Biophysical Reviews' "Meet the Councilor Series"-a profile of Kandala V. R. Chary.

It gives me great pleasure to introduce myself to the readers of Biophysical Reviews. I share a brief account of my career and experiences in biophysical research spanning four decades. For the most of this period, I have worked at the Tata Institute of Fundamental Research (TIFR), Mumbai and Hyderabad.

NMR structure and dynamics of inhibitory repeat domain variant 12, a plant protease inhibitor from Capsicum annuum, and its structural relationship to other plant protease inhibitors.

Although several plant protease inhibitors have been structurally characterized using X-ray crystallography, very few have been studied using NMR techniques. Here, we report an NMR study of the solution structure and dynamics of an inhibitory repeat domain (IRD) variant 12 from the wound-inducible Pin-II type proteinase inhibitor from Capsicum annuum. IRD variant 12 (IRD12) showed strong anti-metabolic activity against the Lepidopteran insect pest, Helicoverpa armigera. The NMR-derived three-dimensional structure of IRD12 reveals a three-stranded anti-parallel ß-sheet rigidly held together by four disulfide bridges and shows structural homology with known IRDs. It is interesting to note that the IRD12 structure containing ∼75% unstructured part still shows substantial amount of rigidity of N-H bond vectors with respect to its molecular motion.Communicated by Ramaswamy H. Sarma.

Statistical analysis of intermolecular interactions in trypsin-inhibitor complexes.

Communicated by Ramaswamy H. Sarma.

A Molecular Dynamics Perspective To Identify Precursors to Aggregation in Human γS-Crystallin Unravels the Mechanism of Childhood Cataracts.

Despite the increasing health risk from infantile cataracts, identifying the mechanism of this disease remains a challenge due to a lack of structural investigations using experimental and computational approaches. Mutations in human γS-crystallin are contingent with childhood cataracts. Our recent high-resolution structural studies using solution NMR spectroscopy established the key role of the G57W mutation in human γS-crystallin (abbreviated hereafter as γS-G57W), promoting structural instability. In order to design therapeutics to delay or upset congenital cataracts, the characterization of the precursors to γS-G57W aggregation is indispensable. In this endeavor, we present microsecond long unbiased atomistic molecular dynamics simulations and principal component analyses that unfold insights into lens crystallin aggregation. An enhanced sampling metadynamics approach was further employed to systematically unravel the molecular dynamics underlying crucial interdomain contacts. Taken together, our experiment-guided computational study in this paper led to the identification of domain-swapped intermediates in γS-G57W to atomic resolution with insights into the aggregation of lens crystallins causing childhood cataracts for the first time with functional consequences.

Structural studies on the individual domains of human γS-crystallin and its G57W mutant unfolds mechanistic insights into childhood cataracts.

Inter-domain interactions tune the exceptional stability of human γS-crystallin (γS-WT) in the eye lens, which lasts a lifetime with no protein turnover. Our recent NMR studies revealed the key role of G57W mutation in γS-WT, as the familial determinate of childhood cataracts. As the unusually exposed W57 is near the inter-domain interface, a recurring theme of this study is the upsetting of inter-domain contacts exposing hydrophobic patches, which may initiate aggregation at crystallin concentrations not so surprising in the eye lens. In this endeavour, to untangle the mechanistic pathways triggering aggregation in the cataract variant γS-G57W, we undertook high-resolution structural characterization of isolated domains vis-a-vis full length γS-crystallin. Here we report for the first time, thermodynamic and kinetic determinants of structural stability with their eccentric shifts under pathological stress employing sophisticated spectroscopy techniques. We propose that domain interface acts as an intrinsic stabilizer for the otherwise floppy N-terminal domain in γS-G57W than in γS-WT where it serves an extrinsic role. Our results present a residue resolved quantitative analysis for differential domain stabilities from non-linear temperature coefficients of 1HN chemical shifts using solution NMR spectroscopy. Consistent with the Ca2+-binding episode that lasted poorly for human lens crystallins, our results show that disease mutants attenuate it further and completely silence it in extreme cases. Overall, our study provides a compelling evidence for the diverse structural evolution of lens crystallins elucidating molecular details to apprehend lens opacification and suggests the scope of therapeutics in reducing the global trauma of cataracts.

On identifying low energy conformational excited states with differential ruggedness in human γS-crystallin promoting severe infantile cataracts.

Transient excited states in proteins can be accurately probed from temperature dependence of amide proton (1HN) chemical shifts displaying significant curvatures. Characterizing these near-native alternative states is of high therapeutic relevance in conformational diseases wherein missense mutations promote structural instability that leads to conformational heterogeneity. Extending the structure-function paradigm from physiology to pathology, we recently reported the solution NMR structure and dynamics of a severe congenital cataract variant, G57W of human γS-crystallin (abbreviated as γS-G57W) which is resistant towards crystallization. In an endeavour to explore the functional consequences of this mutation, here we report for the first time, native state conformational ruggedness in human γS-G57W as compared to its wild-type counterpart from residue resolved nonlinear temperature dependence of backbone 1HN chemical shifts using solution NMR spectroscopy. Our calculations suggest that the simulated chemical shift curvatures are indicative of low energy excited states within 2-4 kcal mol-1 from the native state. Residues accessing alternative conformations populate the N-terminal domain of γS-G57W more than its C-terminal counterpart. Collectively, curvatures retaining native state ensemble on mild denaturation suggest that the free energy landscape in human γS-G57W at the bottom of the folding funnel is sufficiently robust and malleable against such perturbations. Overall, this critical study highlights the functional aspects of such structural malleability promoting infantile cataracts as a global health risk marker.

Enhanced H/D exchange unravels sequential structural excursions in G57W variant of human γS-crystallin with pro-cataractogenic conformations.

Our two recent reports on the high resolution NMR structure and conformational dynamics of G57W variant of human γS-crystallin (abbreviated as γS-G57W) causing severe infantile cataracts, revealed slackening of its N-terminal domain with enhanced local conformational dynamics attributed to mutation. Exploring the biochemistry of infantile cataracts in detail, here we studied structural unfolding in both human γS-WT and γS-G57W at residue level resolution using solution NMR spectroscopy and chemical kinetics and characterized the molecular intermediates with functional consequences. We report, for the first time, that human γS-crystallin unfolds sequentially under H/D exchange. This communication forms the first experimental evidence for non-concerted destabilization of structural foldon units in human γS-G57W. Residues contributing to the compact fold and structural stability exchanged their amide protons with deuterons more readily in γS-G57W compared to γS-WT, displaying differential free energies of exchange. Overall, our results establish a direct conformational link between the structure, dynamics, design and function in human γS-crystallin such that the G57W cataract variant promotes enhanced structural excursions concomitant with increased instability, elucidating very crucial molecular details of cataract formation affecting infants across the globe.

Mechanistic Insights from Replica Exchange Molecular Dynamics Simulations into Mutation Induced Disordered-to-Ordered Transition in Hahellin, a ßγ-Crystallin.

Intrinsically disordered proteins (IDPs) form a special category because they lack a unique well-folded 3D structure under physiological conditions. They play crucial role in cell signaling and regulatory functions and are responsible for several diseases. Although they are abundant in nature, only a small fraction of them have been characterized until date. Such proteins adopt a range of conformations and can undergo transformation from disordered-to-ordered state or vice versa upon binding to ligand. Insights of such conformational transition is perplexing in several cases. In the present study, we characterized disordered as well as ordered states and the interactions contributing the transitions through a mutational study by employing replica exchange molecular dynamics simulation with generalized Born implicit solvent model on a protein from the ßγ-crystallin superfamily. Most of the proteins within this superfamily are inherently ordered and highly stable. However, Hahellin, although a member of the ßγ-crystallin family, is intrinsically disordered in its apo-form which takes a well-ordered ßγ-crystallin fold upon binding to Ca2+. It is intriguing that the mutation at the fifth position of the canonical motif to Arg increases the domain stability in several ordered microbial ßγ-crystallins with concomitant loss in Ca2+ binding affinity. We carried out similar Ser to Arg mutations at fifth position of the canonical motif for the first time in an intrinsically disordered protein to understand the mechanistic insights of conformational transition. Our study revealed that newly formed ionic and hydrogen bonding interactions at the canonical Ca2+ binding sites play a crucial role in transforming the disordered conformation into ordered ßγ-crystallin.

Conformational dynamics study on human γS-crystallin as an efficient route to childhood blindness.

Single point mutants of human γS-crystallin cause dominant congenital cataracts, a recent one of which involves the substitution of highly conserved glycine at 57th position with a bulkier tryptophan. Our high-resolution 3D structure of this G57W mutant (abbreviated hereafter as γS-G57W), reported recently revealed site-specific structural perturbations with higher aggregation and lower stability compared to its wild-type; a structural feature associated with important functional and therapeutic consequences. In this communication, we report for the first time, residue resolved conformational dynamics in both γS-WT and γS-G57W using solution NMR spectroscopy, and suggest how these differences could crucially affect the biochemistry of the mutant. Guided by our critical structural investigations, extensive conformational dynamics and biophysical studies presented here show that loss of structural stability arises from enhanced dynamics in Greek key motif 2 inducing flexibility in the N-terminal domain as opposed to its structurally unperturbed C-terminal counterpart. NMR spectral density correlations and internal dynamics comparisons with the wild-type suggest that the overall thermodynamic instability propagates from the mutated N-terminal ß4-ß5 loop providing a residue level understanding of the structural changes associated with this early onset of lens opacification. Our results highlight the vital role of conserved Greek key motifs in conferring structural stability to crystallins and provide crucial molecular insights into crystallin aggregation in the eye lens, which triggers cataract formation in children. Overall, this critical study provides a residue level understanding of how conformational changes affect the structure and function of crystallins in particular and proteins in general, during health and disease.

Structure of G57W mutant of human γS-crystallin and its involvement in cataract formation.

A recently identified mutant of human γS-crystallin, G57W is associated with dominant congenital cataracts, the familial determinate of childhood blindness worldwide. To investigate the structural and functional changes that mediate the effect of this cataract-related mutant to compromise eye lens transparency and cause lens opacification in children, we recently reported complete sequence-specific resonance assignments of γS-G57W using a suite of heteronuclear NMR experiments. As a follow up, we have determined the 3D structure of γS-G57W and studied its conformational dynamics by solution NMR spectroscopy. Our structural dynamics results reveal greater flexibility of the N-terminal domain, which undergoes site-specific structural changes to accommodate W57, than its C-terminal counterpart. Our structural inferences that the unusual solvent exposure of W57 is associated with rearrangement of the N-terminal domain suggest an efficient pathway for increased aggregation in γS-G57W and illuminates the molecular dynamics underlying cataractogenic aggregation of lens crystallins in particular and aggregation of proteins in general.

1H, 13C and 15N NMR assignments of two plant protease inhibitors (IRD7 and IRD12) from the plant Capsicum annuum.

Helicoverpa species are polyphagous pests, with the larval stages causing major damage to economically valuable crops such as cotton, tomato, corn, sorghum, peas, sunflower, wheat and other pulses. Over the years, Helicoverpa armigera has developed resistance to most classes of chemical insecticides, and consequently it is now largely controlled on cotton plants via the use of Bt transgenic crops that express insecticidal Cry toxins which in-turn expedited resistance development in a number of pest species including H. armigera. In a hope to provide other eco-friendly alternatives solutions to counter the effect of the pest, people have identified a number of protease inhibitors (PIs) from the domesticated capsicum species Capsicum annuum, several of which potently inhibited H. armigera gut proteases and impeded growth of H. armigera larva. With a view to explore and enhance the specific nature or properties of these PIs on the mechanism of inhibition, structural and functional characterization of these PIs are inevitable. Towards this goal, we have carried out complete 1H, 13C and 15N resonance assignments of two of these PIs, identified as IRD7 and IRD12, using a suite of 2D and 3D multi-dimensional and multi-nuclear NMR experiments.

Sequence specific 1H, 13C and 15N resonance assignments of the C-terminal domain of human γS-crystallin.

The high solubility and stability of crystallins present in the human eye lens maintains its transparency and refractive index with negligible protein turnover. Monomeric γ-crystallins and oligomeric ß-crystallins are made up of highly homologous double Greek key domains. These domains are symmetric and possess higher stability as a result of the complex topology of individual Greek key motifs. γS-crystallin is one of the most abundant structural ßγ-crystallins present in the human eye lens. In order to understand the structural stability of individual domains of human γS-crystallin in isolation vis-à-vis full length protein, we set out to structurally characterize its C-terminal domain (abbreviated hereafter as γS-CTD) by solution NMR. In this direction, we have cloned, over-expressed, isolated and purified the γS-CTD. The 2D [15N-1H] HSQC recorded with uniformly 13C/15N labeled γS-CTD showed a highly dispersed spectrum indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific 1H, 13C and 15N resonance assignments of γS-CTD using a suite of heteronuclear 3D NMR experiments.

Structural and functional characterization of a missense mutant of human γS-crystallin associated with dominant infantile cataracts.

Infantile cataracts constitute one of the most important causes of childhood blindness worldwide. Human γS-crystallin is the most abundant protein in the eye lens cortex. A missense mutant of human γS-crystallin, Y67N (abbreviated hereafter as γS-Y67N) is recently reported to be associated with dominant infantile cataracts. To understand the structural basis for γS-Y67N to cause lens opacification, we constructed, expressed and purified γS-Y67N and its wild-type (abbreviated hereafter as γS-WT) and studied the structural and functional differences between them in solution using circular dichroism (CD), differential scanning calorimetry (DSC), fluorescence spectroscopy and extrinsic spectral probes. Extensive equilibrium characterization indicate that replacement of the highly conserved Tyr at 67th position by Asn distorts the conserved Tyr corner at the second Greek key motif in the N-terminal domain (NTD) and leads to substantial loss of structural stability. Our intrinsic fluorescence quenching results reveal differential in-vitro refolding kinetics identifying partially folded kinetic intermediates for both proteins. Extrinsic fluorescence studies further reveal loosening up of the compact structure of γS-crystallin upon mutation associated with enhanced aggregation. As Ca2+ homeostasis is a crucial regulator of lens transparency, we further investigated the Ca2+-binding properties of γS-WT and γS-Y67N by isothermal titration calorimetry (ITC) to identify lens Ca2+ distribution in health and in disease. Overall, our results highlight the vital role of conserved Tyr corners in stabilizing Greek key motifs and provide useful structural and functional insights into the mechanism of cataract formation in humans.

Structural characterization of a novel KH-domain containing plant chloroplast endonuclease.

Chlamydomonas reinhardtii is a single celled alga that undergoes apoptosis in response to UV-C irradiation. UVI31+, a novel UV-inducible DNA endonuclease in C. reinhardtii, which normally localizes near cell wall and pyrenoid regions, gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV-stress. Solution NMR structure of the first putative UV inducible endonuclease UVI31+ revealed an α1-ß1-ß2-α2-α3-ß3 fold similar to BolA and type II KH-domain ubiquitous protein families. Three α-helices of UVI31+ constitute one side of the protein surface, which are packed to the other side, made of three-stranded ß-sheet, with intervening hydrophobic residues. A twenty-three residues long polypeptide stretch (D54-H76) connecting ß1 and ß2 strands is found to be highly flexible. Interestingly, UVI31+ recognizes the DNA primarily through its ß-sheet. We propose that the catalytic triad residues involving Ser114, His95 and Thr116 facilitate DNA endonuclease activity of UVI31+. Further, decreased endonuclease activity of the S114A mutant is consistent with the direct participation of Ser114 in the catalysis. This study provides the first structural description of a plant chloroplast endonuclease that is regulated by UV-stress response.

Sequence specific 1H, 13C and 15N resonance assignments of a cataract-related variant G57W of human γS-crystallin.

γS-crystallin is a major structural component of the human eye lens, which maintains its stability over the lifetime of an organism with negligible turnover. The G57W mutant of human γS-crystallin (abbreviated hereafter as γS-G57W) is associated with dominant congenital cataracts. In order to provide a structural basis for the ability of γS-G57W causing cataract, we have cloned, overexpressed, isolated and purified the protein. The 2D [15N-1H]-HSQC spectrum recorded with uniformly 13C/15N-labelled γS-G57W was highly dispersed indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific 1H, 13C and 15N resonance assignments of γS-G57W using a suite of heteronuclear 3D NMR experiments.

Mechanism of Initiation, Association, and Formation of Amyloid Fibrils Modeled with the N-Terminal Peptide Fragment, IKYLEFIS, of Myoglobin G-Helix.

Some peptides and proteins undergo self-aggregation under certain conditions, leading to amyloid fibrils formation, which is related to many disease conditions. It is important to understand such amyloid fibrils formation to provide mechanistic detail that governs the process. A predominantly α-helical myoglobin has been reported recently to readily form amyloid fibrils at a higher temperature, similar to its G-helix segment. Here, we have investigated the mechanism of amyloid fibrils formation by performing multiple long molecular dynamics simulations (27 µs) on the N-terminal segment of the G-helix of myoglobin. These simulations resulted in the formation of a single-layered tetrameric ß-sheet with mixed parallel and antiparallel ß-strands and this is the most common event irrespective of many different starting structures. Formation of the single-layered tetrameric ß-sheet takes place following three distinctive pathways. The process of fibril initiation is dependent on temperature. Further, this study provides mechanistic insights into the formation of multilayered fibrilar structure, which could be applicable to a wider variety of peptides or proteins to understand the amyloidogenesis.

1H, 13C and 15N NMR assignments of a bacterial immunoglobulin-like domain (group 2) of a protein of a bacterium Paenarthrobacter aurescens TC1.

The bacterial immunoglobulin-like (Big) domain is one of the prevalent domain types, which facilitates cell-cell adhesion by assembling into multi-domain architectures. We selected a four Big_2 domain protein (named 'Arig') from a Gram positive, Paenarthrobacter aurescens TC1 (known earlier as Arthrobacter aurescens TC1). In an attempt to characterize structural and ligand-binding features of individual Big_2 domains, we have cloned, overexpressed, isolated and purified the second Big_2 domain of Arig along with a few of its adjacent Big_2 domain residues (residue 143 to 269) referred to as 'Arig2'. The 13C/15N-doubly-labeled His-tagged Arig2 (133 residues long) showed an ordered conformation as revealed by the well dispersed 2D [15N-1H]-HSQC spectrum. Subsequently, a suite of heteronuclear 3D NMR experiments has enabled almost complete 1H, 13C and 15N NMR resonance assignments of Arig2.

Structure of Ca2+-binding protein-6 from Entamoeba histolytica and its involvement in trophozoite proliferation regulation.

Cell cycle of Entamoeba histolytica, the etiological agent of amoebiasis, follows a novel pathway, which includes nuclear division without the nuclear membrane disassembly. We report a nuclear localized Ca2+-binding protein from E. histolytica (abbreviated hereafter as EhCaBP6), which is associated with microtubules. We determined the 3D solution NMR structure of EhCaBP6, and identified one unusual, one canonical and two non-canonical cryptic EF-hand motifs. The cryptic EF-II and EF-IV pair with the Ca2+-binding EF-I and EF-III, respectively, to form a two-domain structure similar to Calmodulin and Centrin proteins. Downregulation of EhCaBP6 affects cell proliferation by causing delays in transition from G1 to S phase, and inhibition of DNA synthesis and cytokinesis. We also demonstrate that EhCaBP6 modulates microtubule dynamics by increasing the rate of tubulin polymerization. Our results, including structural inferences, suggest that EhCaBP6 is an unusual CaBP involved in regulating cell proliferation in E. histolytica similar to nuclear Calmodulin.

1H, 13C and 15N NMR assignments of an unusual Ca2+-binding protein from Entamoeba histolytica in its apo form.

We report almost complete sequence specific 1H, 13C and 15N NMR assignments of an unusual Ca2+-binding protein from Entamoeba histolytica (EhCaBP6) in its apo form as a prelude to its structural and functional characterization.