A substitution matrix for structural alphabet based on structural alignment of homologous proteins and its applications

Analysis of protein structures based on backbone structural patterns known as structural alphabets have been shown to be very useful. Among them, a set of 16 pentapeptide structural motifs known as protein blocks (PBs) has been identified and upon which backbone model of most protein structures can be built. PBs allows simplification of 3D space onto 1D space in the form of sequence of PBs. Here, for the first time, substitution probabilities of PBs in a large number of aligned homologous protein structures have been studied and are expressed as a simplified 16 x 16 substitution matrix. The matrix was validated by benchmarking how well it can align sequences of PBs rather like amino acid alignment to identify structurally equivalent regions in closely or distantly related proteins using dynamic programming approach. The alignment results obtained are very comparable to well established structure comparison methods like DALI and STAMP. Other interesting applications of the matrix have been investigated. We first show that, in variable regions between two superimposed homologous proteins, one can distinguish between local conformational differences and rigid-body displacement of a conserved motif by comparing the PBs and their substitution scores. Second, we demonstrate, with the example of aspartic proteinases, that PBs can be efficiently used to detect the lobe/domain flexibility in the multidomain proteins. Lastly, using protein kinase as an example, we identify regions of conformational variations and rigid body movements in the enzyme as it is changed to the active state from an inactive state.     

Enzymatic modification of cassava starch by bacterial lipase

Enzymatic modification of starch using long chain fatty acid makes it thermoplastic suitable for a myriad of industrial applications. An industrial lipase preparation produced by Burkholderia cepacia (lipase PS) was used for modification of cassava starch with two acyl donors, lauric acid and palmitic acid. Reactions performed with palmitic acid by liquid-state and microwave esterification gave a degree of substitution (DS) of 62.08% (DS 1.45) and 42.06% (DS 0.98), respectively. Thermogravimetric analysis showed that onset of decomposition is at a higher temperature (above 600°C) for modified starch than the unmodified starch (280°C). Modified starch showed reduction in α-amylase digestibility compared to native starch (76.5–18%). Swelling power lowered for modified starch as esterification renders starch more hydrophobic, making it suitable for biomedical applications as materials for bone fixation and replacements, carriers for controlled release of drugs and bioactive agents. Thus enzymatic esterification is ecofriendly.     

Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications

Collagen is a widely investigated extracellular matrix material with extensive potentials in the field of tissue engineering. This protocol describes a method to prepare reconstituted collagen that can be ready-to-use, storable and suitable for further in vitro and in vivo investigations. Type I collagen was extracted from rat tail tendons and processed in acetic acid solution to obtain sterile soluble collagen. At first, crude collagen was dissolved in acetic acid, then frozen at -20 degrees C and lyophilized to obtain a sponge, which could be stored at -80 degrees C. Lyophilized collagen was then dispersed in acetic acid to obtain a sterile solution of collagen at targeted concentrations. The whole low-cost process from the extraction to the final sterile solution takes around 2-3 weeks. The collagen solution, once neutralized, has the potential to be used to produce gels or scaffolds, to deposit thin films on supports and to develop drug delivery systems.     

Functional Characterization of Trehalose Biosynthesis Genes from <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis>: An Osmolyte Involved in Stress Tolerance

Trehalose (1-α-d-glucopyranosyl-1-α-d-glucopyranoside), a non-reducing disaccharide is a major compatible solute, which maintains fluidity of membranes and protects the biological structure of organisms under stress. In this study, trehalose-6-phosphate synthase (otsA) and trehalose-6-phosphate phosphatase (otsB) genes encoding for trehalose biosynthesis from Escherichia coli was cloned as an operon and expressed in E. coli M15(pREP4). The recombinant E. coli strain showed a threefold increase in the activity of otsBA pathway enzymes, compared to the control strain. The transgenic E. coli accumulated up to 0.86 mg/l of trehalose. The sequence of otsA and otsB genes reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in the altered amino acid sequences of the translated proteins.     

Functional characterization,secondary structure prediction and analysis of ectoine biosynthesis genes from <Emphasis Type="Italic">Bacillus halodurans</Emphasis>: an osmolyte involved in stress tolerance

An open reading frame encoding the ectoine biosynthesis genes was cloned from the Bacillus halodurans genome. An expression plasmid containing the operon was introduced into Escherichia coli cells, and the recombinant ectoine was quantified. The secondary structure of ectoine biosynthesis proteins were predicted and was quite similar to that of reported proteins from eubacteria.     

Discovery of INCB3344, a potent, selective and orally bioavailable antagonist of human and murine CCR2

Rational design based on a pharmacophore of CCR2 antagonists reported in the literature identified lead compound 9a with potent inhibitory activity against human CCR2 (hCCR2) but moderate activity against murine CCR2 (mCCR2). Modification on 9a led to the discovery of a potent CCR2 antagonist 21 (INCB3344) with IC(50) values of 5.1 nM (hCCR2) and 9.5 nM (mCCR2) in binding antagonism and 3.8 nM (hCCR2) and 7.8 nM (mCCR2) in antagonism of chemotaxis activity. INCB3344 exhibited >100-fold selectivity over other homologous chemokine receptors, a free fraction of 24% in human serum and 15% in mouse serum, and an oral bioavailability of 47% in mice, suitable as a tool compound for target validation in rodent models.     

Design,synthesis, and biological evaluation of 4-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives as potent antitubercular agents

Based on stereoelectronic feature analysis using density functional theory (DFT) at B3LYP/3-211G level, a series of 4-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives with low LUMO energies (<?0.10 eV); concentrated over the nitro group, furan moiety and α,β-unsaturated carbonyl bridge were envisaged as potential antitubercular agents. The target compounds were prepared by condensation of 5-nitro-2-furaldehyde with various ketones under acidic condition. The compounds were evaluated for antitubercular activity against Mycobacterium tuberculosis H37Rv and their cytotoxicity in VERO cell line. Several synthesized compounds showed good antitubercular activity of <5 μM along with low cytotoxicity. In particular, compound ((E)-3-(5-nitrofuran-2-yl)-1-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one) (3v) was found to be very potent (MIC: 0.19 μM) with good selectivity index (MIC₉₀/CC₅₀: >1800). Thus, this study shows the potential of stereoelectronic property analysis in developing improved nitroaromatics as antitubercular agents.     

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti‐streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti‐streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV‐AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8‐Anilino‐1‐naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near‐UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular β‐sheet and turn structures between the monomer and aggregate species. Free thiol determination showed ～2.4‐fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4‐fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.