Date of Award

1-1-2010

Document Type

Campus Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

Lukasz Lebioda

Abstract

CHAPTER 1: REPLACEMENT OF VAL3 IN HUMAN THYMIDYLATE SYNTHASE (HTS) AFFECTS ITS KINETIC PROPERTIES AND INTRACELLULAR STABILITY.

Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of about 27 amino acids which is not present in bacterial TSs. The extension has been considered to play a primary role in protein turnover but not in catalytic activity. Two mutants, V3L and V3F, have strongly compromised dUMP binding, with Km,app values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of loop 181-197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position which stabilizes loop 181-197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a binary complex V3F·FdUMP indicates that the high Km,app value is caused by substrate binding in a non- productive, inhibitory site.

CHAPTER 2: COMPLEXES OF SPERM WHALE MYOGLOBIN (MB) G65T WITH PHENOL AND ETHYLENE GLYCOL.

Sperm whale myoglobin (Mb) mutants at position 65 were designed to mimic the heme environment of homologous dehaloperoxidase (DHP). The distance between the distal histidine and heme iron in G65T Mb increased to 4.56Å (it is 4.30Å in aquomet-Mb), and the turnover number for the oxidative dechlorination of 2,4,6-trichlorophenol (TCP) increased 5-fold. The crystal structure of G65T•phenol complex reveals that the phenol molecule binds in the proximal cavity. Soaking G65T crystals in 4-iodophenol solution yielded a different complex: a molecule of ethylene glycol bound in the distal cavity and coordinated to Fe was observed. The His64-Fe distance in G65I crystal structure increased to 5.11Å, the turnover number for the TCP increased 7-fold. Unlike the G65T•phenol complex, the crystals of G65I soaked in phenol did not contain bound phenol but only an acetate ion coordinated to Fe at the distal site. CHAPTER 3: STRUCTURE OF A FRAGMENT OF HUMAN END-BINDING PROTEIN 1 (EB1).

End-binding protein 1 (EB1) in the presence of other proteins regulates microtubule dynamics and chromosome separation. EB1 is composed of three domains; the N-terminal domain which binds to microtubules, an intermediate domain I and the C-terminal domain responsible for dimerization and interactions with other proteins. While attempting to crystallize EB1 we obtained crystals which turned out to contain two molecules per asymmetric part of the unit cell: one with electron density for residues 1-131 and the other for residues 1-132. These molecules constitute the protease resistant N-terminal domain (residues 1-130) with some extension.

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