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124 || AWSAR Awarded Popular Science Stories - 2019
than a tiny marble, our eye lens is densely packed with three different types of a protein called crystallin. 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!
This is Khandekar Jishan Bari and my dialogue in the dark commences as the famous physician Lewis Thomas: (A; Not clear.) The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music. Cataracts are no longer attributed to only senior citizens. Recently, a three-year-old child was diagnosed with severe childhood cataract. Genome sequencing
of crystallins extracted from
his eye lens revealed a novel
mutation in the γS-crystallin
gene (the route to darkness).
Identified for the first time, the
mutant replaced a glycine
amino acid at the 57th position
in the protein with tryptophan
(also called the dark mutant in
this dialogue). The change of a
single amino acid in the protein
was identified as the familial
determinate of childhood
cataract. This form of cataract
has no preventive diagnosis due to its very early onset and lack of surgical measures. Thus, outlining various projects for my doctoral research after a heavy traditional coursework at TIFR, I zeroed in on to unravel the mechanism of this unexplored disease, a real challenge for the pharmaceutical industry.
My dialogue in the dark proceeded. In
this endeavour, our initial biophysical studies instilled remarkable structural instability in the dark mutant in comparison to its non-cataract variant. This led to the coining of my initial hypothesis: The structural instability in the dark mutant may propagate through its 3D structure providing insights into the mechanism of childhood cataracts. I realized that to develop novel drug targets to inhibit or reverse crystallin aggregation, high-resolution structural study of the dark mutant is inevitable. Although X-ray crystallography is traditionally used to solve protein structures in structural biology, the dark mutant was recalcitrant to crystallization. Thus, we resorted to NMR spectroscopy to determine its high-resolution 3D structure. And the dialogue continued through my annual talks and research meetings.
As a prelude to structure determination, we optimized sample preparation for NMR studies and successfully completed resonance
assignments of the dark mutant, a fingerprint strategy in NMR spectroscopy to uniquely assign nuclear spins, (we published our optimized protocols and complete assignments in Biomolecular NMR Assignments). Next few months witnessed intense structure calculation strategies and refinements. Finally, we arrived at its high-resolution NMR structure resolved to 0.5 Å. Investigating the origin of instability, the 3D structure
revealed the location of the 57th tryptophan residue in an unusually solvent-exposed orientation. This geometry resulted in a rare conformation observed in less than 1% of well- resolved crystal structures (from a database called the worldwide Protein Data Bank). We published the 3D structure of this dark mutant in a recent issue of the Journal of Structural
The immediate challenge in
the post-genomic era today is, therefore, to translate this sequence information into useful biochemistry and to decipher structural, functional and evolutionary clues encoded in these genome sequences.