The physiology and behavior of virtually all organisms oscillate with a periodicity of approximately 24 h, which is now known to be the result of an endogenous oscillator called the circadian clock. All circadian clocks studied thus far are based on a common design composed of a transcription/translation feedback loop of clock genes and clock proteins. The activity of a clock protein is modulated by its interactions with other clock proteins and these protein-protein interactions play an important role in the periodicity of the circadian rhythm. As no high-resolution structure of a circadian clock protein has been solved, no firm structural predictions can be made as to how clock proteins interact and, therefore, how their interactions help achieve a normal (or abnormal) circadian periodicity of the oscillator. As a result, the mechanism of the circadian pacemaker at the structural level is still unclear. It is proposed here that significant advances in understanding the structural basis of circadian time keeping can be achieved by investigating the three-dimensional structures and dynamics of the clock proteins of Synechococcus elongatus, SasA, KaiA, KaiB and KaiC using nuclear magnetic resonance spectroscopy (NMR).
The specific aims are to solve the structures of these clock proteins by NMR and then elucidate the protein-protein interactions central to setting the pace of the circadian rhythm. The data base of genetic and biochemical information on the function of the circadian clock of S. elongatus is quite extensive and, if mapped onto the three dimensional architecture of the circadian clock, will allow insights into the molecular mechanism of a circadian pacemaker. For example, whether residues E103, R249 and E274 of KaiA, whose mutation alter the circadian rhythm, are clustered together on the surface or make important hydrogen bonds in the core of the protein, are important questions of the research proposed here. If the residues are clustered together on the surface, NMR studies will ascertain whether they form part of the dimer interface with KaiB, KaiC, and/or SasA. If the residues are buried in the core of the protein, comparisons of the E1O3K, R249H and E274H mutants with wild-type KaiA will reveal aspects of the structures and dynamics that are important to the function of KaiA. The long-range objective is to eventually investigate clock protein complexes by NMR in an effort to understand how specific protein-protein interactions modulate circadian rhythms. Key questions of our long-range objectives are whether interactions between KaiA, KaiB, SasA, and KaiC involve allostery, localized changes in backbone or side chain dynamics, displacement of tightly bound water molecules or rearrangement of crucial hydrogen bonds, and how they play important roles in setting the pace of the circadian rhythm. Although some of the conclusions resulting from the work proposed here will be specific to S. elongatus, others, should have universal ramifications for all organisms, including humans.
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