Thermodynamic and kinetic studies of protein structure and enzymic mechanisms

Minton, Allen P.

Abstract

(i) Methods are being developed for the detection of very low concentrations of flourescently labeled proteins in the presence of very high concentrations of unlabeled proteins and polymers. These techniques will be used to investigate non-specific protein/protein and protein/polymer interactions in solution. (ii) Optical studies on the structures of amyloid peptides and proteins are continuing.We are particularly interested in origin of the very intense CD spectrum which has been associated with the cross-beta structure of amyloid fibrils. (iii) Riboswitches are functional RNA elements typically located in the 5'-region of m-RNAs, involved in modulation of gene expression. A type II riboswitch from proteobacteria binds magnesium and S-adenosyl methionine (SAM). Previous work has shown that binding of these ligands produces a large decrease in molecular volume (Chen et al, in press). We predicted that these reactions should be very sensitive to the presence of molecular crowding agents. Binding of Mg++ and SAM to the riboswitch induces marked changes in it circular dichroism spectrum. High concentrations of the crowder trimethylamine oxide (TMAO) produces similar changes. Using this change for analysis, we can show that increasing concentrations of TMAO facilitates binding of SAM to the riboswitch.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
• Type: Investigator-Initiated Intramural Research Projects (ZIA)
• Project #: 1ZIADK024964-05
• Application #: 8349677
• Support Year: 5
• Fiscal Year: 2011
• Total Cost: $307,736

Institution

• Name: National Institute of Diabetes and Digestive and Kidney Diseases

Publications

McPhie, Peter; Brown, Patrick; Chen, Bin et al. (2016) Modulation of Conformational Equilibria in the S-Adenosylmethionine (SAM) II Riboswitch by SAM, Mg(2+), and Trimethylamine N-Oxide. Biochemistry 55:5010-20

Chagas, Andrezza C; McPhie, Peter; San, Hong et al. (2014) Simplagrin, a platelet aggregation inhibitor from Simulium nigrimanum salivary glands specifically binds to the Von Willebrand factor receptor in collagen and inhibits carotid thrombus formation in vivo. PLoS Negl Trop Dis 8:e2947

Backus, Keriann M; Dolan, Michael A; Barry, Conor S et al. (2014) The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions. J Biol Chem 289:25041-53

Zhang, Min; Khripin, Constantine Y; Fagan, Jeffrey A et al. (2014) Single-step total fractionation of single-wall carbon nanotubes by countercurrent chromatography. Anal Chem 86:3980-4

Chattopadhyay, Manas K; Fernandez, Cristina; Sharma, Deepak et al. (2011) Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex. Biochem Biophys Res Commun 406:177-82

McGlinchey, Ryan P; Shewmaker, Frank; Hu, Kan-nian et al. (2011) Repeat domains of melanosome matrix protein Pmel17 orthologs form amyloid fibrils at the acidic melanosomal pH. J Biol Chem 286:8385-93

Phillips, Robert S; Miles, Edith W; McPhie, Peter et al. (2010) Effects of hydrostatic pressure on the conformational equilibrium of tryptophan synthase from Salmonella typhimurium. Ann N Y Acad Sci 1189:95-103

Calvo, Eric; Tokumasu, Fuyuki; Mizurini, Daniella M et al. (2010) Aegyptin displays high-affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo. FEBS J 277:413-27

Shewmaker, Frank; McGlinchey, Ryan P; Thurber, Kent R et al. (2009) The functional curli amyloid is not based on in-register parallel beta-sheet structure. J Biol Chem 284:25065-76

Fernandez, Cristina; Minton, Allen P (2009) Static light scattering from concentrated protein solutions II: experimental test of theory for protein mixtures and weakly self-associating proteins. Biophys J 96:1992-8