The physical biochemistry research in our group centers on understanding the molecular basis for affinity, specificity, and cooperativity in macromolecular interactions. We apply a wide range of biochemical and biophysical experiments and computational approaches in an effort to obtain a picture that can be integrated with the biological role of each system we study. A unifying feature of our work is the application of ligand-binding theory to quantify molecular interactions. A few of the systems under study at present are highlighted.
TrpR is a transcription factor that regulates bacterial metabolism by sensing intracellular levels of free tryptophan. The dimeric protein is thermostable to >90°C yet displays remarkable flexibility in its tryptophan- and DNA-binding domains. Binding of tryptophan is not cooperative despite the fact that each binding site contains residues from both monomers (1). DNA-binding specificity relies on a complex interplay of affinity and cooperativity (2). These mysteries led us to isolate a temperature-sensitive mutant, L75F with phenylalanine (pink) replacing leucine 75, that has an average NMR structure (left; 3) very similar to that of wildtype TrpR and only modest differences in tryptophan- and DNA-binding affinity and specificity but profoundly altered long-range dynamics (4). Crystal structures (middle and right; 5) suggest that a bistable helix underlies the unusual dynamic properties of the mutant. We are presently testing whether this hypothesis explains the many puzzling NMR observations for wildtype TrpR. A bistable helix affixed to a very stable protein core might suppress long-range intraprotein communication and could explain the lack of cooperativity of tryptophan binding.
WrbA is an FMN-binding tetramer (middle; 6) that can reduce water-soluble quinones in vitro using electrons from NADH. Although it is conserved from bacteria to higher plants (7), its physiological function is unknown. We have suggested it represents a bridge (8) between monomeric bacterial flavodoxins that shuttle single electrons between protein partners via FMN and multimeric mammalian oxidoreductases that shuttle two electrons between substrates in various metabolic processes via FAD. Its cavernous active site (left, right; 9) is connected to the protein surface by a hydrophobic channel that may permit access by membrane-bound substrates. A search for the native physiological substrates of WrbA is under way using biochemical and biological approaches. The redox kinetics of WrbA share with several other NADH-dependent enzymes unusual two-plateau Michaelis-Menten plots (10) that have never been explained in molecular terms. We are presently testing possible explanations for this behavior of WrbA with the aim of understanding the other enzymes as well.
ArgR is another transcription factor that regulates bacterial metabolism, in this case by sensing intracellular levels of free arginine. It is allosterically activated for DNA binding (left; 11, 12) by cooperative binding of arginine to the hexamer (13). Through a novel combination of thermodynamic and computational approaches (14; right) we have recently discovered that the hexamer undergoes a global dynamic process that is interrupted by the bound ligand (15). The mechanism of the allosteric activation signal appears to involve both redistribution of the dynamic conformational ensemble and ligand-induced adaptation, an hypothesis that is presently under investigation.
Selected Recent Publications
- Strawn R, Melichercik M, Green M, Stockner T, Carey J, Ettrich R (2010) "Symmetric allosteric mechanism for hexameric E. coli arginine repressor exploits competition between L-arginine ligands and resident arginine residues." pLOS Comput. Biol. 6, e1000801.
- Jin, L., Xue, W.-F., Fukayama, J.W., Yetter, J., Pickering, M., & Carey, J. (2005) “Asymmetric allosteric activation of the symmetric ArgR hexamer,” J. Mol. Biol. 346, 43-56.
- Sunnerhagen, M.S., Nilges, M., Otting, G., & Carey, J., (1997) "Solution structure of the DNA-binding domain and model for the complex of multifunctional, hexameric arginine repressor with DNA," Nature Structural Biology 4, 819-825.
- Szwajkajzer, D., Dai, L., Fukayama, J.W., Abramczyk, B., Fairman, R., & Carey, J. (2001) "Quantitative analysis of DNA binding by E. coli arginine repressor," J. Mol. Biol. 312, 949-962.
- Kishko, I., Harish, B., Beri, D., Gustavsson, T., Ettrich, R., & Carey, J. manuscript in preparation.
- Wolfová J, Smatanová IK, Brynda J, Mesters JR, Lapkouski M, Kuty M, Natalello A, Chatterjee N, Chern S-Y, Ebbel E, Ricci A, Grandori R, Ettrich R, Carey J. (2009) “Structural organization of WrbA in apo- and holo-protein crystals,” Bioch. Biophys. Acta, 1794, 1288-1298. cover story
- Carey J, Brynda J, Wolfová J, Grandori R, Gustavsson T, Ettrich R, Smatanová IK. (2007) “WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H:quinone oxidoreductases,” Protein Science 16, 2301-2305. cover story
- Grandori, R., & Carey, J. (1994) "Six new candidate members of the alpha/beta twisted open-sheet family detected by sequence similarity to flavodoxins," Prot. Sci. 3, 2185-2193.
- Grandori, R., Khalifah, P., Boice, J.A., Fairman, R., Giovanielli, K., & Carey, J. (1998) "Biochemical characterization of WrbA, founding member of a new family of multimeric flavodoxins," J. Biol. Chem. 273, 20960-20966.
- Carey, J., Benhoff, B., Harish, B., Yuan, L., & Lawson, C.L. manuscript in preparation.
- Jin, L., Fukayama, J.W., Pelczer, I., & Carey, J. (1999) "Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor," J. Mol. Biol. 285, 361-378.
- Tyler, R., Pelczer, I., Carey, J., & Copie, V. (2002) “Three-dimensional solution NMR structure of apo-L75F-TrpR, a temperature-sensitive mutant of the tryptophan repressor protein,” Biochemistry 41, 11954-11962.
- Yang, J., Gunasekera, A., Lavoie, T.A., Jin, L., Lewis, D.E.A., & Carey, J. (1996) "In vivo and in vitro studies of TrpR-DNA interactions," J. Mol. Biol. 258, 37-52.
- Jin, L., Yang, J., & Carey, J. (1993) "Thermodynamic analysis of ligand binding to trp repressor," Biochemistry 32 7302-7309.