Protein misfolding and aggregation:

             We are interested in the study of sequence specificity of protein aggregation and the associated mechanisms. The fluorescence lifetimes and anisotropy decays are studied for extrinsic as well as intrinsic fluorophores in proteins, peptides and their aggregates. The anisotropy decay reveals interesting information about the amyloid core region and is a reliable tool to monitor the molecular level processes.

 

 

 

 

 

 

 

 

  

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Capturing dynamics of protein folding using time-resolved FRET measurements:

A double-kinetic set up, consisting of a stopped-flow mixer with millisecond dead time coupled to detectors like Streak Camera or TCSPC along with a picoseconds laser source, is being used to collect fluorescence intensity decays during protein folding or unfolding.  The lifetimes are then converted to Foster distance between two labeled segments of a protein. The pattern of temporal evolution of such distances in a protein during folding or unfolding describes whether the protein goes through a continuum of intermediates or follows simple two-state model.  

 

Incorporation of non-natural amino acid like 5F-trp into protein and characterization its fluorescence properties:

We are investigating the use of non-natural amino acids as fluorophores in proteins. For example, 5-fluorotryptophan (5F-trp) is known to have homogeneous lifetime, unlike the naturally occurring tryptophan in proteins. So, we have biosynthetically incorporated 5F-trp into protein and measured the lifetimes. Additionally, it has been incorporated in different small peptides to characterize the effect of neighboring residues on its fluorescence lifetime. The results show that the lower environment sensitivity as well lower heterogeneity in lifetimes for the 5F-trp may prove to be advantageous for future studies.

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Dynamics in an RNA thermometer:

The structure and dynamics of regions of RNA are emerging as regulators of expression message encoded in the sequence. We have studied the local dynamics at various strategic locations in the ROSE element (Repression Of heat Shock gene Expression) - an “RNA thermometer”, by using time resolved fluorescence of 2-aminopurine (2AP), a sensitive fluorescent analogue of adenine. The results have shown very high sensitivity of the sequence in the temperature range 25-40ºC.

 

 

 

DNA-Protein Interactions :

DNA is the genetic polymer present in most eukaryotic cells and performs a vital role in the normal development and function with the help of several DNA-binding proteins. These proteins come in the general category of groove binders and find their target location by specific and/or non-specific modes of interaction. In our research group we try to visualize this important area of research using Atomic Force Microscopy in air and liquid mode.  

 

 

 

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Membrane Transport:

Cellular channels usually consist of large protein complexes with multiple transmembrane a-helices. Some amyloid proteins also get inserted into the membranes, aggregate and conduct ions leading to cell death. Here we are studying the interactions of soluble proteins with membranes and whether these lead to channel formation in the bilayer, by observing the fluorescence intensity change of the trapped probe called pyranine inside the liposomes. Till now barstar, lysozyme and BSA (bovine serum albumin) have been studied and the preliminary results indicate that barstar and BSA permeabilise vesicle to H+ ions by forming non-exchangeable channels. Lysozyme did not cause permeabilization of vesicles. Permeabilization of vesicles by soluble proteins    may be related to the extent of α helix content. Lower pI values may be assisting the proteins in the interaction with membrane.

 

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Collaborations:

Correlation between dynamic modes in proteins:

Among the various dynamic modes in proteins, the local motional dynamics of side-chains and motional dynamics of the solvent around the side chains are the most studied. We use site specific fluorescence labeling in the protein barstar in its various structural forms such as native, unfolded, aggregated and protofibril forms. Subsequently we have measured the local motional dynamics of these probes by using time resolved  fluorescence anisotropy decay kinetics and solvation dynamics by monitoring time dependent Stokes shifts. Our research has shown one to one correlation between the two modes in several cases, indicating a mechanistic coupling between these dynamic modes.

(This work was done in collaboration with Jayant Udgaonkar of  NCBS, Bangalore and Tahei Tahara of RIKEN, Japan)

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Correlation of dynamics with function in polytene chromosomes:  

The eukaryotic chromatin shows heterogeneus intensity when labeled with a fluorescent dye. The dim region corresponds to the euchromatin while the bright area represents heterochromatin. We have observed a positive correlation between fluorescence lifetime and intensities - the bright regions of heterochromatin having longer lifetimes while the dim regions showing shorter lifetimes. This can be interpreted as dim regions being more dynamic which was also confirmed by fluorescence anisotropy data.

(This work was done in collaboration with Dr. Sunil Noothi and Prof. B.J. Rao of Dept of Biological Sciences, TIFR)

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Structural transformation during protein unfolding:

In order to probe deeper into the structural changes occurring during unfolding, we have monitored the changes in several intra-molecular distances and the distance distributions during the unfolding kinetics by site specific labeling and time-resolved FRET coupled with MEM for the data analysis.  This work showed that the unfolding process of this protein (monelin) is not a two state process but involves a continuous dissolution of structure.

(This work was done in collaboration Santosh Jha, Prof Jayant Udgaonkar Of NCBS Bangalore and Prof Deepak Dhar)

 

Instruments in the lab:

ØSpectrofluorometer (Fluorolog)

 

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ØUV/ Vis Spectrometer (Perkin Elmer )

 

ØAtomic Force Microscope( Molecular Imaging)

 

ØDynamic Light Scattering (Protein Solutions)
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ØLaser sources:
10W Tsunami pulsed laser: wavelength range 800nm- 1000nm
5W Tsunami Pulsed laser : wavelength range 750nm-800nm
Rhodamine 6G dye laser : wavelength range 580nm-630nm

 

Detection systems
Time Correlated Single Photon Counting setup with L configuration. (for cuvette measurements)
Stopped Flow combined TCSPC in T configuration.
Epifluorescence microscope with TCSPC (for single cell measurements)
Stopped Flow with streak camera for fast time scales detection.

 

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