Number Of Disulfide Bridges Research Articles

ESI-IM-MS reveals the metal binding of three analog methanobactin peptides with different numbers of free Cys at physiological pH

Three analog methanobactin (amb7) peptides containing the primary structure acetyl-Leu1-His2-Cys3-Gly4-Ser5-Cys6-Tyr7-Pro8-His9-Cys10-Ser11-Cys12-Met13 but differing by the number of disulfide bridges, have been studied for their reactivity with Fe(III), Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Ag(I), and Pb(II) using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) and fluorescence quenching. The Pb(II) bound only with the amb7-0 (0 disulfide bonds), Zn(II) only to amb7-0 and amb7-1 (1 disulfide bond), Ni(II) only to amb7-1 and amb7-2 (2 disulfide bonds), and Cu(II) to amb7-2, concomitant with the oxidation of amb7-0 and amb7-1. The reduction of Cu(II) to Cu(I) resulted with 1-4Cu(I) binding to amb7-1 and amb7-0, while Ag(I) also exhibited 1-6 Ag(I) ions binding through a mechanism indicative of labile proton exchange. The binding of the oxidized Co(III/V) and Mn(III/V) were observed at pH 9 and 11, while Fe(III) did not exhibit significant binding. The observed negatively charged complexes were consistent with the deprotonation of the metal sites of Cys, His, carboxyl terminus and indicative of ion-ion interactions with the metal at pH 7, 9, and 11. The positively charged complexes were indicative of ion-dipole metal binding but at pH 9 and pH 11 gave results similar to those of the negative ion analyses. Fluorescence quenching also qualitatively agreed with the ESI-IM-MS results providing evidence that they revealed the solution-phase metal binding behavior of amb7.

A highly divergent α-amylase from Streptomyces spp.: An evolutionary perspective

The present study deals with the genetic changes observed in the protein sequence of an α-amylase from Streptomyces spp. and its structural homologs from Pseudoalteromonas haloplanktis, invertebrates and mammals. The structural homologs are renowned for their important features such as chloride binding triad and a serine-protease like catalytic triad (a triad which is reported to be strictly conserved in all chloride-dependent α-amylases). These conserved regions are essential for allosteric activation of enzyme and conformational stability, respectively. An evaluation of these distinctive features in Streptomyces α-amylases revealed the role of mutations in conserved regions and evolution of chloride-independent α-amylases in Streptomyces spp. Besides, the study also discovers a highly divergent α-amylase from Streptomyces spp. which varies greatly even within the homologs of the same genus. Another very important feature is the number of disulfide bridges in which the structural homologs own eight Cys residues to form four disulfide bridges whereas Streptomyces α-amylases possess only seven Cys to form three disulfide bridges. The study also highlights the unique evolution of carbohydrate binding module 20 domain (CBM20 also known as raw starch binding domain or E domain) in Streptomyces α-amylases which is completely absent in α-amylases of other structural homologs.

Predicting GPCR Promiscuity Using Binding Site Features

G protein-coupled receptors (GPCRs) represent a large family of signaling proteins that includes many therapeutic targets. GPCR ligands include odorants, tastants, and neurotransmitters and vary in size and properties. Dramatic chemical diversity may occur even among ligands of the same receptor. Our goal is to unravel the structural and chemical features that determine GPCRs' promiscuity toward their ligands. We perform statistical analysis using more than 30 descriptors related to the sequence, physicochemical, structural, and energetic properties of the GPCR binding sites-we find that the chemical variability of antagonists significantly correlates with the binding site hydrophobicity and anticorrelates with the number of hydrogen bond donors in the binding site. The number of disulfide bridges in the extracellular region of a receptor anticorrelates with the range of molecular weights of its antagonists, highlighting the role of the entrance pathway in determining the size selectivity for GPCR antagonists. The predictive capability of the model is successfully validated using a separate set of GPCRs, using either X-ray structures or homology models.

Smart polymer mediated purification and recovery of active proteins from inclusion bodies

Obtaining correctly folded proteins from inclusion bodies of recombinant proteins expressed in bacterial hosts requires solubilization with denaturants and a refolding step. Aggregation competes with the second step. Refolding of eight different proteins was carried out by precipitation with smart polymers. These proteins have different molecular weights, different number of disulfide bridges and some of these are known to be highly prone to aggregation. A high throughput refolding screen based upon fluorescence emission maximum around 340nm (for correctly folded proteins) was developed to identify the suitable smart polymer. The proteins could be dissociated and recovered after the refolding step. The refolding could be scaled up and high refolding yields in the range of 8mgL−1 (for CD4D12, the first two domains of human CD4) to 58mgL−1 (for malETrx, thioredoxin fused with signal peptide of maltose binding protein) were obtained. Dynamic light scattering (DLS) showed that polymer if chosen correctly acted as a pseudochaperonin and bound to the proteins. It also showed that the time for maximum binding was about 50min which coincided with the time required for incubation (with the polymer) before precipitation for maximum recovery of folded proteins. The refolded proteins were characterized by fluorescence emission spectra, circular dichroism (CD) spectroscopy, melting temperature (Tm), and surface hydrophobicity measurement by ANS (8-anilino1-naphthalene sulfonic acid) fluorescence. Biological activity assay for thioredoxin and fluorescence based assay in case of maltose binding protein (MBP) were also carried out to confirm correct refolding.

Proteomics and Glycomics Analyses of N-Glycosylated Structures Involved in Toxoplasma gondii-Host Cell Interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

Antibacterial activity of linear peptides spanning the carboxy-terminal β-sheet domain of arthropod defensins

The antibacterial activity of peptides without disulfide bridges, spanning the carboxy-terminal segment of arthropod defensins, has been investigated. Although all the peptides have net positive charges, they exhibited varying antibacterial potencies and spectra. Atomic force and fluorescence microscopic analyses indicate that the peptides exert their activity by permeabilizing the outer and inner membranes of Gram-negative bacteria such as Escherichia coli. It appears that the plasticity observed in the activity of mammalian defensins with respect to sequence, number of disulfide bridges or net positive charge, is also observed in insect defensins.

Antimicrobial Peptides with Unusual Amino Acid Compositions and Unusual Structures

Antimicrobial peptides, which constitute an important component of innate immunity in animal and plant kingdom, are ubiquitously distributed in nature. However, they differ widely in their sizes, sequences and structures. On the basis of their structure they have been broadly classified into three classes: a) linear peptides with propensity for amphiphilic alpha-helical structure, b) peptides with beta or alphabeta structure stabilized by different number of disulfide bridges and c) peptides with over-representation of certain amino acids or unusual structures. Although considerable amount of work has been done on peptides of all the three classes, recent reviews have emphasized on peptides belonging to the first two classes. The present review focuses on the peptides belonging to the third group. The antimicrobial peptides discussed in this article include aromatic amino acid-rich peptides, (Pro-Arg)-rich peptides, unusual defensins and defensin-like molecules, unusual antimicrobial peptides from amphibians, bacteriocins with unusual structure and anionic antimicrobial peptides.

Graph-representation of oxidative folding pathways

BackgroundThe process of oxidative folding combines the formation of native disulfide bond with conformational folding resulting in the native three-dimensional fold. Oxidative folding pathways can be described in terms of disulfide intermediate species (DIS) which can also be isolated and characterized. Each DIS corresponds to a family of folding states (conformations) that the given DIS can adopt in three dimensions.ResultsThe oxidative folding space can be represented as a network of DIS states interconnected by disulfide interchange reactions that can either create/abolish or rearrange disulfide bridges. We propose a simple 3D representation wherein the states having the same number of disulfide bridges are placed on separate planes. In this representation, the shuffling transitions are within the planes, and the redox edges connect adjacent planes. In a number of experimentally studied cases (bovine pancreatic trypsin inhibitor, insulin-like growth factor and epidermal growth factor), the observed intermediates appear as part of contiguous oxidative folding pathways.ConclusionsSuch networks can be used to visualize folding pathways in terms of the experimentally observed intermediates. A simple visualization template written for the Tulip package can be obtained from V.A.

Structure of acutolysin-C, a haemorrhagic toxin from the venom of Agkistrodon acutus, providing further evidence for the mechanism of the pH-dependent proteolytic reaction of zinc metalloproteinases.

The structure of acutolysin-C, a haemorrhagic zinc metalloproteinase from the venom of Agkistrodon acutus, has been analyzed and refined at 2.2 A resolution. The space group of the crystal is P2(1)2(1)2(1), with unit-cell dimensions a = 46.84, b = 49.52, c = 95.34 A. One molecule was found in each asymmetric unit. The phasing problem was solved by the molecular-replacement program AMoRe. Crystallographic refinement was performed using X-PLOR, leading to final R and free R factors of 0.176 and 0.272, respectively. The residue sequence of acutolysin-C was determined mainly by electron density. No density was found for the first residue at the N--terminus and the last two residues at the C-terminus, which was also the case for most other P-I class snake-venom metalloproteinases (SVMPs). Acutolysin-C has two highly conserved characteristic sequences His142-Glu143-X-X-His146-X-X-Gly149-X-X-His15 2 and Cys162-Ile163-Met164. The enzyme has three disulfide bridges: Cys117-Cys195, Cys157-Cys179 and Cys159-Cys162. The entire structure shows good agreement with that of other reported P-I class SVMPs and has two subdomains with a cleft in which one catalytic zinc ion is localized. However, the local conformation (especially the disulfide configurations), the coordination of the catalytic water molecules and some residue side chains differ compared with other P-I class SVMPs. The proteolytic activities of SVMPs are sensitive to the pH value. The molecular superpositions around the proteolytic active sites of all the P-I class SVMP crystal structures show that the distances between the zinc ion and its ligands are not correlated with the crystallization pH values, although the contact distances between the catalytic water molecule and the O atoms of the Glu143 carboxylate group in the neutral and weakly alkaline structures are shorter than those in weakly acidic structures, and the closer the crystallization pH value of one enzyme is to its optimal activity pH value, the shorter the contact distances. Overall, all P-I class SVMPs have similar conformations in the active-site cleft. The size of the active site is not correlated with the crystallization pH values or the proteolytic activities. The disulfide bridge Cys117-Cys195 is conserved in all crystal structures of P-I class SVMPs, whereas the conformation and number of disulfide bridges in the C-terminal subdomain differ. Acutolysin-C has no structural calcium ion, which may not affect the proteolytic activity or haemorrhagic activity directly.

Kinetic behaviour of pancreatic lipase in five species using emulsions and monomolecular films of synthetic glycerides

In the absence of colipase and bile salts, using tributyrin emulsions or monomolecular films of dicaprin at low surface pressure, we observed that no significant lipase activity can be measured with Human Pancreatic Lipase (HuPL), Horse Pancreatic Lipase (HoPL) or Dog Pancreatic Lipase (DPL). Only Porcine Pancreatic Lipase (PPL) and recombinant Guinea Pig Pancreatic Lipase Related Protein of type 2 (r-GPL) hydrolyse pure tributyrin in the absence of any additive, as well as dicaprin films at low surface pressures. The former lipases may lack enzyme activity because of irreversible interfacial denaturation due to the high energy existing at the tributyrin/water interface and at the dicaprin film surface at low surface pressures. The enzyme denaturation cannot be reflected in the number of disulfide bridges, since all the pancreatic lipases tested here contain six disulfide bridges, but behaved very differently at interfaces. We propose to use the surface pressure threshold, as determined using the monomolecular technique, as a criterion for classifying lipases in terms of their sensitivity to interfacial denaturation.

On the Correlation of S-S and C-S Raman Bands Frequencies and Intensities with Conformations of Disulfide Bridges in Proteins

We have studied dependence of the S-S and C-S raman bands frequencies and intensities on the conformation of disulfide bridges. These correlations have been analyzed on the basis of the normal coordinates treatment in the valence force field and from comparison of the raman and X-ray data for the model compounds and proteins. The found regularity of the intensity variations with conformation make it possible to distinguish these spectral perturbations from those associated with the change in the number of disulfide bridges in proteins. Particularly, this was demonstrated on the gamma-irradiated insulin solutions spectra. We also present some data appropriate for estimation of the content of disulfide bridges in proteins using internal intensity standards.