Thermal Denaturation and Domain Stability of NAMPT Protein

Abstract

Nicotinamide phosphoribosyltransferase (NAMPT) is also known as Pre-B cell colony enhancing factor (PBEF) or visceral fat protein (Visfatin). It is a highly conserved 52-kDa protein, found in living species from bacteria to humans [1], and acts as a rate-limiting step in the salvage pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. NAMPT is involved in several inflammatory deceases notably, sepsis, acute lung injury, and rheumatoid arthritis [2]. It is also known to activate insulin receptor causing type II diabetes [3]. Because of such medicinal relevance, NAMPT has been extensively investigated by X-ray crystallography, aiming at developing inhibitors [1]. However, physiologically relevant biochemical characterization including nuclear magnetic resonance (NMR) spectroscopy for NAMPT is scarce. NMR methods are complementary to X-ray crystallography, and provide valuable information on the structure and functional dynamics simultaneously. They offer an important tool for understanding biological functioning [4]. Bacterial expression, biochemical characterization, and thermal stability studies on NAMPT have been undertaken with the objective of optimizing conditions for NMR experiments. Secondary structure estimates using circular dichroism (CD) spectroscopy are consistent with crystallographic data. Thermal unfolding studies indicated an unusually high degree of thermal stability of NAMPT showing a melting temperature of about 90 °C. Results for dynamic light scattering (DLS) experiments showed a salt-dependent multimeric state. Presently, we are optimizing solvent conditions for obtaining the stable oligomeric state relevant to NMR experiments. Calorimetric studies are also under progress. We successfully expressed NAMPT in E.coli and are developing strategies for better yields and isotope labelling. [1] T. Wang et al. (2006) Nat. Struct. Mol. Biol. 13, 661. [2] S. M. Camp et al. (2015) Sci. Rep. 5, 13135. [3] A. Fukuhara et al. (2005) Science 307, 426. [4] T. R. Molugu et al. (2015) Concepts Magn. Reson. (in press).