Student Theses and Dissertations

Date of Award

2008

Document Type

Thesis

RU Laboratory

Young Laboratory

Keywords

circadian clocks, protein period, drosophila

Abstract

Circadian clocks regulate changes in behavior and physiology that occur with a period of approximately 24 hours and are based on negative feedback loops. The molecular components of circadian clocks are conserved among animals, and a key element in all such clocks is the protein Period (PER), a circadian transcription inhibitor. The stable production, posttranslational modification, and nuclear translocation of PER all contribute to the timing of the clock. This work describes the synthesis and purification of various Drosophila PER fragments for biochemical and crystallographic analysis. Several stable PER fragments are identified, including one crystallizable fragment. The structure of the crystallized fragment is provided, and the implications of a critical intermolecular PER-PER interaction are explored. The crystallized region of PER includes the PAS (PER-ARNT-SIM) domain (230-512) as well as two subsequent helices (512-575). Unlike a previously published structure for a similar fragment [1], this structure shows a closed PER homodimer that relies on a flexible hydrophobic interaction between the final helix of one molecule and the PAS domain of its partner. Biochemical and mutational analyses of the crystallized fragment confirm the robust yet dynamic quality of this interaction. A critical residue in the dimerization interaction, Valine 243 is positioned at the center of the hydrophobic interface. Previous research has established that a lengthenedperiod phenotype in flies is caused by a point mutation at this residue, which changes the valine to asparitc acid [2]. Further studies have shown that the long period of flies bearing this mutation (perLong flies) results from a delay in the PER nuclear translocation [3]. Disruption of the hydrophobic molecular interface introduced by V243D suggests the delay in nuclear entry associated with perLong may be related to a defect in the PER self-binding interaction demonstrated in the crystal structure. The proximity of the observed PER-PER binding interface to the proposed PER-TIM binding interfaces also introduces the possibility that PER self binding may affect on the PER-TIM interaction. This work concludes with a proposed model for how disrupting the observed PER-PER intermolecular interaction may delay nuclear entry.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/133

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