Structural and thermodynamic studies on protein stability

Abstract

Proteins are the products of gene sequences. Their form and function depend on their amino acid sequence. The three dimensional structure of a protein, together with its local concentration and spatial location, determines its role in a biological system. The steady progress in molecular biology-wherein genes corresponding to proteins of interest can be cloned and overexpressed-along with advances in instrumentation and computing power, has revolutionized the pace and depth of our understanding of biological processes. The study of proteins encompasses three broad areas:

Translation of an amino acid sequence into a three dimensional structure Dependence of biological function or activity on structure Modulation of function in accordance with biochemical requirements

The investigations reported in this thesis include:

Structural and biophysical characterization of a calcium binding protein (CaBP) from the parasitic amoeboid Entamoeba histolytica. CaBP is a highly soluble, monomeric protein (14.7 kDa) without disulfide bonds or cis prolyl peptide bonds. It binds four Ca² ions per monomer, and both the holo and apo forms are thermostable. CaBP has therefore been used as a model system to study protein structure and stability.

Structural effects of site specific mutations at the dimeric interface of thymidylate synthase (TS) from Lactobacillus casei and triosephosphate isomerase (TIM) from Plasmodium falciparum. The goal was to elucidate structural determinants of dimer stability. Three mutants-two TS mutants (R178F and R218K) and one TIM mutant (Y74C)-were studied. Crystal structures of TS R178F and TIM Y74C were solved. The TS R218K mutant did not crystallize.

The structural and thermodynamic studies on monomeric CaBP and the dimeric TS and TIM are organized across the following chapters:

Chapter 1 - Background This chapter surveys literature relevant to the studies in this thesis, including a brief overview of protein stability. Data on monomeric proteins is compared with that on oligomeric proteins, since unfolding in multimeric proteins may involve sequential or coupled unfolding and dissociation events. A section on protein engineering as a tool to modulate function or stability is included. Literature on the three model systems-CaBP, TS, and TIM-is summarized.

Chapter 2 - Thermodynamics of CaBP This chapter reports thermodynamics of metal ion binding and denaturation of CaBP using isothermal titration calorimetry (ITC) at pH 7.0 and 20 °C. Key findings:

CaBP possesses four Ca² binding sites:

Two low affinity exothermic sites showing positive cooperativity Two high affinity sites, one endothermic and one exothermic, also with positive cooperativity

Binding constants for Ca² were verified using competitive binding with 5,5 Br BAPTA.

Mg² binding is single site, endothermic, but behavior changes when CaBP is pre saturated with magnesium or when calcium is present.

A cleaved N terminal peptide fragment reveals two Ca² specific sites, indicating one high affinity Ca² /Mg² site, one high affinity Ca² specific site, and two low affinity Ca² specific sites.

Effects of GdnHCl induced unfolding were studied:

At 0.25 M GdnHCl, Ca² binding affinity decreases and cooperativity is lost. At >2.5 M GdnHCl (where tertiary unfolding occurs), Ca² binding is completely lost. The domain containing low affinity sites unfolds first.

Chapter 3 - Unfolding of Apo and Holo CaBP Equilibrium denaturation studies show:

Stable intermediates at low denaturant concentrations that bind ANS dye. Hydrodynamic properties studied using size exclusion chromatography. Stopped flow fluorescence used to measure association/dissociation rates of ion binding and unfolding kinetics. Preferential binding of Gdn ions to N terminal cation binding sites influences EF hand sites in the C terminal domain.

Chapter 4 - Crystallographic Studies on CaBP CaBP was crystallized using MPD as precipitant. X ray diffraction data were collected using a MAR imaging plate detector.

Crystals belong to hexagonal space group P6 22, with unit cell parameters: a = b = 96.21 Å, c = 65.48 Å. Molecular replacement suggests a structure similar to calmodulin, with differences in inter domain orientation.

Chapter 5 - TS Interface Mutants R178F and R218K Effects of arginine to phenylalanine (R178F) and arginine to lysine (R218K) mutations:

R178F mutant shows apparent thermal stability and decreased aggregation. R218K is destabilized by ~5 °C and failed to crystallize.

Crystal structure of R178F shows no dramatic changes, suggesting stability differences arise from mutational effects in folded vs. unfolded states. Results also highlight utility of site specific substitution to identify regions involved in non native protein association.

Chapter 6 - Radiation Induced Unit Cell Transformation in TS Crystals Certain TS crystal forms in P6 22 space group undergo unit cell elongation along the c axis upon X ray exposure. This chapter:

Analyzes transformations across two crystal forms of R178F Compares transformed and untransformed structures Concludes that rearrangements involve concerted changes in packing and small adjustments in intersubunit contact regions.

Chapter 7 - Crystal Structure of TIM Y74C The Y74C mutation introduces an intersubunit disulfide bridge between Cys74 (mutant) and native Cys13 of the opposite monomer. Findings:

Bis oxidized TIM Y74C shows reduced activity but similar stability to wild type. The reduced form is significantly less stable. Crystal structure shows effects on interfacial cavities and role of disulfide tethering in stability restoration.

Chapter 8 - Summary Summarizes major findings and outlines future directions motivated by this work.