Protein dynamics control proton transfer from bulk solvent to protein interior: a case study with a green fluorescent protein

The kinetics of proton transfer in Green Fluorescent Protein (GFP) have been studied as a model system for characterizing the correlation between dynamics and function of proteins in general. The kinetics in EGFP (a variant of GFP) were monitored by using a laser-induced pH jump method. The pH was jumped from 8 to 5 by nanosecond flash photolysis of the "caged proton," o-nitrobenzaldehyde, and subsequent proton transfer was monitored by following the decrease in fluorescence intensity. The modulation of proton transfer kinetics by external perturbants such as viscosity, pH, and subdenaturing concentrations of GdnHCl as well as of salts was studied. The rate of proton transfer was inversely proportional to solvent viscosity, suggesting that the rate-limiting step is the transfer of protons through the protein matrix. The rate is accelerated at lower pH values, and measurements of the fluorescence properties of tryptophan 57 suggest that the enhancement in rate is associated with an enhancement in protein dynamics. The rate of proton transfer is nearly independent of temperature, unlike the rate of the reverse process. When the stability of the protein was either decreased or increased by the addition of co-solutes, including the salts KCl, KNO(3), and K(2)SO(4), a significant decrease in the rate of proton transfer was observed in all cases. The lack of correlation between the rate of proton transfer and the stability of the protein suggests that the structure is tuned to ensure maximum efficiency of the dynamics that control the proton transfer function of the protein.     

An efficient route to thioglycosides with the 2,3-anhydro-D-ribo stereochemistry

An improved route for the synthesis of p-tolyl 2,3-anhydro-5-O-benzoyl-1-thio-beta-D-ribofuranoside and its alpha anomer, which are important intermediates in the synthesis of alpha- and beta-D-arabinofuranosides, has been developed. The products are obtained in six steps from D-xylose in 39% overall yield.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.     

Human adenovirus 36 induces adiposity, increases insulin sensitivity, and alters hypothalamic monoamines in rats

Objective: Human adenovirus 36 (Ad‐36) increases adiposity and reduces serum lipids in chicken, mouse, and non‐human primate models, and it is linked to obesity in sero‐epidemiological studies in humans. Involvement of the central nervous system (CNS) or adipose tissue in the mechanism of Ad‐36‐induced adiposity is unknown. The effects of Ad‐36 on adiposity and on the neuroendocrine system were investigated in a rat model. Research Methods and Procedures: Five‐week‐old male Wistar rats were inoculated intraperitoneally with Ad‐36 or medium. Results: Despite similar food intakes, infected rats attained significantly greater body weight and fat pad weight by 30 weeks post‐inoculation. Epididymal‐inguinal, retroperitoneal, and visceral fat pad weights of the infected group were greater by 60%, 46%, and 86%, respectively (p < 0.00001). The fasting serum insulin level and homeostasis model assessment index indicated greater insulin sensitivity in the infected group. Visceral adipose tissue expression of glycerol 3‐phosphate dehydrogenase, peroxisome proliferator‐activated receptor γ, and CCAAT/enhancer‐binding protein α and β was markedly increased in the infected animals compared with controls. Ad‐36 decreased norepinephrine levels significantly in the paraventricular nucleus in infected vs. control rats (mean ± standard error, 8.9 ± 1.1 vs. 12.8 ± 1.2 pg/μg protein; p < 0.05). Ad‐36 markedly decreased serum corticosterone in infected vs. control rats (mean ± standard error, 97 ± 41.0 vs. 221 ± 111 ng/mL; p < 0.005). Discussion: The results suggest that the pro‐adipogenic effect of Ad‐36 may involve peripheral as well as central effects. The male Wistar rat is a good model for the elucidation of metabolic and molecular mechanisms of Ad‐36‐induced adiposity.     

Enhancement of dissolution profile by solid dispersion (kneading) technique

This article investigates enhancement of the dissolution profile of valdecoxib using solid dispersion with PVP. The article also describes the preparation of fast-dissolving tablets of valdecoxib by using a high amount of superdisintegrants. A phase solubility method was used to evaluate the effect of various water-soluble polymers on aqueous solubility of valdecoxib. Polyvinyl pyrrolidone (PVP K-30) was selected and solid dispersions were prepared by the method of kneading. Dissolution studies, using the USP paddle method were performed for solid dispersions of valdecoxib. Infrared (IR) spectroscopy, differential scanning calorimetry (DSC), and x-ray diffractometry (XRD) were performed to identify the physicochemical interaction between drug and carrier, hence its effect on dissolution. Tablets were formulated containing solid dispersion products and compared with commercial products. IR spectroscopy, XRD, and DSC showed no change in the crystal structure of valdecoxib. Dissolution of valdecoxib improved significantly in solid dispersion products (<85% in 5 minutes). Tablets containing solid dispersion exhibited better dissolution profile than commercial tablets. Thus, the solid dispersion technique can be successfully used for improvement of dissolution of valdecoxib. Published: August 18, 2006     

Activation of the NLRP3 inflammasome by intracellular poly I:C

Several RNA viruses can be detected by the inflammasome, which promotes IL-1β and IL-18 secretion, but the underlying mechanisms of detection remain unclear. Cytosolic dsRNA is a replication intermediate of many RNA viruses. We show here that transfection of the dsRNA analogue poly I:C activates the NLRP3 inflammasome via a pathway requiring endosomal acidification. This detection is independent of the other poly I:C sensors: TLR3 and MDA5. These results suggest a mechanism by which cytosolic dsRNA produced during viral infection could activate the NLRP3 inflammasome.     

Motif inference reveals optimal CTL epitopes presented by HLA class I alleles highly prevalent in southern Africa

HIV-specific CTL play a central role in immune control of HIV. The basis for understanding the success or failure of this immune response requires identification of the specific epitopes targeted by CTL. However, in populations most severely affected by the global epidemic, this fundamental knowledge is hindered by the lack of characterization of many of the HLA class I alleles highly prevalent in such populations. Overall, the peptide-binding motif has been determined for a small minority (9%) of HLA class I alleles, with a strong bias toward those alleles prevalent in Caucasoid populations. These studies therefore set out to define, in a South African Zulu/Xhosa population at the epicenter of the epidemic, the epitopes presented by alleles highly prevalent, but for which the peptide-binding motif had not been characterized. Using a method of motif inference, epitopes presented by four such alleles prevalent in the Zulu/Xhosa population of Durban, South Africa, namely, B*3910, B*4201, B*8101, and Cw*1801, are described. Importantly, this approach may additionally facilitate optimization of epitopes in certain instances where conflicting reports in the literature exist regarding the peptide-binding motif, such as for HLA-A*2902, also highly prevalent in southern African populations. These data indicate that the previously anomalous position of HLA-A*2902 among HLA-A alleles, outside any recognized HLA-A supertype, is artifactual, and the true position of the A*2902 motif overlaps those of the A1 and A24 supertypes.     

Differential selection pressure exerted on HIV by CTL targeting identical epitopes but restricted by distinct HLA alleles from the same HLA supertype

HLA diversity is seen as a major challenge to CTL vaccines against HIV. One current approach focuses on "promiscuous" epitopes, presented by multiple HLA alleles from within the same HLA supertype. However, the effectiveness of such supertype vaccines depends upon the functional equivalence of CTL targeting a particular epitope, irrespective of the restricting HLA. In this study, we describe the promiscuous HIV-specific CTL epitopes presented by alleles within the B7 supertype. Substantial differences were observed in the ability of CTL to select for escape mutation when targeting the same epitope but restricted by different HLA. This observation was common to all six promiscuous B7 epitopes identified. Moreover, with one exception, there were no significant differences in the frequency, magnitude, or immunodominance of the CTL responses restricted by different HLA alleles to explain these discrepancies. This suggests that the unique peptide/MHC complexes generated by even closely related HLA induce CTL responses that are qualitatively different. This hypothesis is supported by additional differences observed between CTL targeting identical epitopes but restricted by different HLA: first, the occurrence of distinct, HLA-specific escape mutation; second, the recruitment of distinct TCR repertoires by particular peptide/MHC complexes; and, third, significant differences in the functional avidity of CTL. Taken together, these data indicate that significant functional differences exist between CTL targeting identical epitopes but restricted by different, albeit closely related HLA. These findings are of relevance to vaccine approaches that seek to exploit HLA supertypes to overcome the problem of HLA diversity.     

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP₂. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP₂ binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP₂ binding. The study provides the first structural view of the activated ezrin bound to PIP₂ and to F-actin.     

Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways

Determining whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding to have an upward curvature. In this study, the folding mechanism of heterodimeric monellin [double-chain monellin (dcMN)] has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations, using the intrinsic tryptophan fluorescence of the protein as the probe for the folding reaction. Refolding is shown to occur in multiple kinetic phases. In the first stage of refolding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes. A productive folding intermediate population is identified on one route but not on both of these routes. Two intermediate subpopulations appear to form in a fast kinetic phase, and native dcMN forms in a slow kinetic phase. The chevron arms for both the fast and slow phases of refolding are shown to have upward curvatures, suggesting that at least two pathways each defined by a different intermediate are operational during these kinetic phases of structure formation. Refolding switches from one pathway to the other as the GdnHCl concentration is increased.