Analyzing the fusion process of influenza hemagglutinin by mutagenesis and molecular modeling.
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Animals↗
Biomedical subjects
S R Durell
Publications and source records attributed to S R Durell.
Atomic scale structure and functional models of voltage-gated potassium channels.
Recent mutagenesis experiments have confirmed our hypothesis that a segment between S5 and S6 forms the ion selective portion of voltage-gated ion channels. Based on these and other new data, we have revised previous models of the general folding pattern of voltage-gated channel proteins and have developed atomic scale models of the entire transmembrane region of the Shaker A K+ channel. In these models, the ion selective region is a beta-barrel that spans the outer half of the membrane. The inner half of the pore is larger. The voltage-dependent conformational changes of activation gating are modeled to occur by the "helical screw" mechanism, in which the four S4 segments move along and rotate about their axes. These changes are followed by a voltage-independent conformational change, in which the segments linking S4 to S5 move from blocking the intracellular entrance of the pore to forming part of the lining of the large inner portion of the pore. The NH2-terminal of the protein was modeled as an alpha-helix that plugs the intracellular half of the pore to inactivate the channel.
Amino Acid Sequence↗
Similarities in amino acid sequences of Drosophila eag and cyclic nucleotide-gated channels.
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Amino Acid Sequence↗
Chemical modification of spinach plastocyanin using 4-chloro-3,5-dinitrobenzoic acid: characterization of four singly-modified forms.
Chemical modification of plastocyanin was carried out using 4-chloro-3,5-dinitrobenzoic acid, which has the effect of replacing positive charges on amino groups with negatively charged carboxyl groups. Four singly-modified forms were obtained which were separated using anion exchange FPLC. The four forms were modified at the N-terminal valine and at lysines 54, 71 and 77. The rates of reaction with mammalian cytochrome c were increased for all four modified plastocyanins. In contrast, the rates of reaction with cytochrome f were inhibited for the forms modified at residues 1, 54 and 77, whereas no effect was observed for the form modified at residue 71. Modification had no effect on either the midpoint redox potential or the reaction with K3Fe(CN)6. These results are consistent with a model in which charged residues on plastocyanin located at or near the binding site for cytochrome f recognize the positively-charged binding site on cytochrome f. In contrast, charged residues located at points on plastocyanin distant from the cytochrome f binding site recognize the net negative charge on the cytochrome f molecule. Based on these considerations, Glu-68 may be within the interaction sphere of cytochrome f, suggesting that cytochrome f may donate electrons to plastocyanin at either Tyr-83 or His-87.
Amino Acid Sequence↗
Factor analysis of the near-ultraviolet absorption spectrum of plastocyanin using bilinear, trilinear, and quadrilinear models.
Factor analysis was used to resolve the spectral components in the near-uv absorption spectrum of plastocyanin. The data set was absorption as a function of four variables: wavelength, species of plastocyanin, oxidation state of the copper center, and environmental pH. The data were fit with the traditional bilinear model, as well as with trilinear and quadrilinear models. Trilinear and quadrilinear models have the advantage that they uniquely define the components, avoiding the indeterminacy of bilinear models. Bilinear analysis using the absorption spectra of tyrosine and copper metallothionein as targets resulted in a two-component solution which was nearly identical to that obtained using trilinear and quadrilinear models, for which no targets are required. The two-component models separate the absorption into tyrosine and copper center components. The absorption of tyrosine is found to be pH dependent in reduced plastocyanin, and the absorption magnitude of the reduced copper center is the same in the four different plastocyanin species. Further resolution is provided by a three-component quadrilinear model. The results indicate that there are at least two different electronic transitions which cause the absorption of the reduced copper center and that one of them couples to a tyrosine residue. It is the absorption of this coupled tyrosine residue which is pH dependent. Correlation of the results with previous studies indicates that it is Tyr 83 which is the perturbed residue. The separation of the absorption of the copper center and Tyr 83 provides spectroscopic probes for the conformations of the north pole and east face reaction sites on the plastocyanin protein.
Binding Sites↗
Modeling of the electrostatic potential field of plastocyanin.
The DelPhi computer program is used to calculate the electrostatic potential field of the photosynthetic electron transport protein plastocyanin. Knowledge of the potential field is important for understanding the mechanisms by which plastocyanin interacts with other charged reagents. The program uses a macroscopic, continuum approach in which the protein and solvent are assigned different dielectric constants, the crystal structure of the protein defines the dielectric boundary, and the ionic strength of the solvent is taken into account. The potential field is determined by numerically solving the Poisson-Boltzmann equation. The field surrounding plastocyanin is characterized by a region of positive potential over the copper center active site, and a region of negative potential over the adjacent association site containing tyrosine 83. The shape and magnitude of the potential field shows a strong dependence on the ionic strength and pH of the solvent. The program is able to accurately predict the effect of the copper center oxidation state on the pKa of a tetranitromethane derivative of tyrosine 83 using an intrinsic protein dielectric constant of 2 to 4. Evidence is also presented that the glutamate 68 side chain is exposed to the solvent to a greater extent in the solution structure of plastocyanin than in the crystal structure.
Amino Acid Sequence↗
Analysis of the near-ultraviolet absorption and circular dichroic spectra of parsley plastocyanin for the effects of pH and copper center conformation changes.
The absorption and circular dichroic (CD) spectra of parsley plastocyanin (PC) were measured in order to determine the effects of changes in primary amino acid sequence on both the copper center and protein components of the PC molecule. The near-ultraviolet (uv) absorption and CD spectra of parsley PC were found to be qualitatively similar to those of spinach, poplar, and lettuce PC, except for the near-uv CD spectrum of the reduced form at low pH (ca. pH 5.0). The CD spectrum of reduced parsley PC in the 250-265 nm wavelength region changes from positive to negative ellipticity upon reduction of pH, and is characterized by a pKa value of 5.7. This pKa value is the same as that for the protonation of the histidine 87 copper ligand, observed by NMR, and the change in conformation of the copper center. Similar processes are believed to occur in the other PC species at lower pH values. Thus, the pH-dependent perturbations of the near-uv CD spectra of reduced PC are interpreted as due to transitions in the reduced copper center. The increase in the near-uv absorption spectrum of reduced PC can be divided into pH-independent and pH-dependent portions. The pH-independent portion resembles the absorption spectrum of tetrahedral Cu(I) metallothionein, suggesting the presence of Cu(I)-Cys 84 and/or Cu(I)-Met 92 charge transfer transitions in the near-uv absorption spectra of reduced PC. The pH dependence of the absorption spectrum changes and the pH difference absorption spectrum indicate that tyrosine residues may contribute to at least a part of the pH-dependent portion of the absorption increase of reduced PC.
Chlorophyll↗
What studies of fusion peptides tell us about viral envelope glycoprotein-mediated membrane fusion (review).
This review describes the numerous and innovative methods used to study the structure and function of viral fusion peptides. The systems studied include both intact fusion proteins and synthetic peptides interacting with model membranes. The strategies and methods include dissecting the fusion process into intermediate stages, comparing the effects of sequence mutations, electrophysiological patch clamp methods, hydrophobic photolabelling, video microscopy of the redistribution of both aqueous and lipophilic fluorescent probes between cells, standard optical spectroscopy of peptides in solution (circular dichroism and fluorescence) and attenuated total reflection-Fourier transform infrared spectroscopy of peptides bound to planar bilayers. Although the goal of a detailed picture of the fusion pore has not been achieved for any of the intermediate stages, important properties useful for constraining the development of models are emerging. For example, the presence of alpha-helical structure in at least part of the fusion peptide is strongly correlated with activity; whereas, beta-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion. The functional role of the fusion peptides (which tend to be either nonpolar or aliphatic) is then to bind to and dehydrate the outer bilayers at a localized site; and thus reduce the energy barrier for the formation of highly curved, lipidic 'stalk' intermediates. In addition, the importance of the formation of specific, 'higher-order' fusion peptide complexes has also been shown. Recent crystallographic structures of core domains of two more fusion proteins (in addition to influenza haemagglutinin) has greatly facilitated the development of prototypic models of the fusion site. This latter effort will undoubtedly benefit from the insights and constraints gained from the studies of fusion peptides.
Amino Acid Sequence↗