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Techniques used
Molecular Biology: Cloning of Drk SH3, Mutagenesis of Drk SH3 (A3C, D32C, and D59C)
Biochemistry: Expression and purification of WT and all three variants of Drk SH3 with 15N labeled
Chemistry: Reducing with DTT and reaction with MTSL
NMR: Recorded HSQC spectra with different mixing times, Varian 600 M.Hz spectometer
Spectra processing: NMR pipe, NMRDRAW, Sparky
Fitting relaxation data: Matlab
2. The amide proton exchange is known to occur when it is exposed to solvent H2O/D2O. The amide
proton exchange rates along the protein backbone are useful reporters of accessibility and structural
stability of specific residues and secondary structure elements 1. However the conventional NMR
experiments like COSY and HSQC are limited to slow exchange motions, and we designed NMR pulse
sequence to measure fast amide proton exchange with solvent. Since unfolded protein amide protons are
more exposed and get exchanged on faster time scales, we are using this pulse sequence to get the
structural information of different unfolded states of proteins like ubiquitin, DrkN SH3. Rates of
exchange in the fast time scales are useful in predicting solvent accessibility, ionization states of nearby
groups, and presence and absence of ligand molecule. These studies also predicting that there is a
persistent structure in the unfolded (2M GdnHCl, ph6) drkN SH3.
Techniques used
Molecular biology: Cloning of Ubiquitin
Biochemistry: Expression and purification of ubiquitin and DrkSH3 with 13C, 15N.
NMR: Our new pulse sequence, running series of spectra for different exchange time
Spectrometer : Bruker 800 M.Hz.
Spectra processing: NMR pipe, NMRDRAW, Sparky, Topspin, fitting data with matlab.
3. Proteins are known to bind non-specific DNA before they reach to its specific DNA. It is long debate
issue that proteins transfer via 1dimensional diffusion (sliding) or 3 dimensional diffusion (dissociation
&re-association or direct transfer) models. To study these different mechanisms we plan to use spin
labeled DNA and multi domain DNA binding protein Zif268. I prepared spin labeled DNA with 100%
efficiency (confirmed by ESI-MS) by conjugating nitroxide radical by means of a peptide bond. I
expressed and purified double labeled zif268 protein. The paramagnetic relaxation enhancement (PRE) of
backbone protons of zif268 protein in protein-DNA complex can suggest the orientation of protein on
DNA. (This work is in progress)
Techniques Used
Purification of DNA using mono Q column and FPLC.
15N and 2H Zif268 expression and purification.
A chemical reaction between Thiamine modified DNA and nitroxide spin label.
NMR spectroscopy is one of the best tools to study the dynamics and structure of a protein in solution
state. Though crystal structural studies of proteins are better known, solution state study of proteins is
more advantageous in many fields like drug discovery, protein dynamics and unfolded protein structures.
3. Unfolded protein structures using novel thiol-reactive EDTA derivative paramagnetic tag
Unfolded proteins structure and dynamics are more important to analyze different processes like protein
folding, and amyloid formation, and even in understanding many diseases like Parkinsons, mad cow, and
cancer. It is interesting and important to study the thermo dynamical structure of unfolded proteins. Due
to highly dynamic nature of unfolded protein structures, it is very difficult to find their structures with
conventional methods of NMR or crystal studies. Even paramagnetic relaxation enhancement studies with
regular spin labeling is no more useful; as mtsl electron relaxation time is very long( s-ns), many
motions in the unfolded protein are shorter time scale(ps) compare to electron relaxation time of mtsl.
Therefore, paramagnetic relaxation enhancement methods need to be updated with new EDTA derivative
thiolreactive tags (MTS-EDTA). These tags can react with different kinds of paramagnetic metal ions like
Ni, Mn, Co, and lanthanides. The electron relaxation time of those metal ions is in the picoseconds time
scale with less curie and inter molecular relaxation. We can introduce this paramagnetic tag through
disulfide bond to mutated cystein at desired position in the protein to study the fast dynamics of unfolded
protein.
References:
1. Bai, Y., milne, J.S., Englander, S.W. Proteins: Structure, Function, and Genetics, 1993, 17, 75-86.
2. Ellis, R.J., van der Vies, S.M. Annual Review of Biochemistry, 1991, 60, 321-347.