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Research Proposal

Recent research projects:

1. Characterization of unfolded proteins structure and dynamics is crucial in understanding the


mechanism of many diseases like Alzheimers, Parkinson's, and formation of amyloid fibrils. The
dynamic heterogeneity of unfolded protein ensembles remains major obstacle in finding their structural
information. Still there is a significant progress in finding residual structure of disordered proteins at
atomic resolution, especially with various NMR techniques. In this regard, we use site-directed spin
labeling (SDSL), in which a paramagnetic moiety is introduced into the polypeptide chain by cysteine
mutagenesis and chemical linkage. We are working on N-terminal SH3 domain of Drosophila protein
(drkN SH3) under denatured conditions (2M Guanidine hydrochloride, pH 6). We introduced cysteine
mutation at three different positions (A3C, D32C, and D59C) of the protein. MTSL
(methanethiosulfonate), a paramagnetic spin label was chemically reacted to cysteine residue and attached
through disulfide bond. Optical spectroscopy and NMR chemical shift data proven that these mutations
and spin label does not change the structure of unfolded protein. The measured Paramagnetic relaxation
enhancement (R2p-R2) of amide proton, suggests that there is persistent structure in the unfolded protein,
which resembles both native and non-native long range interactions. These long range contacts have key
role in initiating chain collapse and folding of a protein.

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.

Future research plans

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.

1. Inter molecular interactions using paramagnetic relaxation enhancement


Protein-protein interactions are crucial in many biological processes, like in programmed cell death
(apoptosis), and cell signaling. 1H paramagnetic relaxation enhancement (PRE) studies of protein
backbones using site directed spin labeling of nitroxyl radical or paramagnetic metal bind MTS-EDTA
would provide impending knowledge about protein-protein, protein-ligand, and protein-DNA interactions.
As preliminary experiments, I would use optical spectroscopy like UV-Vis absorbance, fluorescence, CD,
and iso thermal calorimetry (ITC) to study the thermodynamics of these macromolecules, at later stage I
would use NMR paramagnetic relaxation enhancement to decode the protein-protein interactions at
atomic level. These experiments would able to answer many ambiguous questions like which part of a
protein involved in the interaction with other protein or drug or DNA/RNA.

2. Protein-ligand interactions using engineered proteins and simple NMR methods


Ligand binding makes considerable change in the conformation of the protein. I would be interesting in
finding the protein-ligand interactions by NMR using engineered proteins like methylation of lysines,
esterfication of glutamic or adipic acids. If we use the additive groups with 13C, those additive13C labeled
methyl groups may exhibit rapid localized motion, these motions could be faster than the overall tumbling
motion of whole molecule. Thus NMR investigations of these additive groups attached to side chains of
larger proteins are more beneficial when we compare with the conventional methods. Advantages of this
method include low protein concentration, unlabeled protein, easy and fast experiments. In contrast to the
conventional methods, it can deal with larger proteins, with less experimental time.

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.

4. Role of chaperone in protein folding


The most challenging problem in the field of molecular biophysics is protein folding. It is difficult to
understand how proteins fold to their active three dimensional structures, after they are synthesized from
ribosome. It is known that invivo chaperones will assists nascent polypeptide to fold into correct
conformation2. Molecular chaperones are known to interact with unfolded protein and stop the
aggregation process and catalyze the folding process. As unfolded structure is more dynamic with random
motions, it is hard to define the interaction between chaperone and unfolded protein using conventional
methods. I will be using novel paramagnetic tag with suitable metal ion to understand the interaction
between chaperone and unfolded protein. It would explore the so far unknown mechanisms of dynamical
and structural changes during chaperone assisted folding of proteins.

5. How protein reaches its specific DNA sequence using PRE


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. Although all mechanisms are possible in the translocation of
protein from non-specific DNA to specific DNA, as DNA concentration in the cell is very high, among
the above all mechanisms the direct transfer mechanism is more relevant in the cell. To test the direct
transfer mechanism I will construct two different DNA molecules with one protein binding site on one
and the other binding site on the other with slightly different environment next to the protein binding site.
I will use the protein with more than one DNA-binding domain. If the direct transfer mechanism is true
then protein should bind to two DNA molecules, since environment next to the binding site is different
NMR chemical shift for protein backbone protons will be different. With that I will able to confirm that
protein translocation using direct transfer mechanism. If I attach spin label to anyone of the DNA I would
extract the distance and geometry/orientation of interaction between protein and DNA.

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.

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