The aims of these ongoing integrated cross-disciplinary mutational, biochemical, and structural investigations of NOS are to (1) determine the basis for functional domain interactions, cofactor recognition, and how the NOS enzymes tune these cofactors to perform complex catalytic and redox chemical reactions; (2) identify and apply the features that distinguish among the 3 isozymes to the design of isozyme-specific inhibitors; and (3) understand the diverse regulatory mechanisms that differentially control the 3 isozymes.
In other research, we are investigating photoactive chromophores that enable proteins to translate light energy into defined conformational changes or fluorescence to send biological signals. We are characterizing the mechanisms of light-induced protein activities in the family of green and red fluorescent proteins used as powerful biological markers; in the blue-light receptor photoactive yellow protein; and in the cryptochrome flavoproteins, which are components of circadian clocks in animals and humans. For green and red fluorescent proteins, our high-resolution crystallographic structures are providing important new insights into the mechanisms by which 3 sequential component amino acid residues are spontaneously modified after translation into fluorescent chromophores of different colors. We have determined structures of trapped intermediates in fluorophore synthesis, proposed a novel conjugation-trapping mechanism for fluorophore synthesis, and identified features key to fluorophore formation and spectral tuning.
The structures of red fluorescent protein
clarify differences between green-emitting immature and red-emitting mature chromophores
and the surrounding protein environments and highlight the additional reaction steps involved
in red-shifting the fluorescence of the red fluorescent protein (Fig. 2).
For photoactive yellow protein, we applied ultra-high-resolution crystallography and other biophysical techniques combined with computational chemistry to define mechanisms for photochemical tuning, elucidate intermediates in the light cycle, resolve controversies arising from spectroscopic studies, identify active-site dynamics that favor photoisomerization of the chromophore, and characterize the conformational changes that occur during signal transduction.
For cryptochrome, we sequenced a new gene, identified a new cryptochrome protein family, and determined the first crystal structure. The structure contains the redox-active FAD cofactor bound in an unusual U-shaped conformation with a surrounding positive electrostatic surface consistent with a function in DNA binding. Through structural and functional studies of diverse members of the cryptochrome and homologous DNA-repairing photolyase protein families, we are deciphering how their similarities and differences direct the same cofactor and protein fold to produce different biological responses to light.
Macromolecular Assemblies in Human Health and Disease
We are examining the consequences for human health of appropriate macromolecular recognition and assembly. In collaboration with J. Tainer, the Skaggs Institute, we focus on NOS and superoxide dismutase (SOD), the enzymes that control reactive oxygen species, and the pilus virulence factors responsible for the attachment of pathogenic bacteria to human hosts. For human SOD, we are investigating the mechanisms by which many different, naturally occurring, single-site mutations cause the fatal neurodegenerative disease amyotrophic lateral sclerosis or Lou Gehrig disease. Our structural and biochemical studies of SOD proteins incorporating these human genetic defects have revealed the molecular basis for the disease-causing defect. We are now applying our discovery that the mutant proteins are architecturally destabilized and can form amyloid-like aggregates, resembling the aggregates found in the motor neurons of patients with amyotrophic lateral sclerosis, to strategies for developing therapeutic agents. For pili systems from pathogenic bacteria that cause gonorrhea, meningitis, pneumonia, and cholera, we are characterizing how pili assemble, function, and help these bacteria evade the immune response in humans.
Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry. Biochemistry 44:1960, 2005.
Dunn, A.R., Belliston-Bittner, W., Winkler, J.R., Getzoff, E.D., Stuehr, D.J., Gray, H.B. Luminescent ruthenium(II)- and rhenium(I)-diimine wires bind nitric oxide synthase. J. Am. Chem. Soc. 127:5169, 2005.
Hitomi, K., Oyama, T., Han, S., Arvai, A.S., Getzoff, E.D. Tetrameric architecture of the circadian clock protein KaiB: a novel interface for intermolecular interactions and its impact on the circadian rhythm. J. Biol. Chem. 280:19127, 2005.
Stuehr, D.J., Wei, C.C., Santolini, J., Wang, Z., Aoyagi, M., Getzoff, E.D. Radical reactions of nitric oxide synthases. In: Free Radicals: Enzymology, Signaling, and Disease. Cooper, C.E., Wilson, M.T., Darley-Usmar, V.H. (Eds.). Portland Press, London, 2004, p. 39. Biochemical Society Symposia, Vol. 71.
Tiso, M., Konas, D.W., Panda, K., Garcin, E.D., Sharma, M., Getzoff, E.D., Stuehr, D.J. C-terminal tail residue Arg1400 enables NADPH to regulate electron transfer in neuronal nitric oxide synthase. J. Biol. Chem. 280:39208, 2005.
Tubbs, J.L., Tainer, J.A., Getzoff, E.D. Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation. Biochemistry 44:9833, 2005.
Wei, C.C., Wang, Z.Q., Durra, D., Hemann, C., Hille, R., Garcin, E.D., Getzoff, E.D., Stuehr, D.J. The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation. J. Biol. Chem. 280:8929, 2005.
Wood, T.I., Barondeau, D.P., Hitomi, C., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis. Biochemistry 44:16211, 2005.