Petety V. Balaji

Understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coli

Solubility of proteins on overexpression in Escherichia coli is a manifestation of the net effect of several sequence‐dependent and sequence‐independent factors. This study aims to delineate the relationship between the primary structure and solubility on overexpression. The amino acid sequences of proteins reported to be soluble or to form inclusion bodies on overexpression in E. coli under normal growth conditions were analyzed. The results show a positive correlation between thermostability and solubility of proteins, and an inverse correlation between the in vivo half‐life of proteins and solubility. The amino acid (Asn, Thr, Tyr) composition and the tripeptide frequency of the protein were also found to influence its solubility on overexpression. The amino acids that were seen to be present in a comparatively higher frequency in inclusion body‐forming proteins have a higher sheet propensity, whereas those that are seen more in soluble proteins have a higher helix propensity; this is indicative of a possible correlation between sheet propensity and inclusion body formation. Thus, the present analysis shows that thermostability, in vivo half‐life, Asn, Thr, and Tyr content, and tripeptide composition of a protein are correlated to the propensity of a protein to be soluble on overexpression in E. coli. The precise mechanism by which these properties affect the solubility status of the overexpressed protein remains to be understood.

A support vector machine-based method for predicting the propensity of a protein to be soluble or to form inclusion body on overexpression in Escherichia coli

MOTIVATION Inclusion body formation has been a major deterrent for overexpression studies since a large number of proteins form insoluble inclusion bodies when overexpressed in Escherichia coli. The formation of inclusion bodies is known to be an outcome of improper protein folding; thus the composition and arrangement of amino acids in the proteins would be a major influencing factor in deciding its aggregation propensity. There is a significant need for a prediction algorithm that would enable the rational identification of both mutants and also the ideal protein candidates for mutations that would confer higher solubility-on-overexpression instead of the presently used trial-and-error procedures. RESULTS Six physicochemical properties together with residue and dipeptide-compositions have been used to develop a support vector machine-based classifier to predict the overexpression status in E.coli. The prediction accuracy is approximately 72% suggesting that it performs reasonably well in predicting the propensity of a protein to be soluble or to form inclusion bodies. The algorithm could also correctly predict the change in solubility for most of the point mutations reported in literature. This algorithm can be a useful tool in screening protein libraries to identify soluble variants of proteins.

Identification of common structural features of binding sites in galactose-specific proteins

Galactose‐binding proteins characterize an important subgroup of sugar‐binding proteins that are involved in a variety of biological processes. Structural studies have shown that the Gal‐specific proteins encompass a diverse range of primary and tertiary structures. The binding sites for galactose also seem to vary in different protein‐galactose complexes. No common binding site features that are shared by the Gal‐specific proteins to achieve ligand specificity are so far known. With the assumption that common recognition principles will exist for common substrate recognition, the present study was undertaken to identify and characterize any unique galactose‐binding site signature by analyzing the three‐dimensional (3D) structures of 18 protein‐galactose complexes. These proteins belong to 7 nonhomologous families; thus, there is no sequence or structural similarity across the families. Within each family, the binding site residues and their relative distances were well conserved, but there were no similarities across families. A novel, yet simple, approach was adopted to characterize the binding site residues by representing their relative spatial dispositions in polar coordinates. A combination of the deduced geometrical features with the structural characteristics, such as solvent accessibility and secondary structure type, furnished a potential galactose‐binding site signature. The signature was evaluated by incorporation into the program COTRAN to search for potential galactose‐binding sites in proteins that share the same fold as the known galactose‐binding proteins. COTRAN is able to detect galactose‐binding sites with a very high specificity and sensitivity. The deduced galactose‐binding site signature is strongly validated and can be used to search for galactose‐binding sites in proteins. PROSITE‐type signature sequences have also been inferred for galectin and C‐type animal lectin‐like fold families of Gal‐binding proteins. Proteins 2004;55:44–65.

Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine

BackgroundGriseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug.MethodsThe effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied in vitro by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1.ResultsGriseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the αβ intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation.ConclusionsThe study provided evidence suggesting that griseofulvin shares its binding site in tubulin with paclitaxel and kinetically suppresses microtubule dynamics in a similar manner. The results revealed the antimitotic mechanism of action of griseofulvin and provided evidence suggesting that griseofulvin alone and/or in combination with vinblastine may have promising role in breast cancer chemotherapy.

Energetics of galactose- and glucose-aromatic amino acid interactions: implications for binding in galactose-specific proteins

An aromatic amino acid is present in the binding site of a number of sugar binding proteins. The interaction of the saccharide with the aromatic residue is determined by their relative position as well as orientation. The position‐orientation of the saccharide relative to the aromatic residue was found to vary in different sugar‐binding proteins. In the present study, interaction energies of the complexes of galactose (Gal) and of glucose (Glc) with aromatic residue analogs have been calculated by ab initio density functional (U‐B3LYP/ 6‐31G**) theory. The position‐orientations of the saccharide with respect to the aromatic residue observed in various Gal‐, Glc‐, and mannose–protein complexes were chosen for the interaction energy calculations. The results of these calculations show that galactose can interact with the aromatic residue with similar interaction energies in a number of position‐orientations. The interaction energy of Gal–aromatic residue analog complex in position‐orientations observed for the bound saccharide in Glc/Man–protein complexes is comparable to the Glc–aromatic residue analog complex in the same position‐orientation. In contrast, there is a large variation in interaction energies of complexes of Glc‐ and of Gal‐ with the aromatic residue analog in position‐orientations observed in Gal–protein complexes. Furthermore, the conformation wherein the O6 atom is away from the aromatic residue is preferred for the exocyclic —CH2OH group in Gal–aromatic residue analog complexes. The implications of these results for saccharide binding in Gal‐specific proteins and the possible role of the aromatic amino acid to ensure proper positioning and orientation of galactose in the binding site have been discussed.

Characterization of symmetric and asymmetric lipid bilayers composed of varying concentrations of ganglioside GM1 and DPPC.

Gangliosides are a group of structurally diverse, sialic acid containing glycosphingolipids embedded into the membrane via their hydrophobic ceramide moiety. To gain atomic level insights into the structural perturbations caused by Galbeta3GalNAcbeta4(NeuAcalpha3)Galbeta4Glc1Cer (GM1), molecular dynamics (MD) simulations of a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayer containing GM1 at five different concentrations have been performed. Biological membranes contain GM1 only on the exoplasmic leaflet. However, vesicles prepared in the laboratory contain GM1 in both the leaflets albeit unequally. Hence, simulations were performed with GM1 present in only one (asymmetric bilayers) or in both of the leaflets (symmetric bilayers) of the bilayer. In symmetric bilayers, there is a decrease in surface area, an increase in deuterium order parameter, and an increase in peak-to-peak distance of DPPC with increasing concentration of GM1. Thus, the overall area of the lipid bilayer decreases (condensation effect) and the thickness increases with increasing concentrations of GM1. Even in asymmetric systems, decrease in surface area and increase in deuterium order parameter of hydrocarbon chains of DPPC are observed. However, the decrease in bilayer area and the increase in bilayer thickness are not as much as in the symmetric bilayer.

E93R Substitution of Escherichia coli FtsZ Induces Bundling of Protofilaments, Reduces GTPase Activity, and Impairs Bacterial Cytokinesis

Recently, we found that divalent calcium has no detectable effect on the assembly of Mycobacterium tuberculosis FtsZ (MtbFtsZ), whereas it strongly promoted the assembly of Escherichia coli FtsZ (EcFtsZ). While looking for potential calcium binding residues in EcFtsZ, we found a mutation (E93R) that strongly promoted the assembly of EcFtsZ. The mutation increased the stability and bundling of the FtsZ protofilaments and produced a dominating effect on the assembly of the wild type FtsZ (WT-FtsZ). Although E93R-FtsZ was found to bind to GTP similarly to the WT-FtsZ, it displayed lower GTPase activity than the WT-FtsZ. E93R-FtsZ complemented for its wild type counterpart as observed by a complementation test using JKD7–1/pKD3 cells. However, the bacterial cells became elongated upon overexpression of the mutant allele. We modeled the structure of E93R-FtsZ using the structures of MtbFtsZ/Methanococcus jannaschi FtsZ (MjFtsZ) dimers as templates. The MtbFtsZ-based structure suggests that the Arg93-Glu138 salt bridge provides the additional stability, whereas the effect of mutation appears to be indirect (allosteric) if the EcFtsZ dimer is similar to that of MjFtsZ. The data presented in this study suggest that an increase in the stability of the FtsZ protofilaments is detrimental for the bacterial cytokinesis.

Functional domains of bovine beta-1,4 galactosyltransferase.

A number of N- and C-terminal deletion and point mutants of bovine β-1,4 galactosyltransferase (β-1,4GT) were expressed inE. coli to determine the binding regions of the enzyme that interact withN-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1–129, do not show any significant difference in the apparentKms toward NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15mm NAG and 50mm EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130–402 of bovine β-1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130–257 of the β-1,4GT, binds to, and elutes with 15mm NAG and 50mm EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15mm NAG. The C-terminus fragment GT-257UDP, containing residues 258–402 of β-1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5–10% of the bound protein, can be eluted from the UDP-agarose column with 50mm EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of β-1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn2+.

C-H…pi interactions in proteins: prevalence, pattern of occurrence, residue propensities, location, and contribution to protein stability.

AbstractC-H…pi interactions are a class of non-covalent interactions found in different molecular systems including organic crystals, proteins and nucleic acids. High-resolution protein structures have been analyzed in the present study to delineate various aspects of C-H…pi interactions. Additionally, to determine the extent to which redundancy of a database biases the outcome, two datasets differing from each other in the level of redundancy have been analyzed. On average, only one out of six {with C-H(Aro) group} or eight {with C-H(Ali) group} residues in a protein participate as C-H group donors. Neither the frequency of occurrence in proteins nor the number of C-H groups present in it is correlated to the propensity of an amino acid to participate in C-H…pi interactions. Most of the residues that participate in C-H…pi interactions are solvent-shielded. Solvent shielded nature of most of the C-H…pi interactions and prevalence of intra- as well as inter-secondary structural element C-H…pi interactions suggest that the contribution of these interactions to the enthalpy of folded form will be significant. The separation in the primary structure between donor and acceptor residues is found to be correlated to secondary structure type. Other insights obtained from this study include the presence of networks of C-H…pi interactions spanning multiple secondary structural elements. To our knowledge this has not been reported so far. A substantial number of residues involved in C-H…pi interactions are found in catalytic and ligand binding sites suggesting their possible role in maintaining active site geometry. No significant differences of C-H…pi interactions in the two datasets are found for any of the parameters/features analyzed. FigureC-H…pi interactions in proteins

Size, orientation and organization of oligomers that nucleate amyloid fibrils: Clues from MD simulations of pre-formed aggregates

All-atom MD simulations of pre-formed aggregates of GNNQQNY with variable size (5 to 16 peptides), orientation (parallel or anti-parallel), organization (single or double sheet, with or without twist), charge status of termini and temperature (300 and 330K) have been performed for 50ns each (68 simulations; total time=3.4μs). Double-layer systems are stable irrespective of whether the peptides within the sheet are oriented parallel or anti-parallel. The lifetime of single sheet systems is determined by the protonation status, nature of association of peptides and the size of the aggregates. For example, single sheet 8-mers are stable with parallel arrangement and neutral termini, or with anti-parallel arrangement and charged termini. This suggests that the residues flanking the amyloidogenic sequence also play an important role in determining the organization of peptides in an aggregate. Twist of the cross-beta sheets is found to be intrinsic to the aggregates. Main chain H-bonds are key determinants of stability and loss of these H-bonds is followed by disorder and/or dissociation of the peptide despite the presence of side chain hydrogen bonds. Aggregates are inherently asymmetric along the fiber axis and dissociation from the C-edge is observed more often. An aggregate can disintegrate into smaller-sized oligomers or the edge peptides can dissociate sequentially. A variety of dissociation and disintegration events are observed pointing to the existence of multiple pathways for association during nucleation. It appears that a heterogeneous mixture of oligomers of different sizes exist prior to the formation of the critical nucleus.