanism from a Covalent Complex of the Flavoenzyme Trapped during Turnover" Biochemistry 45, 1138-1150 (2006)

Fitzpatrick, P.F., Orville, A.M., Nagpal, A., & Valley, M.P. "Nitroalkane oxidase, a carbanion-forming flavoprotein homologous to acyl-CoA dehydrogenase" Arch. Biochem. Biophys. 433 157-165 (2005)

Lountos, G.T., Mitchell, K.H., Studts, J.M., Fox B.G. & Orville, A.M., "Crystal structures and functional studies of T4moD, the toluene 4-monooxygenase catalytic effector protein" Biochemistry 44, 7131-7142 (2005)