Sulfhydryls of tubulin

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 ° C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 ° C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects

Structure–function analyses involving palindromic analogs of tritrypticin suggest autonomy of anti-endotoxin and antibacterial activities

Neutralization of invading pathogens by gene-encoded peptide antibiotics has been suggested to manifest in a variety of different modes. Some of these modes require internalization of the peptide through a pathway that involves LPS-mediated uptake of the peptide antibiotics. Many proline/tryptophan-rich cationic peptides for which this mode has been invoked do, indeed, show LPS (endotoxin) binding. If the mechanism of antibiotic action involves the LPS-mediated pathway, a positive correlation ought to manifest between the binding to LPS, its neutralization, and the bacterial killing. No such correlation was evident based on our studies involving minimal active analogs of tritrypticin. The anti-endotoxin activities of these analogs appear not to relate directly to their antibiotic potential. The two palindromic analogs of tritrypticin, NT7 (RRFPWWW) and CT7 (WWWPFRR), showed comparable antibacterial activities. However, while NT7 exhibited anti-endotoxin activity, CT7 did not. The LPS binding of two tritrypticin analogs correlated with their corresponding structures, but the antibacterial activities did not. Further structure–function analysis indicated specific structural implications of the antibacterial activity at the molecular level. Studies involving designed analogs of NT7 incorporating either rigid or flexible linkers between the specifically distanced hydrophobic and cationic clusters modulate the LPS binding. On the other hand, not knowing the target receptor for antibacterial activity is a drawback since the precise epitope for antibacterial activity is not definable. It is apparent that the anti-endotoxin and antibacterial activities represent two independent functions of tritrypticin, consistent with the emerging multifunctionality in the nature of cathelicidins

Stabilizing exposure of conserved epitopes by structure guided insertion of disulfide bond in HIV-1 envelope glycoprotein.

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will 'snap' Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to 'lock' gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules

Schematic representation of layered organization of the gp120 inner domain.

<p>Ribbon diagram representation showing the inner domain of gp120 and its organization into three layers: layer 1 (magenta), 2 (green) and 3 (orange). The outer domain of gp120 is shown in white/gray. The representation is based on RCSB PDB ID - 3JWO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076139#pone.0076139-Pancera1" target="_blank">[6]</a>.</p

Expression and purification of disulfide-stabilized SF162 gp120 and gp140. The

<p>two disulfide-stabilized proteins are referred to as gp120 L1-SS-L2 and gp140 L1-SS-L2. The SS refers to the disulfide bond. SDS-PAGE followed by coomassie-staining to show purity of GNA-lectin affinity column and DEAE-column purified (A) Disulfide stabilized SF162 gp120 L1-SS-L2 (lane 1) and wild-type gp120 (lane 2), and (B) Disulfide stabilized SF162 gp140 L1-SS-L2 (lane 3) and wild-type gp140 (lane 4). MW refers to Molecular Weight standard/marker. The gp120 s and gp140 s are indicated by the red arrow.</p

Binding analysis of serum antibodies post-immunization.

<p>Env binding analysis of serum antibodies from an immunogenicity study in rabbits using a gp120/gp140 protein-only regimen with Carbopol971P + MF59 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076139#pone.0076139-Dey7" target="_blank">[54]</a> as adjuvant. (A) Geometric mean (binding) titers and (B) Avidity of anti-Env antibodies in sera collected before the onset of immunization (pre-bleed) and at 2wp2, 2wp3, 4wp3 and 8wp3 (wp - weeks post) immunization time-points.</p

Dissection of epitope-specific recognition of antibodies from vaccine sera.

<p>Analysis of 2wp3-sera from rabbits for dissecting specificity of epitopes recognized (expressed as ‘% epitope-directed binding’) by the antibodies elicited when immunized with (A) wild-type gp120 (red solid symbols) or gp120 L1-SS-L2 (red open symbols) or (B) wild-type gp140 (blue solid symbols) or gp140 L1-SS-L2 (blue open symbols). The percent epitope-directed response is calculated as follows: EC50(mutant)/EC50(wild-type)x100. ns – non-significant (p>0.05); * <0.05; ** <0.001.</p