Structural studies on thymidylate synthase and triosephosphate isomerase

Abstract

In recent years, technological advances have led to dramatic increases in the rate at which structural information can be obtained. Simultaneously, there has been an increase in the size of the macromolecules that can be studied. Protein crystallography, as applied to studies on biological macromolecules, has matured tremendously over the last four decades and has helped discover the fundamental principles governing the structure and interactions of biological macromolecules and their roles in life processes. Today, the detailed three dimensional information obtained using X ray crystallography is being used in the design of therapeutic leads, vaccine development, and the rational design of mutations to alter protein stability or activity in desired directions. The investigations reported in this thesis mainly concentrate on two aspects:

To investigate the structural effects of specifically designed interface mutations in the dimeric protein L. casei thymidylate synthase. These mutations were designed to stabilize the enzyme against thermal denaturation by introducing disulfide bridges.

To determine the structure of triosephosphate isomerase from the malarial parasite Plasmodium falciparum and compare it with human triosephosphate isomerase, with the goal of identifying subtle differences that may be exploited in the design of specific inhibitors that target the activity and assembly of the parasite enzyme.

To determine the three dimensional structure of a biological macromolecule by X ray crystallography, it is necessary to obtain a well ordered single crystal. In the present study, we crystallized a triple mutant of L. casei thymidylate synthase and triosephosphate isomerase from Plasmodium falciparum. Both proteins yielded crystals suitable for structure determination by single crystal X ray diffraction.

Chapter 1 - Background Chapter 1 covers the background literature relevant to this thesis. It emphasizes the important contributions made by crystallography to rational protein engineering, including:

elucidation of structure–function relationships, stabilization of industrially important proteins against denaturation, design of fusion proteins to selectively bind disease causing cells or extracellular targets.

A description of the catalytic mechanism of thymidylate synthase (TS) and a brief review of available structural information are presented. This is followed by a discussion of crystallography’s role in rational drug design. Next, a brief description of the malarial parasite Plasmodium falciparum is presented, highlighting:

the importance of glycolysis in its life cycle, the relevance of structural studies of parasite glycolytic enzymes.

Structural features and the catalytic mechanism of triosephosphate isomerase (TIM) are also discussed.

Chapter 2 - Purification and Crystallization Chapter 2 describes purification and crystallization procedures for the TS triple mutant and TIM.

TS purification: hydroxyapatite chromatography ammonium sulfate precipitation gel filtration. TIM purification: ammonium sulfate precipitation anion exchange chromatography.

Both proteins were crystallized using the hanging drop vapor diffusion method:

TS crystals: 10 mg/mL protein, 50 mM potassium phosphate buffer pH 5.8–6.2, 15 mM ammonium sulfate. PfTIM crystals: 10 mg/mL protein in HEPES pH 7.5, 1 mM DTT, 1 mM EDTA, 12% PEG 6000; equilibrated against 24% PEG 6000.

Chapter 3 - X ray Data Collection X ray diffraction data were collected initially using a multiwire area detector and later with an imaging plate system. Key points:

Lyophilized TS crystals underwent unit cell transformation upon X ray exposure; cooling to ~13 °C prevented this. TIM yielded only two good crystals, each in a different space group; a complete dataset was collected from a single crystal.

Chapter 4 - Phase Determination (Molecular Replacement) Phase information, essential for computing electron density maps, cannot be directly measured. Chapter 4 explains:

the molecular replacement method in detail, other phase determination methods, specific strategies used for the structures determined in this thesis.

Chapter 5 - Model Building and Refinement Since diffraction from large macromolecules rarely reaches atomic resolution, initial electron density maps are imperfect. This chapter describes:

manual interpretation of electron density, refinement protocols, structure validation procedures, use of the program O for efficient map fitting.

Solvent flattening improved maps for the lyophilized TS mutant crystals.

Chapter 6 - Lyophilization-Induced Thermostability in TS Lyophilization can alter protein structure; usually these changes are reversible. Here, an unusual trapping of a thermostable non native structure was achieved by lyophilizing a covalently bridged TS mutant. Key findings:

TS triple mutant (E155C/T188C/C244T) becomes remarkably thermostable after lyophilization (~30 °C increase in melting temperature). Lyophilized protein retains ~15% of wild type activity, while unlyophilized protein is fully active. Structural changes appear irreversible, likely due to disulfide bridges trapping altered conformations. Thermal precipitation is abolished, presumably due to protection of aggregation competent regions.

These findings have implications for pharmaceutical and industrial applications of lyophilized proteins.

Chapter 7 - Structure of Plasmodium falciparum TIM P. falciparum causes malignant malaria, affecting over 120 million people annually. The parasite relies solely on glycolysis, making its enzymes attractive drug targets. PfTIM was crystallized and its structure determined. Key features:

TIM is a homodimeric enzyme with ~250 residues per subunit (PfTIM) and adopts the / barrel (TIM barrel) fold. Structural comparison between PfTIM and human TIM reveals subtle but important differences:

surface hydrophobicity near residue 183, parasite residues Cys13 and Phe96 correspond to Met and Ser in human TIM, these residues lie near the active site and at the dimer interface.

Such differences may be exploited to design specific inhibitors targeting PfTIM without affecting human TIM. TIM is implicated as a surface antigen in schistosomal and malarial infections. The structure pinpoints regions that may interact with membrane components or immune factors.

Conclusion The thesis concludes with a summary of major contributions and outlines prospects for future work.