Somatic Embryogenesis and Plant Regeneration from Tissue Cultures of Hyoscyamus muticus L.

Somatic embryogenesis and regeneration of plantlets was achieved In callus cultures derived from cotyledonary leaf pieces of Hyoscyamus muticus L on MS medium enriched with 2 mg/l 2,4–0 and 0.5 mg/l BAP. For embryogenesis and organogenesis varying concentrations of NAA with or without BAP were added In the medium. Organogenesis was also achieved when callus was transferred to the hormone free medium.     

Molecular characterization and diversity analysis of bacterial communities associated with Dialeurolonga malleswaramensis (Hemiptera: Aleyrodidae) adults using 16S rDNA amplicon pyrosequencing and FISH

Dialeurolonga malleswaramensis Sundararaj (Hemiptera: Aleyrodidae) is a phytophagous sap sucking insect. It infests Polyalthia longifolia, an important avenue tree of India, effective in alleviating noise pollution and having immense medicinal importance. Samples of this insect were collected from Polyalthia longifolia. The cytochrome c oxidase subunit I gene (mtCO1) helped in the molecular characterization of the insect. This study reports the bacterial diversity in D. malleswaramensis adults by high throughput 16S rDNA amplicon pyrosequencing. The major genera identified were Portiera and Arsenophonus. Other bacterial genera detected were uncultured alpha proteobacterium, Sphingopyxis and Methylobacterium. We also employed fluorescence in situ hybridization (FISH) in whole mount samples to confirm the presence of dominant endosymbionts Portiera and Arsenophonus to the bacteriocyte of D. malleswaramensis. This study concludes that combining techniques like 16S rDNA amplicon pyrosequencing and FISH reveal both dominant and rare bacteria. The data also predict the evolutionary position of this pest with respect to other whitefly species using a mitochondrial marker.     

Modeling the binding sites of anti-hen egg white lysozyme antibodies HyHEL-8 and HyHEL-26: an insight into the molecular basis of antibody cross-reactivity and specificity

Three antibodies, HyHEL-8 (HH8), HyHEL-10 (HH10), and HyHEL-26 (HH26) are specific for the same epitope on hen egg white lysozyme (HEL), and share >90% sequence homology. Their affinities vary by several orders of magnitude, and among the three antibodies, HH8 is the most cross-reactive with kinetics of binding that are relatively invariable compared to HH26, which is highly specific and has quite variable kinetics. To investigate structural correlates of these functional variations, the Fv regions of HH8 and HH26 were homology-modeled using the x-ray structure of the well-characterized HH10-HEL complex as template. The binding site of HH26 is most charged, least hydrophobic, and has the greatest number of intramolecular salt bridges, whereas that of HH8 is the least charged, most hydrophobic and has the fewest intramolecular salt bridges. The modeled HH26-HEL structure predicts the recently determined x-ray structure of HH26, (Li et al., 2003, Nat. Struct. Biol. 10:482-488) with a root-mean-square deviation of 1.03 A. It is likely that the binding site of HH26 is rendered rigid by a network of intramolecular salt bridges whereas that of HH8 is flexible due to their absence. HH26 also has the most intermolecular contacts with the antigen whereas HH8 has the least. HH10 has these properties intermediate to HH8 and HH26. The structurally rigid binding site with numerous specific contacts bestows specificity on HH26 whereas the flexible binding site with correspondingly fewer contacts enables HH8 to be cross-reactive. Results suggest that affinity maturation may select for high affinity antibodies with either "lock-and-key" preconfigured binding sites, or "preconfigured flexibility" by modulating combining site flexibility.     

Protein structure to function via dynamics

Protein folding, binding, catalytic activity and molecular recognition all involve molecular movements, with varying extents. The molecular movements are brought upon via flexible regions. Stemming from sequence, a fine tuning of electrostatic and hydrophobic properties of the protein fold determine flexible and rigid regions. Studies show flexible regions usually lack electrostatic interactions, such as salt-bridges and hydrogen-bonds, while the rigid regions often have larger number of such electrostatic interactions. Protein flexible regions are not simply an outcome of looser packing or instability, rather they are evolutionally selected. In this review article we highlight the significance of protein flexibilities in folding, binding and function, and their structural and thermodynamic determinants. Our electrostatic calculations and molecular dynamic simulations on an antibody-antigen complex further illustrate the importance of protein flexibilities in binding and function.     

Spectrum of <Emphasis Type="Italic">CREBBP</Emphasis> mutations in Indian patients with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome (RSTS), a developmental disorder comprising abnormalities that include mental retardation, an unusual facial appearance, broad thumbs and big toes is frequently associated with molecular lesions in the CREB-binding protein gene, CREBBP. The objective of the present study was to identify and analyse CREBBP mutations in Indian RSTS patients on which there are no data. Direct sequencing of CREBBP performed in 13 RSTS patients identified the three zinc fingers (CH1, CH2, CH3) and HAT domain as mutational hotspots in which ten novel pathogenic mutations were localized. Functional analysis revealed that three of these mutations affecting amino acids Glu1459, Leu1668 and Glu1724 were critical for histone acetyltransferase activity. Twenty-eight novel CREBBP single-nucleotide polymorphisms (SNPs) were also identified in the Indian population. Linkage disequilibrium studies revealed associations between (i) SNP (rs129974/c.3836-206G>C) and mutation (p.Asp1340Ala); (ii) (rs130002) with mutation (p.Asn435Lys) and (iii) SNPs rs129974, rs130002 and SNP (c.3836-206G>C) signifying a disease affection status. In conclusion, the present study reports the highest detection rate of CREBBP mutations (76.9%) in RSTS patients to date, of which ten are predicted to be pathogenic and three critical for histone acetyltransferase activity. Moreover, identification of the association of CREBBP polymorphisms with disease susceptibility could be an important risk factor for the pathogenesis of RSTS.     

Understanding antibody–antigen associations by molecular dynamics simulations: Detection of important intra- and inter-molecular salt bridges

1 NSec molecular dynamics (MD) simulation of anti-hen egg white antibody, HyHEL63 (HH63), complexed with HEL reveals important molecular interactions, not revealed in its X-ray crystal structure. These molecular interactions were predicted to be critical for the complex formation, based on structure–function studies of this complex and 3-other anti-HEL antibodies, HH8, HH10 and HH26, HEL complexes. All four antibodies belong to the same structural family, referred to here as HH10 family. Ala scanning results show that they recognize ‘coincident epitopes’. 1 NSec explicit, with periodic boundary condition, MD simulation of HH63-HEL reveals the presence of functionally important salt-bridges. Around 200 ps in vacuo and an additional 20 ps explicit simulation agree with the observations from 1 Nsec simulation. Intra-molecular salt-bridges predicted to play significant roles in the complex formation, were revealed during MD simulation. A very stabilizing salt-bridge network, and another intra-molecular salt-bridge, at the binding site of HEL, revealed during the MD simulation, is proposed to predipose binding site geometry for specific binding. All the revealed salt-bridges are present in one or more of the other three complexes and/or involve “hot-spot” epitope and paratope residues. Most of these charged epitope residues make large contribution to the binding free energy. The “hot spot” epitope residue Lys97_Y, which significantly contributes to the free energy of binding in all the complexes, forms an intermolecular salt-bridge in several MD conformers. Our earlier computations have shown that this inter-molecular salt-bridge plays a significant role in determining specificity and flexibility of binding in the HH8-HEL and HH26-HEL complexes. Using a robust criterion of salt-bridge detection, this inter-molecular salt-bridge was detected in the native structures of the HH8-HEL and HH26-HEL complexes, but was not revealed in the crystal structure of HH63-HEL complex. The electrostatic strength of this revealed salt-bridge was very strong. During 1 Nsec MD simulation this salt-bridge networks with another inter-molecular salt-bridge to form an inter-molecular salt-bridge triad. Participation of Lys97_Y in the formation of inter-molecular triad further validates the functional importance of Lys97_Y in HH63-HEL associations. These results demonstrate that many important structural details of biomolecular interactions can be better understood when studied in a dynamic environment, and that MD simulations can complement and expand information obtained from static X-ray structure. This study also highlights “hot-spot” molecular interactions in HyHEL63-HEL complex. The publisher or recipient acknowledges right of the U.S. Government to retain a non exclusive, royalty-free license in and to any copyright covering the article.     

Molecular dynamics simulation of a high-affinity antibody-protein complex

One nanosecond molecular dynamic (MD) simulation of anti-hen egg white lysozyme (HEL) antibody HyHEL63 (HH63) complexed with HEL reveals rigid and flexible regions of the HH63 binding site. Fifty conformations, extracted from the MD trajectory at regular time intervals were superimposed on HH63-HEL X-ray crystal structure, and the root mean squared deviations (RMSDs) and deviations in Calpha atom positions between the X-ray structure and the MD conformer were measured. Residue positions showing the large deviations in both light chain and heavy chain of the antibody were same in all the MD conformers. The residue positions showing smallest deviations were same for all the conformers in the case of light chain, whereas relatively variable in the heavy chain. Positions of large and small deviations fell in the complementarity determining regions (CDRs), for both heavy and light chains. The larger deviations were in CDR-2 of light and CDR-1 of heavy chain. Smaller deviations were in CDR-3 of light and CDR-2 and CDR-3 of heavy chains. The large and small deviating regions highlight flexible and rigid regions of HH63 binding site and suggest a mosaic binding mechanism, including both "induced fit" and preconfigured "lock-and-key" type of binding. Combined "induced fit" and "lock-and-key" binding would be a better definition for the formation of large complexes, which bury larger surface area on binding, as in the case of antibody-HEL complex. We further show that flexible regions, comprising mostly charged and polar residues, form intermolecular interactions with HEL, whereas rigid regions do not. Electrostatic complementarity between HH63 and HEL also imply optimized binding affinity. Flexible and rigid regions of a high-affinity antibody are selected during the affinity maturation of the antibody and have specific functional significance. The functional importance of local inherently flexible regions is to establish intermolecular contacts or they play a key role in molecular recognition, whereas local rigid regions provide the structural framework.