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Conformational Changes in Phosphorylated Proteins


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 affect 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 form which is essential in centrosomal duplication during cell division.

Our aims are:

  • To bacterially over-express and isolate Human centrin 1 and 2 allowing for an abundant source of protein required for its biophysical study.
  • To determine the calcium binding mechanism and relative affinity towards calcium in the terminal domains of centrin using attenuated total reflectance (ATR) Fourier transform infrared (FT-IR) spectroscopy and two-dimensional correlation analysis.
  • To determine the extent of self-association of this calcium binding protein.
  • To investigate the structural changes and relative stability of centrin upon phosphorylation as compared to the unphosphorylated form of the protein using FT-IR spectroscopy and differential scanning calorimetry (DSC).
  • We aim to define the protein/protein interactions and determine the structural and dynamic changes that occur during this interaction.
  • To perform molecular modeling based on the spectroscopic and thermodynamic data obtained in the experiments proposed.

This interdisciplinary approach to the study of phosphorylated and unphosphorylated 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 solution structure of these proteins via NMR.


Belinda Pastrana-Rios.
"Simulation of FT-IR spectra and 2D-COS analysis for the thermal perturbation of apo-centrin." Journal of Molecular Structure,799, Issues 1-3, 6 November 2006, Pages 163-16. more info...

Zuleika Sanoguet, Muriel Campbell, Sindia Ramos, Christina Seda, Luis Pérez Moreno and Belinda Pastrana-Rios.
"Effects of Phosphorylation in Chlamydomonas Centrin Ser 167". Calcium Binding Proteins. volume 1. april/may/june 2006. Pages: 108 - 114. more info...

Susan M. Meyn, Christina Seda, Muriel Campbell, Kevin L. Weiss, Haitao Hua, Belinda Pastrana-Rios and Walter J. Chazin.
"The biochemical effect of Ser167 phosphorylation on Chlamydomonas reinhardtii centrin.". Biochem Biophys Res Commun.2006 Mar 31;342(1):342-348. Epub 2006 Feb 7. more info...

Ortiz M, Sanoguet Z, Hu H, Chazin WJ, McMurray C, Salisbury JL, Pastrana-Rios B.
"Dynamics of hydrogen-deuterium exchange in Chlamydomonas centrin". Biochemistry. 2005 Feb 22;44(7):2409-18. more info...

Pastrana-Rios, B.
"Exploring Structure/Function Relationship of Macromolecules at the Undergraduate Level". 2004 J. Chem. Educ. 81, 837-840. more info...

Mayra Ocasio, Johnny R. Maury-Evertsz, Belinda Pastrana-Ríos, and Gustavo E. López
"Exploring repulsive interactions in a model helical peptide: A parallel tempering Monte Carlo study" 2003 J. Chem. Phys. 119, 9274-9279. more info...

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