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1.
Ding-Kwo Chang Shu-Fang Cheng Chi-Hui Lin EricAssen B. Kantchev Cheng-Wei Wu 《生物化学与生物物理学报:生物膜》2005,1712(1):37-51
Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pKa values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CγH, whereas the deviation of pKa from the reference value for Glu4 and Glu8 CγH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3. 相似文献
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The envelope glycoprotein gp41 of HIV-1 undergoes structural rearrangement to form a helix hairpin during the virus-mediated fusion. Previous studies to investigate the folding and stability of hairpin did not monitor the end-to-end distance of the molecule. To directly probe the distance change, rhodamine dye was conjugated to the gp41 recombinant near the N- and C-terminal regions to detect the UV absorption and fluorescence intensity changes induced by the chemical denaturant guanidinium chloride (GdmCl). Using the singly- and doubly-labeled constructs allowed us to distinguish between the hairpin formation and protein oligomerization. A biphasic transition of helical structure for the wild type protein was revealed by circular dichroism measurements while unfolding of the hairpin occurred at 6M GdmCl. The relevance of our study to the fusion inhibitor for HIV-1 was borne out by results on the mutants at the positions within the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions. A monophasic transition at lower denaturant concentration was detected for the NHR mutant supporting the concept of differential stability of NHR and CHR helical structure. The conclusion that the observed unstacking of doubly-labeled variant arises principally from the intra-molecular dimers was drawn from the unstacking of the protein labeled in the loop. Remarkably, it is deduced that the hairpin is more stable than the CHR helical structure. A model for denaturation of the helix hairpin bundle was proposed from these results. The biological implications of the findings and further applications of the distance-based approach were discussed. 相似文献
3.
Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed. 相似文献
4.
Ding-Kwo Chang Shu-Fang Cheng Eric Aseen B Kantchev Chi-Hui Lin Yu-Tsan Liu 《BMC biology》2008,6(1):2
Background
To study the organization and interaction with the fusion domain (or fusion peptide, FP) of the transmembrane domain (TMD) of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion. 相似文献5.
Biogeochemistry - The coupling between chemical weathering (including silicate, carbonate and pyrite weathering) and physical erosion is of high interest in active mountain belts. Participation of... 相似文献
6.
Crk-associated substrate (Cas) is highly phosphorylated by v-Src and plays a critical role in v-Src-induced cell transformation. In this study, we found that the Src homology (SH) 3 domain of Cas blocked v-Src-stimulated anchorage-independent cell growth, Matrigel invasion, and tumor growth in nude mice. Biochemical analysis revealed that the Cas SH3 domain selectively inhibited v-Src-stimulated activations of AKT and JNK, but not ERK and STAT3. Attenuation of the AKT pathway by the Cas SH3 domain rendered v-Src-transformed cells susceptible to apoptosis. Inhibition of the JNK pathway by the Cas SH3 domain led to suppression of v-Src-stimulated invasion. Taken together, our results indicate that the Cas SH3 domain has an anti-tumor function, which severely impairs the transforming potential of v-Src. 相似文献
7.
Lin CH Lin CH Chang CC Wei TS Cheng SF Chen SS Chang DK 《Journal of biotechnology》2011,153(1-2):48-55
We demonstrated a high level expression and purification of recombinant human immunodeficiency virus type 1 gp41 ectodomain (gp41e-FP) using glass bead approach with a final yield of 12±2mg/L bacterial culture. The proper folding of gp41e-FP encompassing the fusion peptide (FP) was ascertained by circular dichroism (CD) measurement and recognition by NC-1 antibody. The latter assay revealed stabilization of the gp41 coiled coil structure in the presence of liposome dispersion. The differential affinity of gp41e-FP and gp41e (devoid of FP) by NC-1 suggested an aggregated state for gp41e-FP and/or possible proximity of the fusion peptide domain to the coiled coil structure of gp41 ectodomain. Perfluorooctanoate (PFO)-PAGE electrophoresis experiment revealed the trimeric propensity of the recombinant gp41e-FP. In comparison to gp41e, the lipid mixing activity of gp41e-FP was two-fold higher suggesting a role of FP in promoting membrane fusion. The present approach to efficiently and quantitatively preparing the functional full-length recombinant gp41 ectodomain protein can be employed for structural and biomedical investigations and the extraction of other inclusion body-embedded recombinant proteins. 相似文献
8.
Mycopathologia - Corynespora cassiicola is a common plant pathogen, but C. cassiicola infection in human hosts is extremely rare. In this report, we present an 84-year-old male with long-term use... 相似文献
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10.
Joseph G. Jardine Devin Sok Jean-Philippe Julien Bryan Briney Anita Sarkar Chi-Hui Liang Erin A. Scherer Carole J. Henry Dunand Yumiko Adachi Devan Diwanji Jessica Hsueh Meaghan Jones Oleksandr Kalyuzhniy Michael Kubitz Skye Spencer Matthias Pauthner Karen L. Saye-Francisco Fabian Sesterhenn Patrick C. Wilson Denise M. Galloway Robyn L. Stanfield Ian A. Wilson Dennis R. Burton William R. Schief 《PLoS pathogens》2016,12(8)
An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens. 相似文献