Page 149 - AWSAR 2.0

Biology in February 2019.
In our study, we examined the internal
symmetry between the two domains (protein domains are sub-structural units which evolve independently) in the
dark mutant. Our structural
analysis led to identification of
its N-terminal domain as floppy
compared to its C-terminal.
We thought if it introduced
site-specific flexibility, this
would show up in increased
dynamics. Inspired by this,
we undertook conformational
dynamics analysis using
solution NMR spectroscopy.
These experiments confirmed
the enhanced flexibility of
the N-terminal domain in
the dark mutant at a single
residue level, a resolution that
cannot be achieved using
other biophysical techniques.
These results were published
in the issue of Biochemical and Biophysical Research Communications in March 2019.
A dialogue in the dark at TIFR Hyderabad with the dark mutant.
My dialogue in the dark continued in the interval. With complementary ‘High Tea’, we recall that crystallins accommodate themselves in very high concentrations inside a tiny transparent marble (our eye lens). As a consequence, they are evolutionarily adapted to highly folded conformations with extreme
Mr. Khandekar Jishan Bari || 125
solubility and stability. We know from high school chemistry that amide hydrogens (in proteins) have the ability to exchange with the solvent in which they are kept, demonstrating
a phenomenon called chemical exchange. As NMR spectroscopy is ideally poised to probe molecular motions at residue level, we went on to investigate the exchange dynamics of the dark mutant.
And after every interval, the story grew intense. As expected, the flexible N-terminal domain displayed enhanced exchange in comparison to the non- cataract variant. Surprisingly, the dark mutant demonstrated sequential exchange of its N-terminal hydrogens followed by its C-terminal. This study was followed by thermal titration NMR studies, which characterized occasional excursions of the
dark mutant to transient excited states invisible to traditional biophysical techniques. Closing in on the climax of the dialogue, I dissected the dark mutant into its two individual domains and tested our biophysical and NMR experiments on them. Finally, we arrived at our conclusion that the N-terminal domain of the dark mutant is the real culprit in generating hotspots for large-scale aggregation and hence cataract formation. Our findings in these aspects were further published in three successive papers appearing in the Biochemical and Biophysical Research Communications journal from May- July 2019.
Then came the flashback. Revisiting our initialhypothesis,wereframedourconclusions through multiple approaches: The remarkable structural instability in the dark mutant propagates sequentially through its N-terminal domain leaving the C-terminal unperturbed.
Unlike proteins in other cells, lens crystallins do not enjoy vacations and perform the task for a lifetime to provide transparency or clear vision. And of course, this does not come for free. Genetic mutations (or carriers of darkness) in crystallins result in insoluble protein aggregates in the eye lens and promote cataract or blindness, that is, change in a single gene and darkness for a lifetime!