1. School / Centre / Department / Group
TCIS, TIFRH
2. Highlights
Using a Combination of NMR spectroscopy and Molecular Dynamics simulations we
have shown that the free energy barrier for T4 lysozyme to interconvert between its
major and minor states is only 5RT. To the best of our knowledge this is the first report
of a near barrierless transition between two compact conformations of a protein
molecule.
3. Text
As the experimental facilities are yet to arrive, I have been performing computational
studies of protein conformational dynamics.
Mechanism of the interconversion between the major and minor states of T4
Lysozyme L99A: At room temperature T4L L99A populates two conformations a major
(~99%) and a minor state with a population of ~1% and a lifetime of ~1ms. We have
developed new NMR methods [1] and determined the structure of this minor conformer
[2] (Figure 1). However the mechanism of interconversion is still not known. Using a
Combination of NMR spectroscopy and Molecular Dynamics simulations we have
determined the mechanism of interconversion. The simulations are in qualitative
agreement with the experimental results suggesting that the forcefield models the
underlying free energy surface reasonably well. We find that the free energy barrier for
the interconvertion between the two states is only 5RT. To the best of our knowledge
this is the first report of a near barrierless transition between two compact
conformations of a protein molecule.
1. Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11766-71.
2. Nature. 2011 Aug 21;477(7362):111-4.
4. Figure / Photograph Captions (Please indicate position of the figure in
Text)
Fig. 1. Exchange between the Phe114 exposed (E) and buried (B) conformers can be studied using
CPMG relaxation dispersion (RD) NMR spectroscopy. A) The Phe 114 exposed and buried structures of
T4L L99A. Helix f (residues 107-113) is in red and helix g (residues 115-123) is in green. Phe 114 is in
blue. In the buried form Phe114 moves into a helical conformation with its sidechain buried in the protein
and helices f and g now form one large helix. The exposed structure was determined by crystallography
while the structure of the buried form, which is populated to only ~3% was determined by CPMG NMR. B)
15N-1H Trosy spectrum of T4L L99A,G113A,R119P recoded at 35 oC. This experiment correlates amide
15N and 1H resonances and peaks are labeled according to the residue from which they arise. In the case
of this mutant studied here the exposed form is the minor structure while the buried form is the major
structure and the peaks in the spectrum correspond to the buried form. Peaks that are aliased in the
indirect 15N dimension are shown in blue. C) Amide 15N CPMG RD curves of Lys 135 recorded at 1, 5.3,
10.4 and 15.4 oC on a 500 MHz spectrometer. Red dots correspond to the measured relaxation rates and
the blue line is best fit to the data. Analysis of these curves provides information regarding the kinetics,
thermodynamics of the Buried to Exposed exchange processes and can be used to reconstruct the
spectrum of the invisible exposed form. The dispersions decrease in size with increasing temperature as
the processes is endothermic and the exchange regime is moving from intermediate to fast. D, E)
Arrhenius plots of the forward and reverse rate constants obtained from the CPMG data recorded at five
temperatures. The experimental rates are shown as red circles with errorbars and the bestfit Arrhenius
curve is shown in blue.
5. Staff List
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Date, Short Description)
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a) In Journals [Authors, Title, Journal, Volume No., Page, Year]
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Page, Year]
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e) Books/Book Reviews
f) Technical Reports/Internal Reports
13. Lectures / Lecture Courses Given Elsewhere
14. Lectures by Visitors
Organized the following two lectures:
1) Junctions of Dirac Materials by Prof. Krishnendu Sengupta, IACS Kolkata on
Apr 1st 2015.
2) Regulation of kinesin and dynein mediated transport by tubulin posttranslational modifications by Dr. Minhaj Sirajuddin, NCBS Bangalore on Feb
18th 2015.
15. Graduate Courses
Topics in Biophysics (being taught with Dr. Kanchan Garai)
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awarding the degree)
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