Rajaram Swaminathan

Professor

rsw[AT*]iitg.ac.in

Research Areas:

A. Biophotonics

B. Protein Structure and  Dynamics

C. Proteome Analysis

D. Consequences of molecular crowding inside living cells

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The journey leading to discovery of Protein Charge Transfer Spectra

 

 

Protein Charge Transfer Spectra (ProCharTS) is now established as an innovative experimental technique that is sensitive to interaction/proximity between charged atoms in a protein. This enables one to observe structural transitions in moderately charged proteins. Monitoring changes in protein structure conventionally require expensive instrumental setups like CD spectrometer, spectrofluorometer or NMR spectrometer. However, using ProCharTS we have shown that such transitions can be observed by measuring electronic absorption using a simple UV-Visible spectrophotometer. The most useful aspect of using ProCharTS is that it is a LABEL-FREE technique. This facilitates its application to observe proteins in their natural state inside living cells or in a test tube. Here I describe our journey leading to discovery of ProCharTS which began at IIT Guwahati in the year 2000.

 

Ever since 1963, it has been a widely accepted view that electronic absorption among naked proteins in the near ultraviolet region (250—350 nm) arises chiefly from aromatic chromophores in the side chains of Trp, Tyr and Phe. Proteins that lack aromatic amino acids, disulphide bonds, metal ions or coenzymes were expected to be optically silent beyond 250 nm. Thus, the simple observation of the electronic absorption of a protein was believed to be uninformative about changes in protein structure.

 

Work from our lab over the past twenty years has now shown that this acceptance is not true.

 

In 2001, work by Ms. Lopamudra Homchaudhuri first reported (http://dx.doi.org/10.1246/cl.2001.844) electronic absorption (~270 nm) and visible luminescence (~430 nm) in dense aqueous solutions of L-Lysine.HCl. Later, in 2004 (http://dx.doi.org/10.1246/bcsj.77.765) we showed the presence of significant electronic absorption spectra in dilute aqueous solutions of Lysine rich proteins like human serum albumin and calf thymus histone beyond 320 nm that was sensitive to protein oligomerization.

 

The scientific community was skeptical of this discovery because the chromophore behind this light absorption was not obvious. We searched for a model protein for our work that was rich in Lys residues but lacks aromatic residues. With efforts of Mr. Shubham Singh, a summer intern in our lab, we found such a protein alpha3C. In 2014, we entered into a collaboration with Dr. Ravi Venkatramani at TIFR Mumbai to obtain a theoretical rationale for the observed spectra in Lysine rich proteins like alpha3C.

 

Further work during 2014—17, involving Ms. Saumya Prasad and myself from our lab in IIT Guwahati along with Ms. Imon Mandal and Dr. Ravi Venkatramani at TIFR Mumbai, employing Time-Dependent Density Functional Theory (TD-DFT) calculations on the different snapshots of alpha3C structure sampled from Molecular Dynamics simulations, revealed photoinduced electron transfer as the origin of these spectra. In 2017, we proposed that proteins rich in charged amino acids like Lys or Glu can undergo photoinduced electron transfer from donors like amide groups in protein backbone or anionic glutamate to acceptors like cationic lysine or peptide backbone (http://dx.doi.org/10.1039/c7sc00880e). Such novel spectra which were prominent in alpha3C protein between 250—800 nm, were termed as Protein Charge Transfer Spectra or ProCharTS.

 

In the recent past, our lab has shown that ProCharTS is sensitive to: a) changes in spatial proximities of charged headgroups in the protein, and b) presence of salt ions in the aqueous media (http://dx.doi.org/10.1039/c7fd00194k). Changes in conformation of several intrinsically disordered proteins induced by pH or temperature were shown to affect the ProCharTS intensity. Further, our lab showed that events like protein aggregation which bring two protein surfaces in close proximity, can enhance ProCharTS significantly enough to track protein aggregation process as it happens (http://dx.doi.org/10.1039/c7fd00194k). Recently, our group has measured the luminescence arising from ProCharTS bands in proteins. Our work demonstrated the strong influence of ProCharTS absorption coefficient in controlling the luminescence intensity given that ProCharTS luminescence quantum yield were low (< 0.1). Interestingly, ProCharTS molar extinction coefficient had a direct correlation with spatial proximity between charged atoms in the 3D fold of the protein (http://dx.doi.org/10.1021/acs.jpcb.9b10071).

Click here to read recent article in Biophotonics (July/Aug 2021 issue) on ProCharTS.

In addition to ProCharTS, our lab is also working on:

  • Investigating the effect of crowding on enzyme-substrate reactions inside living cells
  • Analysing whole proteome protein sequence databases by assigning prime number to each amino acid

Bachelors in Chemistry (1988): University of Madras

 

Masters in Biotechnology (1990): Indian Institute of Technology Bombay

 

Doctor of Philosophy (1996): Tata Institute of Fundamental Research, Mumbai (Mumbai University)

Thesis Title: Time-resolved fluorescence of Biological Macromolecules

Thesis Supervisors: Prof. G. Krishnamoorthy, Prof. N. Periasamy and Prof. Jayant B. Udgaonkar

1995-1998: Postdoctoral Fellow in the lab of Prof. Alan S. Verkman in Cardiovascular Research Institute, University of California, San Francisco, USA.

1998-1999: Research Associate at the National Centre for Ultrafast Processes, University of Madras, Chennai

1999: Joined Chemistry Department, at Indian Institute of Technology Guwahati as Assistant Professor

2002: Joined Biotechnology Department at Indian Institute of Technology Guwahati as Assistant Professor

2003-2006: Head, Biotechnology Department, IIT Guwahati.

2004: Appointed as Associate Professor, Biotechnology Department, IIT Guwahati

2009: Appointed as Professor, Biotechnology Department, IIT Guwahati

 

 

Title of the Course taught

Level

Experimental Spectroscopy

Ph.D. Chem

Chemical Kinetics and Electrochemistry

M.Sc Chem

Computers and Management in Chemistry*

M.Sc Chem

Chemical Biology

M.Sc Chem

Physical Methods in Chemistry*

M.Sc Chem

Introduction to Computing#

B. Tech Biotech

Chemistry II*

B. Tech Biotech

Modern Biology*

B. Tech Biotech

Biophysics

B. Tech Biotech

Frontiers in Biotechnology*

B. Tech Biotech

Fluorescence Techniques in Biotechnology

B. Tech Biotech

Biochemistry*

B.Tech Biotech

Analytical Biotechnology*

Ph. D. Biotech

Molecular Biophysics

Ph. D. Biotech

Biotechniques

M. Tech Biotech

* These courses were shared among other colleagues

# Only the tutorial classes were taken by me.

.

Journals


1. Mohd. Ziauddin Ansari, Shah Ekramul Alom and Rajaram Swaminathan "Ordered structure induced in human c-Myc PEST region upon forming a disulphide bonded dimer", Journal of Chemical Science. DOI.10.1007/s12039-021-01889-3, Vol.133, , P.P - , (2021)

2. Mohd. Ziauddin Ansari, Rajaram Swaminathan "Structure and dynamics at N- and C-terminal regions of intrinsically disordered human c-Myc PEST degron reveal a pH-induced transition. ", Proteins: Structure Function and Bioinformatics. DOI.10.1002/prot.25880, Vol.88, 7, P.P 889-909 , (2020)

3. Amrendra Kumar, Dileep Ahari, Anurag Priyadarshi, Mohd. Ziauddin Ansari and Rajaram Swaminathan "Weak Intrinsic Luminescence in Monomeric Proteins Arising From Charge Recombination", Journal of Physical Chemistry B. DOI.10.1021/acs.jpcb.9b10071, Vol.124, , P.P 2731-2746 , (2020)

4. Anand, R., Agrawal, M., Mattaparthi, V. K. S., Swaminathan, R., Santra, S. B. "Consequences of heterogeneous crowding on an enzymatic reaction: A residence time Monte Carlo approach.", ACS Omega. DOI., Vol.4, , P.P 727-736 , (2019)

5. Ansari, Mohd. Z., A. Kumar, D. Ahari, A. Priyadarshi, L. Padmavathi, R. Bhandari, R. Swaminathan "Protein charge transfer absorption spectra: An intrinsic probe to monitor structural and oligomeric transitions in proteins.", Faraday Discussions. DOI., Vol.207, , P.P 91-113 , (2018)

Conference Publication


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Title of Invention: COST EFFECTIVE, PORTABLE OPTOELECTRONIC INSTRUMENT TO MEASURE STEADY STATE FLUORESCENCE AND ITS SET UP METHOD

Patent No. : 310875

Application No. : 1136/KOL/2015

Date of Filing : 07/11/2015

Patentee : KULKARNI ALARK SHRIPAD, HARSHAL B. NEMADE and RAJARAM SWAMINATHAN

 

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