Educational Background:

PhD, The Scripps Research Institute 1996 BSc, University of Manitoba, Canada, 1990

Awards:

•2005 Alfred P. Sloan Research Foundation Fellow •Searle Scholar •NSF CAREER Award •Camille and Henry Dreyfus Young Faculty Award •Research Innovation Award, Research Corporation •Helen Hay Whitney Postdoctoral Fellow •Skaggs Foundation Research Fellow •1967 Predoctoral Fellow, Natural Sciences and Engineering Research Council of Canada

Research Description:

Research in the Crane Group is directed towards understanding interactions among proteins, electrons, and photons. Specifically, we are interested in how metalloenzymes stabilize transient intermediates during catalysis, how protein structure controls long-range electron transfer, and how photo and redox processes are used in biological information transfer. To correlate protein structure directly with reactivity we combine genetic and chemical manipulation of proteins, atomic-resolution structure determination, and novel photochemically triggered experiments in single crystals.

Understanding How Structure Controls Electron Transfer and Activated States in Metalloenzymes
We combine photochemistry and X-ray crystallography to study long-range electron transfer reactions between protein partners. Our goal is to directly correlate redox reactivity with atomic structure. Catalytically key metalloenzyme redox states can be difficult to characterize because they are often unstable and generated transiently in situ. We uniformly stimulate chemical reactions in single protein crystals by electron transfer to and from transition metal active centers at rates where important species can be observed by time-resolved crystallography or captured by cryocrystallography. Systems of interest include the production of nitric oxide by mammalian nitric oxide synthases, a heme-peroxide intermediate important in the generation of reactive oxidants for biosynthesis and detoxification, and intermediates in the six-electron reductions of sulfite to sulfide and nitrite to ammonia by sulfite and nitrite reductases.

Bacterial Nitric Oxide Synthases:
Mammalian nitric oxide synthases (NOSs) catalyze oxidation of arginine to the potent cytotoxic agent and intercellular signal nitric oxide. This chemistry is conserved by homologous prokaryotic NOS proteins despite these proteins functioning in the unexpected role of nitrating secondary metabolites. We undertake coupled biochemical and crystallographic studies of bacterial NOS proteins to understand fundamental properties governing NO synthesis and NOS-mediated nitration reactions. Investigation of bacterial NOS substrates, products, and reaction intermediates probe the mechanism of their selective biosynthetic nitration reactions. In particular, we study NOSs from certain Streptomyces strains that function to nitrate a tryptophanyl-moiety of an important class of plant toxins and NOS from the radiation resistant bacterium Deinococcus radiodurans that forms a functional complex with an unusual tryptophanyl tRNA synthetase induced during DNA repair responses.

Light and Redox Sensing
The ability to sense and respond to the environment is a primary requirement of any living organism. We are interested in the biophysical mechanisms that allow organisms to monitor energy in their surroundings. Specifically, we are studying proteins involved in bacterial taxis and mammalian circadian clocks. In these systems, light or reducing energy is trapped by cofactors within sensory proteins. Through unknown mechanisms this captured energy is transduced to the production of new interactions among response proteins within the cell. We aim to determine structures of sensory proteins in different redox states and in association with target response proteins. We also intend to characterize electron transfer mechanisms that allow energy conversion among components of signaling pathways.

Bacterial Chemotaxis
During bacterial chemotaxis ligand binding to transmembrane chemoreceptors regulates the activity of the histidine kinase CheA. CheA initiates cytoplasmic signaling by phosphorylating the response regulator CheY, which diffuses from the membrane to modulate the flagellar motor. How receptors regulate CheA activity is a central question in understanding prokaryotic signal transduction. We aim to understand in molecular detail how the receptor:kinase complexes propagate signals and then adapt to those excitations. Thus, we determine structures of the individual proteins and their domains with high-resolution X-ray diffraction, use small-angle X-ray scattering (SAXS) to construct low-resolution envelopes of high molecular weight complexes and apply resonance energy transfer measurements to track associations and domain motions in solution.

Figure 1:
The interfaces of the yCc:ZnCcP (A) and hCc:ZnCcP (B). Photochemically driven electron transfer reactions in crystals of these protein complexes reveal how protein structure and molecular recognition mediate long-range electron tunneling reactions.

Figure 2:
³ZnCcP decay kinetics monitored at 460 nm. (Normalized D-absorbance) for Fe(III)yCc:ZnCcP (red) and Fe(II)yCc:ZnCcP (green) as monitored in single crystals using laser pump-probe spectroscopy. Inset, Kinetics of hCc:ZnCcP ET. Rate of ZnCcP⁺ formation at 680 nm (blue) matches rate of ³ZnCcP decay at 460 nm (yellow) in crystals.

Figure 3:
Overall structures and surface properties of bacterial NOS (A) and mammalian inducible NOS oxygenase domain (B). Solvent accessible surfaces for bsNOS and iNOS (C and D) colored by electrostatic potential. Key regions for binding substrate, cofactors and reductase proteins are indicated (I, II and III, respectively).

Selected Publications:

Zoltowski, B.D., Schwerdtfeger, C., Widom, J. Loros, J.J., Bilwes, A. M., Dunlap, J.,C. & Crane, B.R. (2007) Conformational switching in the fungal light sensor Vivid. Science, 316 1054-1057.

Park, S. Y., Borbat, P. P., Gonzalez-Bonet, G., Bhatnagar, J., Freed, J. H., Bilwes, A. M. & Crane, B. R. (2006). Reconstruction of the chemotaxis receptor:kinase assembly Nat. Struct. Mol. Biol., 13, 400-407.

Chao, X., Muff, T., Park, S., Zhang, S., Pollard, A. M., Ordal, G., Bilwes, A. M. & Crane, B.R. (2006). A receptor-modifying deamidase in complex with a signaling phosphatase reveals a mechanism of reciprocal regulation. Cell 124, 561-571.

Kang, S. A. & Crane, B.R. (2005). Effects of interface mutations on association modes and electron transfer rates between proteins. Proc. Natl. Acad. Sci. USA, 102, 15465-15470.

Kers, J. A., Wach, M. J., Krasnoff, S. B., Widom, J., Cameron, K. D., Bukhalid, R. A., Gibson, D. M., Crane, B. R. & Loria, R. (2004). Nitration of a peptide phytotoxin by bacterial nitric oxide synthase. Nature 429, 79-82.