Motility of triton-demembranated sea urchin sperm flagella during digestion by trypsin

The survival curves for a population of reactivated spermatozoa exposed to digestion by trypsin indicate that a large number of trypsin-sensitive targets must be digested before the flagellum disintegrates. Changes in flagellar movement during trypsin digestion can be very small, especially when the spermatozoa are reactivated at 0.25 M KCl. They are not the changes which would be expected if elastic resistance of the trypsin-sensitive structures responsible for maintaining the integrity of the axoneme is a significant determinant of flagellar bend amplitude. By carrying out trypsin digestion under a variety of conditions, at least six distinct effects of trypsin digestion on parameters of flagellar movement have been detected. These include a gradual increase in the rate of sliding between tubules, gradual and abrupt changes in beat frequency accompanied by reciprocal decreases in bend angle, changes in the symmetry and planarity of bending, and selective interference with mechanisms for bend initiation and bend propagation

Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica.

Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-microns-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy. Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al. 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali-stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles. The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor

Use of 18O Labels to Monitor Deamidation during Protein and Peptide Sample Processing

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c· + 58 and z − 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)(n−1)+· − 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of 18O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (̃4 h) trypsin digestion process generates no additional detectable peptide deamidations

Explorations into the nature of insulin binding to oxidized dextran : this thesis was presented in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at Massey University

The results reported in this thesis comprise an investigation into the conjugation of insulin to oxidized dextran, various release studies from the conjugates, and an attempt to interpret the binding nature of the conjugates. A model system involving the sustained release from insulin-dextran conjugates has been employed in this study. For insulin, up to 3 potential sites only (A1-Gly. B1-Phe and B29-Lys) were expected to bind to oxidized dextran. The rate of release and the maintenance of activity of the released protein are vital to such systems. Success in the interpretation of the binding nature of the conjugate will allow us to investigate its relationship to the rate of release. The desired rate of release for the sustained release of protein could then be achieved, once the projected binding could be obtained. Activation of dextran was achieved by periodate oxidation to give levels of 8%, 16% and 27% oxidized dextran. Insulin was chosen for its relatively 'uncomplicated' structure and few possible sites available for binding with activated dextran. Insulin was bound to the dextran through imine bonds. Complex formation was examined under a wide range of conditions. Initial studies were begun with the determination of a desirable basic molar ratio. A molar ratio of insulin to 8% activated dextran of 10 : 1 arose from this set of experiments. Insulin was bound to 27% activated dextran at pH 7.4, pH 9 and pH 10. In the cases of pH 9 and pH 10, many more lower MW complexes were formed than at pH 7.4. It seemed that the higher the pH of formation, the more crosslinks occurred between an insulin molecule and dextran molecules in the lower MW range. Approximate physiological pHs (pH 7.1-7.8) were used for complex formation in all subsequent experiments. Release studies were carried out under approximate physiological conditions (pH 7.4, 37°C). Immediate release was observed upon isolation by size exclusion chromatography. The greatest release occurred in the first 24 hours for all three activation levels. The higher the activation level of dextran, the lower the level of release. An equilibrium was established after several days' release and studies at 37°C produced the expected result: greater release relative to ambient. A number of studies were carried out with complex after sodium cyanoborohydride had been used to reduce the imine bonds. The first set of experiments on the reduced complexes was enzymatic cleavage studies, which employed trypsin and α-chvmotrypsin. The results for trypsin digestion of the reduced insulin-27% oxidized dextran complex indicated partial binding had occurred at B29-Lys, in combination with full binding at B1 and/or Al. Amino acid analysis results of the isolated complex after trypsin digestion indicated about 90% binding occurred at B29-Lys for the complex, which formed at pH 7.1. The results of α-chymotrypsin digestion study were shown questionable due to its incomplete cleavage. The reduced complexes were analyzed by amino acid analysis. The insulin-27% activated dextran complexes formed at pH 7.4, pH 9 and pH 10 showed similar extents of binding at B1-Phe, indicating B1 might be the prime binding site. There was more binding at B29 and A1 for the pH 9 than at pH 7.4 case. At pH 10 abnormal values arose. The studies for the complexes of insulin with 16% and 27% activated dextran indicated the more highly activated the dextran, the greater the binding at B29 and A1. Trials with the 2, 4-dinitrophenyl-derivativatization method proved to be a useful way to examine the degree of B1 and B29 binding from the amino acid analysis results of complex. The insulin-16% activated dextran complex formed at pH 7.1 was found to be about 100% binding at B1, 60% at A1 and 50% at B29. Oxidative and reductive cleavage studies of A and B chains of insulin and the complex were carried out to investigate the level of A1 binding. After chemical cleavage of the three disulfide bonds in insulin and subsequent chromatography, the amino acid analysis results for the treated complexes indicated a significant proportion of A chain had bound to dextran, i.e. at A1. An estimation of 60-70% of A1 binding was achieved for this study. This exploratory study has shown that varied complex formation conditions such as the level of activation of dextran, pH, and temperature could alter the extent of binding between insulin and dextran molecules. Amino acid analysis of the reduced complex was a useful method to interpret the binding

Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones

Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for,10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e. g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 A crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5

Deamidation at Asparagine and Glutamine As a Major Modification upon Deterioration/Aging of Proteinaceous Binders in MuralPaintings

Proteomic strategies are herein proved to be a complementary approach to the well established amino acid composition analysis for the characterization of the aging and deterioration phenomena occurring to proteinaceous materials in works-of-art. Amino acid analyses on several samples demonstrated that proteins in the frescoes from the Camposanto Monumentale in Pisa are deteriorated as revealed by the decrease in Met, Lys, and Tyr content and by the presence in all the samples of amino malonic acid as a result of Ser, Phe, and Cys oxidation. Proteomic analysis identified deamidation at Asn and Gln as a further major event occurred. This work paves the way to the exploitation of proteomic strategies for the investigation of the molecular effects of aging and deterioration in historical objects. Results show that proteomic searches for deamidation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) could constitute a routine analysis for paintings or any artistic and historic objects where proteins are present. Peptides that can be used as molecular markers when casein is present were identified

The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection

Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß-ß-a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel β-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated

Structural analysis of Salmonella enterica effector protein SopD

Salmonella outer protein D (SopD) is a type III secreted virulence effector protein from Salmonella enterica. Full-length SopD and SopD lacking 16 amino acids at the N-terminus (SopDDeltaN) have been expressed as fusions with GST in Escherichia coli, purified with a typical yield of 20-30 mg per litre of cell culture and crystallized. Biophysical characterization has been carried out mainly on SopDDeltaN. Analytical size exclusion chromatography shows that SopDDeltaN is monomeric and probably globular in aqueous solution. The secondary structure composition, calculated from the CD spectrum, is mixed (38% alpha-helix and 26% beta-strand). Sequence analysis indicates that SopD contains a coiled coil motif, as found in numerous other type III secretion system-associated proteins. This suggests that SopD has the potential for one or more heterotypic protein-protein interactions. Limited trypsin digestion of SopDDeltaN, monitored by both one-dimensional proton NMR spectroscopy and SDS-PAGE, shows that the protein has a large, protease-resistant core domain of 286 amino acid residues. This single-domain architecture suggests that SopD lacks a cognate chaperone. In crystallization trials, SopDDeltaN produced better crystals than either full-length SopD or trypsin-digested SopDDeltaN. Diffraction to 3.0 Angstrom resolution has so far been obtained from crystals of SopDDeltaN