T.K.S. Kumar

Structurally Homologous All β-Barrel Proteins Adopt Different Mechanisms of Folding

Acidic fibroblast growth factors from human (hFGF-1) and newt (nFGF-1) (Notopthalamus viridescens) are 16-kDa, all beta-sheet proteins with nearly identical three-dimensional structures. Guanidine hydrochloride (GdnHCl)-induced unfolding of hFGF-1 and nFGF-1 monitored by fluorescence and far-UV circular dichroism (CD) shows that the FGF-1 isoforms differ significantly in their thermodynamic stabilities. GdnHCl-induced unfolding of nFGF-1 follows a two-state (Native state to Denatured state(s)) mechanism without detectable intermediate(s). By contrast, unfolding of hFGF-1 monitored by fluorescence, far-UV circular dichroism, size-exclusion chromatography, and NMR spectroscopy shows that the unfolding process is noncooperative and proceeds with the accumulation of stable intermediate(s) at 0.96 M GdnHCl. The intermediate (in hFGF-1) populated maximally at 0.96 M GdnHCl has molten globule-like properties and shows strong binding affinity to the hydrophobic dye, 1-Anilino-8-naphthalene sulfonate (ANS). Refolding kinetics of hFGF-1 and nFGF-1 monitored by stopped-flow fluorescence reveal that hFGF-1 and nFGF-1 adopts different folding mechanisms. The observed differences in the folding/unfolding mechanisms of nFGF-1 and hFGF-1 are proposed to be either due to differential stabilizing effects of the charged denaturant (Gdn(+) Cl(-)) on the intermediate state(s) and/or due to differences in the structural interactions stabilizing the native conformation(s) of the FGF-1 isoforms.

Production of an anti-Candida peptide via fed batch and ion exchange chromatography.

Interest in peptides as diagnostic and therapeutic materials require their manufacture via either a recombinant or synthetic route. This study examined the former, where a recombinant fusion consisting of an antifungal peptide was expressed and isolated from Escherichia coli. Fed batch fermentation with E. coli harboring an arabinose‐inducible plasmid produced the 12 residue anti‐Candida peptide fused to the N‐terminal of Green Fluorescent Protein (GFPUV). The purification of the fusion protein, using ion‐exchange chromatography, was monitored by using the intrinsic fluorescence of GFPUV. The recombinant antifungal peptide was successfully released by cyanogen bromide‐induced cleavage of the fusion protein. The recombinant peptide showed the expected antifungal activity.

Understanding the Structural Determinants of the Extreme Thermal Stability of Rubredoxin

Rubredoxins are a class of redox-active, iron-sulfur proteins found in strict anaerobes. These 6-7 kDa proteins remain stable at very high temperatures. Rd functions as part of the oxygen detoxification system with a melting temperature near to 200°C. Rd structure consists of a three-stranded β-sheet, a hydrophobic core, a middle loop, and a hydrophilic tail. The β-strands are joined by two iron-coordinating loops each containing two Cys residues, which are liganded to an iron ion to form a tetrahedral iron-sulfur complex. In the present study, Rd gene from Pyrococcus furiosus was successfully cloned into pET22b, E.coli expression vector and the recombinant Rd was successfully purified to homogeneity. While structural information about Rd is known, this study aims to gain a more comprehensive understanding of the structural stability through various biochemical and biophysical studies under different experimental conditions such as change in pH, metal and ionic strength. The details of these results will be discussed in depth.

Understanding the Structural Determinants for the Stability of Human Fibroblast Growth Factor

Human fibroblast growth factor (FGF) is an important protein that plays a role in morphogenesis, angiogenesis and wound healing. Because of these capabilities this protein is of medical interest. Unfortunately the lack of stability of FGF poses a problem to further research. Understanding the structure of FGF at the atomic level is necessary for designing novel FGF variants with enhanced stability and wound healing properties. The specific aim of this study is to test and record the structural stability of FGF through a gradient of salt concentrations. Through thermal denaturation, it was shown that the stability increased slightly with the increased salt concentration. The structure of hFGF-1 has been investigated using a variety of biophysical techniques such as trypsin digestion and circular dichroism. To ensure that the structure was maintained, N-15 enriched human fibroblast growth factor-1 was over expressed and purified. This sample will be used to test the structural stability of hFGF-1 by Heteronuclear Single Quantum Coherence (HSQC) using NMR spectroscopy. The hope of this study is to identify key properties that will allow the optimization of FGF stability. These data can then be used to develop novel mutants with enhanced stability and wound healing properties.

Investigation of the Structural Stability of cpSRP43 Chromodomain2 by Hydrogen-Deuterium Exchange

The essential pathway by which light-harvesting chlorophyll binding proteins are inserted into the thylakoid of the chloroplast is mediated by a unique signal recognition particle (cpSRP). Its novel 43kDa subunit (cpSRP43) contains three chromodomains (CDs). CD2 has been shown to be significant in various aspects of the cpSRP-mediated pathway. While structural information about CD2 is known, this study aims to gain a more comprehensive understanding of the structural stability through analysis of hydrogen-deuterium exchange. 15N-labeled CD2 samples will be produced from over-expression of CD2 in E. coli cultured in labeled media. Subsequently, the readily-occurring exchange between amide hydrogen and solvent deuterium will be monitored by NMR spectroscopy. The kinetics of the exchange can provide useful information about the free energy of exchange and thus the stability.

A Study of Fibroblast Growth Factor and Its Receptor Complex Using Light Scattering

Dynamical light scattering technique was used to study the interaction of fibroblast growth factor and its receptor proteins in solution.