Ravi Pratap Barnwal

Solution Structure and Calcium-Binding Properties of M-Crystallin, A Primordial βγ-Crystallin from Archaea

The lens betagamma-crystallin superfamily has many diverse but topologically related members belonging to various taxa. Based on structural topology, these proteins are considered to be evolutionarily related to lens crystallins, suggesting their origin from a common ancestor. Proteins with betagamma-crystallin domains, although found in some eukaryotes and eubacteria, have not yet been reported in archaea. Sequence searches in the genome of the archaebacterium Methanosarcina acetivorans revealed the presence of a protein annotated as a betagamma-crystallin family protein, named M-crystallin. Solution structure of this protein indicates a typical betagamma-crystallin fold with a paired Greek-key motif. Among the known structures of betagamma-crystallin members, M-crystallin was found to be structurally similar to the vertebrate lens betagamma-crystallins. The Ca(2+)-binding properties of this primordial protein are somewhat more similar to those of vertebrate betagamma-crystallins than to those of bacterial homologues. These observations, taken together, suggest that amphibian and vertebrate betagamma-crystallin domains are evolutionarily more related to archaeal homologues than to bacterial homologues. Additionally, identification of a betagamma-crystallin homologue in archaea allows us to demonstrate the presence of this domain in all the three domains of life.

Designed α-sheet peptides inhibit amyloid formation by targeting toxic oligomers

Previous studies suggest that the toxic soluble-oligomeric form of different amyloid proteins share a common backbone conformation, but the amorphous nature of this oligomer prevents its structural characterization by experiment. Based on molecular dynamics simulations we proposed that toxic intermediates of different amyloid proteins adopt a common, nonstandard secondary structure, called α-sheet. Here we report the experimental characterization of peptides designed to be complementary to the α-sheet conformation observed in the simulations. We demonstrate inhibition of aggregation in two different amyloid systems, β-amyloid peptide (Aβ) and transthyretin, by these designed α-sheet peptides. When immobilized the α-sheet designs preferentially bind species from solutions enriched in the toxic conformer compared with non-aggregated, nontoxic species or mature fibrils. The designs display characteristic spectroscopic signatures distinguishing them from conventional secondary structures, supporting α-sheet as a structure involved in the toxic oligomer stage of amyloid formation and paving the way for novel therapeutics and diagnostics. DOI: http://dx.doi.org/10.7554/eLife.01681.001

Identification of C-terminal neighbours of amino acid residues without an aliphatic 13Cγ as an aid to NMR assignments in proteins

We propose a methodology that uses GFT (3,2)D CB(CACO)NNH experiment to rapidly collect the data and readily identify six amino acid residue types (Ala, Asn, Asp, Cys, Gly and Ser) in any given protein. Further, the experiment can distinguish the redox state of Cys residues. The proposed experiment in its two forms will have wide range of applications in resonance assignment strategies and structure determination of proteins.

Chemical shift based editing of CH3 groups in fractionally 13C-labelled proteins using GFT (3, 2)D CT-HCCH-COSY: stereospecific assignments of CH3 groups of Val and Leu residues.

We propose a (3, 2)D CT-HCCH-COSY experiment to rapidly collect the data and provide significant dispersion in the spectral region containing 13C–1H cross peaks of CH3 groups belonging to Ala, Ile, Leu, Met, Thr and Val residues. This enables one to carry out chemical shift based editing and grouping of all the 13C–1H cross peaks of CH3 groups belonging to Ala, Ile, Leu, Met, Thr and Val residues in fractionally (10%) 13C-labelled proteins, which in turn aids in the sequence-specific resonance assignments in general and side-chain resonance assignments in particular, in any given protein. Further, we demonstrate the utility of this experiment for stereospecific assignments of the pro-R and pro-S methyl groups belonging to the Leu and Val residues in fractionally (10%) 13C-labelled proteins. The proposed experiment opens up a wide range of applications in resonance assignment strategies and structure determination of proteins.

Structure and mechanism of a molecular rheostat, an RNA thermometer that modulates immune evasion by Neisseria meningitidis.

Neisseria meningitidis causes bacterial meningitis and septicemia. It evades the host complement system by upregulating expression of immune evasion factors in response to changes in temperature. RNA thermometers within mRNAs control expression of bacterial immune evasion factors, including CssA, in the 5′-untranslated region of the operon for capsule biosynthesis. We dissect the molecular mechanisms of thermoregulation and report the structure of the CssA thermometer. We show that the RNA thermometer acts as a rheostat, whose stability is optimized to respond in a small temperature range around 37°C as occur within the upper airways during infection. Small increases in temperature gradually open up the structure to allow progressively increased access to the ribosome binding site. Even small changes in stability induced by mutations of imperfect base pairs, as in naturally occurring polymorphisms, shift the thermometer response outside of the desired temperature range, suggesting that its activity could be modulated by pharmacological intervention.

Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I

The accuracy of the 3′-end processing by cleavage and polyadenylation is essential for mRNA biogenesis and transcription termination. In yeast, two poorly conserved neighboring elements upstream of cleavage sites are important for accuracy and efficiency of this process. These two RNA sequences are recognized by the RNA binding proteins Hrp1 and Rna15, but efficient processing in vivo requires a bridging protein (Rna14), which forms a stable dimer of hetero-dimers with Rna15 to stabilize the RNA–protein complex. We earlier reported the structure of the ternary complex of Rna15 and Hrp1 bound to the RNA processing element. We now report the use of solution NMR to study the interaction of Hrp1 with the Rna14–Rna15 heterodimer in the presence and absence of 3′-end processing signals. By using methyl selective labeling on Hrp1, in vivo activity and pull-down assays, we were able to study this complex of several hundred kDa, identify the interface within Hrp1 responsible for recruitment of Rna14 and validate the functional significance of this interaction through structure-driven mutational analysis.

Interaction of Follicle‐Stimulating Hormone (FSH) Receptor Binding Inhibitor‐8: A Novel FSH‐Binding Inhibitor, with FSH and its Receptor

Follicle‐stimulating hormone (FSH) receptor binding inhibitor (FRBI‐8) is a novel octapeptide purified from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing follicles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI‐8 and understand its mechanism of inhibiting interaction of FSH to its receptors. Homology modeling predicted that the FRBI‐8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI‐8 with reported FSH–FSHR hormone binding (FSHRHB) domain complex using zdock algorithm revealed that the FRBI‐8 binds to FSHβL2–FSHRHB binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI‐8 analogs were designed by replacing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI‐8D6A) had a higher binding affinity than the native FRBI‐8. In vitro radioreceptor assay with FRBI‐8D6A showed 50% lower IC50 compared with the FRBI‐8, confirming the in silico observations. Thus, the study reveals that both FRBI‐8 and FRBI‐8D6A interfered with the binding of FSH to its receptor.

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double- stranded RNA binding domain of protein kinase R

Double‐stranded RNA binding domains of human protein kinase R (dsRBD‐PKR) regulate distinct cellular functions and the fate of an RNA molecule in the cell. This highly homologous domains present in multiple copies in a number of species, exhibit individual and specific functional specificity. Number of NMR and X‐ray crystallographic structural studies reveals that such domains take a common α‐β‐β‐β‐α tertiary fold. However, the functional specificities of these domains could be due to the dynamics of the individual amino acid residues, as has been shown earlier in the case of backbone dynamics of 15N‐1H of dsRNA binding motifs (dsRBMs) of human protein kinase R (PKR) (Nanduri S, Rahman F, Williams BRG, Qin J. EMBO J 2000;19:5567–5574 ). To further investigate if the differences in dynamics of the two dsRBMs are restricted to only backbone, or if the side‐chain motions are also different to the extent of influencing their packing of the two hydrophobic cores, we have investigated the methyl group dynamics using 13C‐methyl relaxation measurements. The results show that the hydrophobic core of dsRBM1 is more tightly packed than dsRBM2, and it seems to undergo less fast scale motions in the subnanosecond regime. Proteins 2006.

Temperature‐dependent oligomerization in M‐crystallin: Lead or lag toward cataract, an NMR perspective

The oligomerization and/or aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from abnormal disease states like Alzheimers and Parkinsons disease to the production of inclusion bodies, stability, and delivery of protein drugs. In the case of eye‐lens proteins, oligomerization is implicated in cataract formation. In the present study, we have investigated the temperature driven oligomerization of M‐crystallin, a close homologue of eye‐lens proteins, using NMR spectroscopy and dynamic‐light scattering (DLS). The NMR data primarily included R1, R2 relaxation rates and nOes of the backbone amide groups recorded at three different temperatures, 25, 20, and 15° C. The major outcome of the study is the two fold increase in the overall tumbling time (τc) of M‐crystallin on lowering the temperature from 25 to 15° C. An extrapolation of τc to a further lower temperature (5° C) may lead to a τc of ∼19 ns that would correspond to a τc value of a tetrameric M‐crystallin. These results also validate the observed changes in the hydrodynamic radius of M‐crystallin, determined using DLS data. Further, the temperature‐dependent protein dynamics of M‐crystallin reveal considerable variation at/near the Ca2+‐binding sites. A concerted analysis of the temperature dependent relaxation parameters and DLS data reveals that the self‐association of the protein is not only a monomer‐dimer equilibrium, but also goes to tetramers or other multimeric states. These higher states may co‐exist in fast exchange with the monomeric and dimeric M‐crystallin at milli‐molar to sub‐millimolar concentrations and at lower temperature. Proteins 2011.

Guanidine-HCl dependent structural unfolding of M-crystallin: fluctuating native state like topologies and intermolecular association.

Numerous experimental techniques and computational studies, proposed in recent times, have revolutionized the understanding of protein-folding paradigm. The complete understanding of protein folding and intermediates are of medical relevance, as the aggregation of misfolding proteins underlies various diseases, including some neurodegenerative disorders. Here, we describe the unfolding of M-crystallin, a βγ-crystallin homologue protein from archaea, from its native state to its denatured state using multidimensional NMR and other biophysical techniques. The protein, which was earlier characterized to be a predominantly β-sheet protein in its native state, shows different structural propensities (α and β), under different denaturing conditions. In 2 M GdmCl, the protein starts showing two distinct sets of peaks, with one arising from a partially unfolded state and the other from a completely folded state. The native secondary structural elements start disappearing as the denaturant concentration approaches 4 M. Subsequently, the protein is completely unfolded when the denaturant concentration is 6 M. The 15N relaxation data (T1/T2), heteronuclear 1H-15N Overhauser effects (nOes), NOESY data, and other biophysical data taken together indicate that the protein shows a consistent, gradual change in its structural and motional preferences with increasing GdmCl concentration.