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  • 1
    ISSN: 0887-3585
    Keywords: protein design ; alpha-helical bundle ; x-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Twelve- and sixteen-residue peptides have been designed to form tetrameric alphahelical bundles. Both peptides are capable of folding into amphiphilic alpha-helices, with leucyl residues along one face and glutamyl and lysyl residues along the opposite face. Four such amphiphilic alpha-helices are capable of forming a noncovalently bonded tetramer. Neighboring helices run in antiparallel directions in the design, so that the complex has 222 symmetry. In the designed tetramer, the leucyl side chains interdigitate in the center in a hydrophobic interaction, and charged side chains are exposed to the solvent. The designed 12-mer(ALPHA-1) has been synthesized, and it forms helical aggregates in aqueous solution as judged by circular dichroic spectroscopy. It has also been crystallized and characterized by x-ray diffraction. The crystal symmetry is compatible with (but does not prove) the design. The design can be extended to a four-alpha-helical bundle formed from a single polypeptide by adding three peptide linkers.
    Additional Material: 6 Ill.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: membrane biomimetic system ; reverse micelles ; interfacial water ; myelin proteins ; solid enzyme activity ; organic solvents ; biotechnology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0887-3585
    Keywords: protein domain ; polymerase ; 3′-5′ exonuclease ; artificial gene ; expression vector ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3′-5′ exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3′-5′ exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3′-5′ exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.
    Additional Material: 5 Ill.
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  • 4
    ISSN: 0887-3585
    Keywords: protein stability ; protein denaturation ; denatured state ; structural intermediates ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m-the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration - these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified - interactions which appear to involve hydrophobic groups.
    Additional Material: 5 Ill.
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  • 5
    ISSN: 0887-3585
    Keywords: regulation ; prokaryotic bacteria ; leucine uptake ; leucyl tRNA corepressor ; cell physiology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The livR gene encoding the repressor for high-affinity branched-chain amino acid transport in Escherichia coli has been cloned from a library prepared from the episome F106. The inserted DNA fragment from the initial cloned plasmid, pANT1, complemented two independent, spontaneously derived, regulatory mutations. Subcloning as well as the creation of deletions with Bal31 exonuclease revealed that the entire regulatory region is contained within a 1.1-kb RsaI-SalI fragment. Expression of the pANT plasmids in E. coli minicells showed that the regulatory region encodes one detectable protein with an apparent molecular weight of 21,000. DNA sequencing revealed one open reading frame of 501 bp encoding a protein with a calculated MW of 19,155. The potential secondary structure of the regulatory protein has been predicted and it suggests that the carboxy terminus may fold into three consecutive alpha helices. These results suggests that the livR gene encodes a repressor which plays a role in the regulation of expression of the livJ and the livK transport genes.
    Additional Material: 8 Ill.
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  • 6
    ISSN: 0887-3585
    Keywords: protein conformation ; energy calculations ; protein modeling ; loop conformation ; surface area ; homologous proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The feasibility of determining the conformation of segments of polypeptide chain up to six residues in length in globular proteins by means of systematic search through the possibles conformations has been investigated. Trial conformations are generated by using representative sets of φ, ψ, and χ angels that have been derived from an examination of the distributions of these angles in refined protein structures. A set of filters based on simple rules that protein structures obey is used to reduce the number of conformations to a manageable total. The most important filters are the maintenance of chain integrity and the avoidance of tooshort van der Waals contacts with the rest of the protein and with other portions of the segment under construction. The procedure is intended to be used with approximate models so that allowance is made throughout for errors in the rest of the structure. All possible main chains are first constructed and then all possible side-chain conformations are built onto each of these. The electrostatic energy, including a solvent screening term, and the exposed hyrophobic area are evaluated for each accepted conformation. The method has been tested on two segments of chain in the trypsin like enzyme from Streptomyces griseus. It is found that there is a wide spread of energies among the accepted conformations, and the lowest energy ones have satisfactorily small root mean square deviations from the X-ray structure.
    Additional Material: 5 Ill.
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  • 7
    ISSN: 0887-3585
    Keywords: G-protein ; phototransduction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The GTP-binding subunit of transducin (Tα) activates the cGMP phosphodiesterase (PDE) of bovine retinal rods by relieving the constraint imposed by the inhibitory subunit PDEγ. We have isolated and characterized the complex Tα.GTPγS-PDEγ formed when Tα is activated by the nonhydrolyzable analog GTPγS. Sedimentation and light-scattering techniques demonstrate that, in contrast to free Tγ.GTPγS, which is soluble, the Tα.GTPγS-PDEγ complex, as well as Tα.GTP-PDEγ, is membrane bound at cytosolic ionic strength. It is eluted from the membrane at low ionic strength as a monomeric and 1:1 stoichiometric complex. The relative affinities of PDEγ for PDEαβ and for Tα.GTP are discussed.
    Additional Material: 5 Ill.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: FV fragment ; space filling hapten ; reconstitution ; scratched analysis ; equilibrium dialysis ; contact residues ; hypervariable loops ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: IgG Gar, a human myeloma protein that binds riboflavin with a high affinity, was used to derive variable region fragments from the heavy chain and the light chain. Riboflavin binding ability of the active site generated by V(H) and light chain and the active site generated by V(H) and V(L) was compared to riboflavin binding by the F(ab) fragment. The riboflavin binding ability of the F(ab) fragment is the same as the intact molecule, while the binding ability of the active site formed by V(H) and light chain is lowered by two to three orders of magnitude, indicating that the removal of C(H1) domain decreases the interaction between riboflavin and the amino acids that is important in tight binding of riboflavin. Removal of the third hypervariable region and the constant region domain from the light chain further lowers the binding constant by one order of magnitude. The results indicate that the V(H) and V(L) segments of IgG Gar can reconstitute a riboflavin binding site. The decrease in affinity probably reflects a decrease in the rigidity with which the hypervariable loops are held together to place the contact amino acid residues in optimal contact with the hapten.
    Additional Material: 5 Ill.
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  • 9
    ISSN: 0887-3585
    Keywords: antibody affinity ; ELISA ; β-endorphin models ; melittin model ; amphiphilic α-helix ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Monoclonal antibodies against human apolipoprotein A-I (apoA-I) were generated by the hybridoma technique. Clone G-10 was selected on the basis of its highest titer. The affinity of this antibody toward a series of synthetic peptides differing in length, amino acid composition, and amphiphilicity was tested by using both the indirect and the competitive enzyme-linked immunosorbent techniques (ELISA). From these measurements we calculated dissociation constants of the complexes of the antibody with apoA-I bound to the surface of the microtiter plate, apoA-I in solution, and any of the several peptides in solution. The dissociation constant (Kd) of the immobilized apoA-I/anti-apoA-I-complex, Kd = 2 × 10-9 M, was significantly lower than that of the complex resulting from the interaction between anti-apoA-I and either apoA-I in solution or any of the several amphiphilic helical peptides in solution. Peptides devoid of amphiphilic secondary structure were inert. These data are consistent with the proposal that monoclonal G-10 recognizes in antigenic peptides an α-helical secondary structure of defined hydrophilic-lipophilic balance and comparatively less the specific amino acid side chains. We propose that the highest contribution to the free energy of binding (8 Kcal/mole) is derived from the docking of the helix to the antibody. It follows that in probing the specificity of a monoclonal antibody the conformation and the physical environment of the interacting antigen must be taken into account.
    Additional Material: 2 Ill.
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  • 10
    ISSN: 0887-3585
    Keywords: primary sequence homology ; primary structure ; secondary structure ; quaternary structure ; gene cloning ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
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