Staining the Nucleus in Yeasts

Yeast cells kept young by repeated subculturing were centrifuged, washed twice in distilled water and smeared on slides coated with a little egg albumen. The cells were treated with 0.002 M 8-hydroxyquinoline for 1 hr, fixed first in OsO, vapour for 30 sec and then in chloroform for 30 sec. The slides were passed through descending grades of alcohol, washed in distilled water, then immersed in 0.17 M NaCl solution for 2 hr. at 57°C. They were again washed in distilled water and later hydrolysed in 1 N HCl at 60°C for 5-7 min. This was followed by washing in distilled water and in buffer. The slides were then kept for 3 hr in Giemsa stain comprising 96 ml of phosphate buffer of pH 7.0 and 4 ml of the stain. After dehydration, mounting was done in balsam. Nuclei were brightly stained and well differentiated; centrosomes were clear, and the process of nuclear division and movement to daughter cells could be studied. Pretreatment with 8-hydroxyquinoline increased the viscosity of the cytoplasm, while NaCl treatment and acid hydrolysis led to the complete removal of ribonucleic acid and basophilic material. A selective staining of chromatin was thus achieved. Structures resembling chromosomes could be seen when fixed and stained cells were squashed, soon after the removal of the slides from the stain, under a cover glass by applying uniform pressure with a rubber stopper. Fixation in osmic acid vapor and chloroform followed by acid hydrolysis and staining in leucobasic fuchsin also helps to obtain bright staining of the nucleus; however, the preparations are inferior to those obtained after 8-hydroxyquinoline, NaCl treatment and Giemsa staining.     

Structural basis of typhoid: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N‐terminal 25 amino acid deleted S. typhi native PilS protein (ΔPilS), which makes the pilus, was determined at 1.9 Å resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of ΔPilS and a target CFTR peptide, determined at 1.8 Å, confirms that residues 113–117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch

The gene for a membrane-bound, halophilic, and thermostable α-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove—the N-C groove—some 30 Å away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 Å resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with α-cyclodextrin and maltoheptaose at 2.2 Å resolution, a maltotetraose molecule is bound in subsites − 1 to + 3, spanning the cleavage point at − 1/+ 1, with the − 1 glucosyl residue present as a ²S_o skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across − 1/+ 1 and ring distortion. We observe three methionine side chains that serve as “binding platforms” for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.     

How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates?

The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0-24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (approximately 5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1-2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.     

Statistical optimization of lipase catalyzed hydrolysis of methyloleate by response surface methodology

Hydrolysis of methyloleate was optimized using lipase from Chromobacterium viscosum immobilized on IRC-50. The optimization was studied by a statistical methodology using response surface methodology (RSM). The cumulative interactive effect of substrate, water concentration and time was studied in optimizing the hydrolysis of methyloleate. The interactive effect of substrate-time was found to be significant compared to substrate-water and time-water interactions. A well correlation was observed between the optimum values obtained from the response surface contour plots and from the quadratic regression model equation. The optimal values obtained for substrate, water and time were found to be in the experimental range chosen.     

Structural comparison of oxidized and reduced FKBP13 from Arabidopsis thaliana

AtFKBP13, an immunophilin in the chloroplast thylakoid lumen, participates in redox-regulatory processes via a pair of conserved disulfide bonds that are present at the N- and C-termini of the protein. Characterization of this protein by structural and biochemical analysis has revealed a novel mechanism of redox regulation in the thylakoid lumen. The protein is active in its oxidized form but is inactivated after reduction by the thioredoxin system. This is in sharp contrast with the regulation of biosynthetic enzymes in the stroma of the chloroplast, where reduction of enzymes by thioredoxin activates their function. To understand how the reduced form of AtFKBP13 is stabilized and how reduction of the cysteine residues affects the molecular properties of the enzyme, we determined the crystal structure of reduced AtFKBP13 at 1.88 A. Comparison of the reduced structure and the oxidized form that we published earlier shows rearrangements in redox site regions, readjustments of hydrogen-bonding interactions and the secondary structure of the active site residues 50-53, and reduced accessibility of the catalytic residues involved in the peptidyl proline isomerase (PPIase) activity of this enzyme. We propose that redox-linked changes in the secondary structure of the PPIase domain are responsible for significant functional differences in this protein in the reduced and oxidized states.     

High level expression of the light chain of botulinum neurotoxin serotype C1 and an efficient HPLC assay to monitor its proteolytic activity

Botulinum neurotoxins (serotypes BoNT/A–BoNT/G) induce botulism, a disease leading to flaccid paralysis. These serotypes are highly specific in their proteolytic cleavage of SNAP-25 (synaptosomal-associated protein of 25 kDa), VAMP (vesicle associated membrane protein) or syntaxin. The catalytic domain (light chain, LC) of the neurotoxin has a Zn²⁺ dependent endopeptidase activity. In order to design drugs and inhibitors against these toxins, high level overexpression and characterization of LC of BoNTs along with the development of assays to monitor their proteolytic activity becomes important. Using the auto-induction method, we attained a high level expression of BoNT/C1(1–430) yielding more than 30 mg protein per 500 ml culture. We also developed an efficient assay to measure the activity of serotype C1 based on a HPLC method. SNAP-25 with varying peptide length has been reported in literature as substrates for BoNT/C1 proteolysis signifying the importance of remote exosites in BoNT/C1 required for activity. Here, we show that a 17-mer peptide corresponding to residues 187–203 of SNAP-25, which has earlier been shown to be a substrate for BoNT/A, can be used as a substrate for quantifying the activity of BoNT/C1(1–430). There was no pH dependence for the proteolysis, however the presence of dithiothreitol is essential for the reaction. Although the 17-mer substrate bound 110-fold less tightly to BoNT/C1(1–430) than SNAP-25, the optimal assay conditions facilitated an increase in the catalytic efficiency of the enzyme by about 5-fold.