My group determines the atomic structures of proteins and seeks to understand how protein structure gives rise to protein function – both in vitro and in living cells. The problems we study have at their heart a fundamental structure-function question, but also address questions of importance to human health. Our approaches include X-ray crystallography, rapid kinetic measurements, spectroscopy, theory, protein expression, drug discovery, molecular genetics and related techniques.

We are particularly interested in nitric oxide signaling mechanisms. Nitric oxide (NO) is a small reactive molecule produced by all higher organisms for the regulation of an immensely varied physiology, including blood pressure regulation, memory formation, tissue development and programmed cell death. We are interested in two NO signaling mechanisms: binding of NO to heme and the nitrosylation (nitrosation) of cysteines. NO, produced by NO synthase, binds to soluble guanylate cyclase (sGC) at a ferrous heme center, either in the same cell or in nearby cells. Binding leads to conformational changes in heme and protein, and to induction of the protein’s catalytic function and the production cGMP. NO can also react with cysteine residues in proteins, giving rise to S-nitroso (SNO) groups that can alter protein function. We are studying the mechanistic details surrounding cGMP and SNO production, and the signaling consequences of their formation.

For reversible Fe-NO chemistry we are studying soluble guanylate cyclase and the nitrophorins, a family of NO transport proteins from blood-sucking insects. Our crystal structures of nitrophorin 4 extend to resolutions beyond 0.9 angstroms, allowing us to view hydrogens, multiple residue conformations and subtle changes in heme deformation. For reversible SNO chemistry, we are studying thioredoxin, glutathione S-nitroso reductase (GSNOR) and also sGC. For regulation in the cell, we have constructed a model cell system based on a human fibrosarcoma called HT-1080, where sGC, NO synthase, thioredoxin and GSNOR can be manipulated in a functional cellular environment. With these tools, we are exploring the molecular details of NO signaling and whole-cell physiology.

• Kondrashov DA, and Montfort WR. Nonequilibrium dynamics simulations of nitric oxide release: comparative study of nitrophorin and myoglobin. J. Phys. Chem. B (2007), in press.

• Amoia AM and Montfort WR. Apo-Nitrophorin 4 at Atomic Resolution. Protein Sci. (2007), in press.

• Weichsel A, Brailey JL, and Montfort WR. (2007) Buried S-Nitrosocysteine Revealed in Crystal Structures of Human Thioredoxin, Biochemistry 46, 1219-1227.

• Weichsel A, Maes EM, Andersen JF, Valenzuela JG, Shokhireva T, Walker FA, and Montfort WR. (2005) Heme-assisted S-nitrosation of a proximal thiolate in a nitric oxide transport protein, Proc. Natl. Acad. Sci. USA 102, 594-599.

• Maes EM, Roberts SA, Weichsel A, and Montfort WR. (2005) Ultrahigh Resolution Structures of Nitrophorin 4: Heme Distortion in Ferrous CO and NO Complexes, Biochemistry 44, 12690-12699.

• Singh SK, Grass G, Rensing C, and Montfort WR. (2004) Cuprous oxidase activity of CueO from Escherichia coli, J. Bacteriol. 186, 7815-7817.

• Kondrashov DA, Roberts SA, Weichsel A, and Montfort WR. (2004) Protein functional cycle viewed at atomic resolution: conformational change and mobility in nitrophorin 4 as a function of pH and NO binding, Biochemistry 43, 13637-13647.

• Roberts SA, Weichsel A, Grass G, Thakali K, Hazzard JT, Tollin G, Rensing C, and Montfort WR. (2002) Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli, Proc. Natl. Acad. Sci. USA 99, 2766-2771.

• Weichsel A, Andersen JF, Roberts SA., and Montfort WR. (2000) Reversible nitric oxide binding to nitrophorin 4 from Rhodnius prolixus involves complete distal pocket burial, Nat. Struct. Biol. 7, 551-554.