A study of the occurrence of regulator secondary structures in globular proteins

The distribution of regular secondary structures, viz. α-helices and β-strands, along the length of over 70 properties whose secondary structural details have been reported, has been analysed. The occurrence of these regular structures tends to be a maximum at the N- and C-termini. Our analysis suggests that both these free ends could possibly serve as nucleating centers for secondary structures and could play an important role in the folding of proteins.     

Structural requirements for a primitive adaptor molecule

Adaptor properties of linear hairpin helices have been examined. The analysis suggests that neither right nor left handed hairpin helices can simultaneously read a comma free messenger and align aminoacyl residues for peptide condensation. Comparison of these studies with the model of the present day peptidyl transfer intermediate suggests that the "L" shaped folding of the present day tRNAs may be a prerequisite for adaptor function. Therefore, the three-dimensional organization of the ancestral adaptor molecule must have had structural features similar to its present day counterpart.     

Fibronectin glycosylation modulates fibroblast adhesion and spreading

The role of the carbohydrate residues of fibronectin concerning the specificities of that glycoprotein to interact with fibroblastic cell surfaces, gelatin, and heparin was examined. Tunicamycin was used to produce carbohydrate-depleted fibronectin; it was synthesized by cultured fibroblasts. Unglycosylated and glycosylated fibronectins were analyzed for their ability to bind gelatin and heparin, using affinity columns. Fibronectin-coated surfaces were used to quantitatively measure cell adhesion and spreading. The results showed that the lack of carbohydrates significantly increased the interaction of the protein with gelatin and markedly enhanced its ability to promote adhesion and spreading of fibroblasts. In contrast, the binding of fibronectin to heparin was not influenced by glycosylation. The composite data indicate that the Asn-linked oligosaccharides of fibronectin act as modulators of biological functions of the glycoprotein.     

Modeling the ion channel structure of cecropin.

Atomic-scale computer models were developed for how cecropin peptides may assemble in membranes to form two types of ion channels. The models are based on experimental data and physiochemical principles. Initially, cecropin peptides, in a helix-bend-helix motif, were arranged as antiparallel dimers to position conserved residues of adjacent monomers in contact. The dimers were postulated to bind to the membrane with the NH2-terminal helices sunken into the head-group layer and the COOH-terminal helices spanning the hydrophobic core. This causes a thinning of the top lipid layer of the membrane. A collection of the membrane bound dimers were then used to form the type I channel structure, with the pore formed by the transmembrane COOH-terminal helices. Type I channels were then assembled into a hexagonal lattice to explain the large number of peptides that bind to the bacterium. A concerted conformational change of a type I channel leads to the larger type II channel, in which the pore is formed by the NH2-terminal helices. By having the dimers move together, the NH2-terminal helices are inserted into the hydrophobic core without having to desolvate the charged residues. It is also shown how this could bring lipid head-groups into the pore lining.     

A quantitating reagent for methanethiolation of protein sulfhydryl groups

p-Nitrophenoxycarbonyl methyl disulfide has been synthesized for use as a quantitating agent for methanethiolation of protein sulfhydryl groups. This reagent reacts specifically and quantitatively with cysteine residues of proteins to yield an unsymmetrical disulfide containing a CH₃S group and concomitantly releases the chromophore, p-nitrophenol. Titration of the sulfhydryl groups of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with this reagent has been studied. Incorporation of CH₃S as measured by the release of p-nitrophenol paralleled the loss of sulfhydryl group dependent activity of the enzyme. The enzyme was found inactive on modification of four of the eight sulfhydryl groups present in the enzyme. Stability of p-nitrophenoxycarbonyl methyl disulfide has also been studied in different buffer systems. The rate of decomposition of the p-nitrophenyl ester due to hydrolysis was found negligible below a pH of 8.0 compared to its rate of reaction with free sulfhydryl groups.     

Effect of pH on the phase behavior of DMPC bilayers

We have studied the effect of acidic pH on the phase behavior of the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) using differential scanning calorimetry and x-ray scattering. Dispersions of DMPC in HCl solutions of pH = 4 and 3 behave identical to dispersions in water. The main transition temperature increases sharply and the pre-transition disappears at lower pH. An untilted gel phase is observed at pH = 2 and 1, in contrast to the tilted gel phase found at higher pH. The relatively large periodicity of the untilted gel phase, in comparison to that of the tilted gel phase occurring near neutral pH, clearly demonstrates the simultaneous charging and dehydration of the headgroups as the pH approaches the pK of the phosphate group. Headgroup dehydration at low pH also leads to the formation of DMPC crystallites and the inverted hexagonal phase at low and high temperatures, respectively, after a few days of incubation. These results show the significant effect of acidic pH on the phase behavior of zwitterionic lipids.     

Characterization of a subunit structure and stability of the recombinant porin fromNeisseria gonorrhoeae

An outer membrane PIA protein fromNeisseria gonorrhoeae strain FA19 was expressed inEscherichia coli and refoldedin vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50°C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80°C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind withK _d=80 and 130 μM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.     

Synthesis and antibacterial property of quinolines with potent DNA gyrase activity

Synthesis of a series of novel tetrahydroquinoline annulated heterocycles has been accomplished by intramolecular imino and bisimino Diels–Alder reaction. These compounds were evaluated for their antibacterial activity. All the synthetic compounds, exhibited good antibacterial activity against microorganisms of which one of them 7 was found to be as active as the antibiotic ciplofloxacin and is found to have MIC value of 2.5 mg/mL against Escherichia coli.     

Production and characterization of haloalkaline protease from ascidian-associated <Emphasis Type="Italic">Virgibacillus halodenitrificans</Emphasis> RSK CAS1 using marine wastes

A marine ascidian-associated bacterium, Virgibacillus halodenitrificans RSK CAS1, was optimized for protease production by response surface methodology using marine waste as substrate. The central composite design was employed, and the optimal medium constituents for maximum protease production (1461.11 U/ml) were determined to be shrimp shell powder (15.32 g/l), casein (5.37 g/l), MgSO₄ (3.0 g/l) and NaCl (55.31 g/l). The protease was purified from the culture supernatant to homogeneity in a three-step procedure consisting of ammonium sulfate precipitation, ion exchange chromatography (DEAE-cellulose column) and gel-filtration chromatography (Sephadex G-75 column), resulting in a 8.7-fold-change in purified protein. This protein had a specific activity of 1,086.78 U/mg and a molecular weight of 21 kDa. It exhibited optimal activity at 50 °C, pH 9 and 25 % NaCl. The significant stability of this protein at higher levels of salt, metal ions, organic solvents and commercial detergents and at higher, temperature, as well as its application as a cleaning additive in blood stain removal, suggests its possible use the laundry detergent industry.     

Stereoselective synthesis of hexahydro-3-methyl-1-arylchromeno[3,4-b]pyrrole and its annulated heterocycles as potent antimicrobial agents for human pathogens

Synthesis of a series of novel hexahydrochromenopyrrole analogues has been accomplished through an intramolecular 1,3-dipolar cycloaddition (1,3-DC reaction) of azomethine ylides, generated by the aldehyde induced decarboxylation of secondary amino acids. These compounds were screened for antibacterial and antifungal activities against six human pathogenic bacteria and three human pathogenic fungi and found to have good antimicrobial properties against most of the microorganisms.