CHANGES IN CONFORMATION OF PHOSPHORYLATED PROTEINS

University Of Puerto Rico Mayaguez, Mayaguez PR

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Abstract

Protein phosphorylation is known to control a wide range of cellular events such as: metabolism, cell division, membrane transport and gene expression among others. However, the Biophysical basis of regulation by phosphorylation has only been studied in two systems. A combined spectroscopic-molecular modeling approach will provide the necessary structural information to aid in the understanding of the inhibitory or activating effect of phosphorylation in protein function. One possible mechanism for the inhibitory or activating effects on protein function is that phosphorylation may induce a change in conformation and thus effect its interaction with other proteins or biological molecules. A model protein used in this study is centrin. Centrin has been found to be a ubiquitous protein associated with centrioles/basal bodies, centrosomes, and mitotic spindle poles and therefore is an integral component of the microtubule organizing center (MTOC) which is responsible for distributing the chromosomes to the daughter cells. Recent findings suggests an activating effect on centrin in its phosphorylated forms which is essential in centrosomal duplication during cell division. Our aims are: (1) To over-express Human 1, 2 and Chlamydomonas centrin in a bacterial system allowing for an abundant source of protein required for the biophysical study. (2) To isolate and characterize centrin using various chromatographic techniques and conform its purity via SDS-PAGE, western blot analysis, amino acid sequence and time of flight (TOF) mass spectrometry. (3) To phosphorylate centrin in vitro using protein kinase A (PKA) and adenosine triphosphate (ATP) in sufficient quantities for Biophysical characterization. (4) To investigate phosphorylated and un-phosphorylated centrin conformation using Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD). (5) To use two-dimensional infrared correlation for the comparison of spectra obtained for proteins in their phosphorylation and un-phosphorylated states. (6) To model these results via molecular modeling techniques. This inter-disciplinary approach to the study of phosphorylated centrin will enable us to establish a relationship between conformation and function in a protein regulated by phosphorylation. The implications of such a study will be to contribute molecular level information which will aid in the understanding of the cellular events studied such as cell division and uncontrolled cell division such as in cell carcinomas. Long-term goals are to study variant proteins generated by site-specific mutagenesis using vibrational spectroscopy and to determine the structure of these proteins via NMR.