Assistant University
Resume:
Meera Bhanu Kolayarattil
Storrs, CT 06268
860-***-**** (Cell)
*****.*****@*****.***
PROFILE:
Specialization in recombinant protein expression and purification, molecular
biology, protein biochemistry and biophysics, microbiology and fluorescence
spectroscopy.
RELEVANT SKILLS:
• PCR sub-cloning, QuikChange mutagenesis and DNA extraction
• Recombinant protein expression and purification in E. coli
• Gel filtration/size exclusion, affinity, ion exchange and hydrophobic
interaction chromatography
• High Performance Liquid Chromatography (HPLC) peptide purification
• Liposome preparation for membrane-mimetic in-vitro studies and analyses
• Electrophoresis and immuno-blotting
• Substituted Cysteine Accessibility Method (SCAM)
• In vitro UV cross-linking and proteolytic digestion assays
• Tryptophan fluorescence spectroscopic analysis and Fluorescence
Resonance Energy Transfer (FRET) detection and analysis for enzyme-
ligand binding and interaction
EDUCATION:
• Doctor of Philosophy, Biochemistry (Anticipated Sept 2013)
University of Connecticut, Storrs, CT
• Master of Science, Biochemistry, 2009
University of Connecticut, Storrs, CT
• Bachelor of Science, Microbiology, Chemistry, Botany, 2005
St. Joseph’s College, Bangalore University, Bangalore, India
MEERA BHANU KOLAYARATTIL
WORK EXPERIENCE:
University of Connecticut- Storrs, CT
Graduate Research Assistant January 2007- present
Molecular and Cell Biology Department
Advisor: Dr. Debra A. Kendall
• Extensive experience with recombinant protein expression and purification.
• Elucidation of protein-ligand binding sites and mapping of the 3-D binding
domain for Escherichia coli SecA and Signal Peptidase I (SPase I) with signal
peptides and truncated preproteins using SCAM and fluorescence spectroscopy
• Identification of the dimeric interface of SecA
• Analysis of the effect of signal peptide hydrophobicity on binding to SecA.
• Lipid composition analysis and its significance in secretory pathways of E. coli.
• Knowledge of OriginLab, Graph Pad Prism, ImageJ and PyMol software.
HONORS/SCHOLARSHIPS:
2003 Certificate of Academic Excellence, St. Joseph’s College,
Bangalore University
2005 Certificate of Academic Excellence, St. Joseph’s College,
Bangalore University
2008 Suraj Manrao Award for Best Poster Presentation,
North East Structural Symposium, University of Connecticut
2009 UConn Molecular & Cell Biology Department
Summer Research Fellowship
2010 UConn Graduate School Doctoral Dissertation Fellowship
MEERA BHANU KOLAYARATTIL
PUBLICATIONS:
Bhanu M.K., and Kendall D.A. (2011) Membrane Protein Biogenesis and
Assembly at the Endoplasmic Reticulum Membrane, in The Structure of Biological
Membranes, 3rd Edition. Edited by Philip Yeagle. CRC Press.
Auclair, S. M., Bhanu, M. K. and Kendall, D. A. (2012), Signal peptidase I:
Cleaving the way to mature proteins. Protein Science, 21: 13–25.
Bhanu M.K., Zhao P., and Kendall D.A. (2013) Mapping of the SecA signal
peptide binding site and dimeric interface using substituted cysteine accessibility
method. (Manuscript in press at the Journal of Bacteriology).
Bhanu M.K., and Kendall D.A. (2013) Fluorescence spectroscopy of soluble
E. coli SPase I Δ2-75 reveals conformational changes in response to ligand binding
(Manuscript under review at Proteins: Structure, Function, and Bioinformatics).
INVITED GUEST LECTURE:
Department of Biology, Connecticut College, New London, CT, “The Secretory
Pathway”, 03/2010.
RESEARCH SUMMARY:
My research is focused on the Sec-dependent protein transport system
of bacterial cells, which is essential for the translocation of several vital
periplasmic and membrane-embedded proteins into or across the bacterial
inner membrane. Components of the Sec pathway include the SecA ATPase,
the SecYEG transmembrane channel and the signal peptidase enzyme (SPase
I).
MEERA BHANU KOLAYARATTIL
One of the important unanswered questions in the field is: Where does the
secretory preprotein bind on SecA? To study this crucial step of translocation,
we generated a library of mono-cysteine SecA mutants; accessibility of each
cysteine to a sulfhydryl reactive biotinylation reagent was analyzed in the
presence and absence of a secretory signal peptide, to locate its binding site on
SecA. We identified a binding region on SecA that provides a hydrophobic
groove for housing signal peptides, this site can also “open” or “close” based
on the presence of additional ligands. The dimeric interface and orientation of
SecA protomers were also examined through disulfide bond redox reactions,
which revealed that SecA exists as a parallel head-to-head dimer and that
monomerization of SecA is dictated by the presence of different translocation
ligands, indicating that the monomer-dimer equilibrium is shifted during
different stages of protein export.
SPase I is the enzyme responsible for cleaving signal peptides off of
mature preproteins following their transport across the inner membrane. We
utilized tryptophan fluorescence spectroscopy for analyzing the dynamic
conformational changes that E. coli SPase I undergoes as it encounters different
environments and ligands. We report the first successful purification of SPase I
Δ2-75 from the bacterial lysate fraction, and generated four single Trp mutants.
Our work established that contrary to earlier studies, a soluble SPase I is
purifiable and active. In addition, we established the conformational changes
SPase I undergoes upon interaction with lipids and signal peptides. One of the
key issues that remain unresolved in the field is the spatial location and
orientation of the signal peptide as it interacts with the SPase I enzyme. Using
FRET, we determined the Kd of signal peptide binding to SPase I and also
determined the orientation of the peptide with respect to the enzyme and the
membrane. We also identified distinct sites on SPase I that underwent marked
structural perturbations upon ligand binding, thereby providing a better
understanding of the enzyme ligand binding reaction and cleavage mechanisms.
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