Determinants of Heparin Cofactor II Specificity for Thrombin. Pp. 215-224.
Angelina V. Ciaccia and Frank C. Church
[Abstract]
Binding of Mutant HIV-I Proteases with Junction B Peptides Containing Methyleneamino Isostere Replacements. Pp. 225-236.
L. Zhang, M. W. Pennington, P. Baur, M.E. Byrnes, J. de Chastonay, S.I. Wilson,
J. Kay and B.M. Dunn
[Abstract]
Papers
The Effect of Truncation on SHK Toxin: Elimination of the Amino-Carboxyl Terminal (3-35) Disulfide Linkage Stabilizing the Amino and Carboxyl Terminal Segments. Pp. 237-242.
M.W. Pennington, V.M. Mahnir, P. Baur, C. T. McVaugh, D. Behm and W.R. Kem
[Abstract]
Human alpha-Fetoprotein is in the Molten Globule State Under Conditions Modelling Protein Environment Near the Membrane Surface. Pp. 243-250.
Natalya V. Narizhneva and Vladimir N. Uversky
[Abstract]
Sequence-Specific 1H-NMR Assignments and Secondary Structure of Neuropeptide Y Analogs in Aqueous Solution. Pp. 251-258.
S.D. Samarasinghe, L. Kar, A. Balasubramaniam and M.E. Johnson
[Abstract]
Use of TNBS Colorimetric Assay to Analyze Viral Protease Inhibitors. Pp. 259-264.
Q. May Wang and Robert B. Johnson
[Abstract]
Additivity of Lytic Activities for Mutant Lysozymes. Pp. 265-270.
Yoshio Hashimoto, Kazuhide Tokuyama, Yuji Ito and Taiji Imoto
[Abstract]
CNBR-Induced Formation of Tryptathionine in Proteins. Pp. 271-276.
L.G. Sparrow, C.P. Robinson, A. Kirkpatrick, D.E. Rivett and J.J. Gorman
[Abstract]
Protein Association and Stability of Thermophilic Enzymes. Pp. 277-280.
Raffaele Ragone
[Abstract]
spartic Protease Inhibitors: Synthesis of a Radiolabelled Fatty Acid Linked Pepstatin. Pp. 281-286.
Michel Bessodes, Kostas Antonakis, Christian Rougeot, Françoise Capony, Marcel Garcia and Henri Rochefort
[Abstract]
Crystallisation Report
Preliminary X-Ray Crystallographic Analysis and Molecular Replacement Studies with 2,5-Diketo-D-Gluconate Reductase. Pp. 287-290.
Sumit Khurana, David B. Powers, Stephen Anderson and Michael Blaber
[Abstract]
[Back to top] Determinants of Heparin Cofactor II Specificity for Thrombin. Angelina V. Ciaccia and Frank C. Church.
Hemostasis is a critical element of the host defense system. The "balance" in hemostasis is achieved by the tight regulation of both procoagulant and anticoagulant proteins. The focus of this short review is on one particular anticoagulant protein, heparin cofactor II (HCII), a member of the serine proteinase (serpin) superfamily of proteins. A brief overview of serpins and the mechanism by which HCII specifically regulates the activity of the most critical procoagulant proteinase thrombin are addressed.
[Back to top] Binding of Mutant HIV-I Proteases with Junction B Peptides Containing Methyleneamino Isostere Replacements. L. Zhang, M. W. Pennington, P. Baur, M.E. Byrnes, J. de Chastonay, S.I. Wilson, J. Kay and B.M. Dunn.
A series of peptidomimetics (with a -CH2-NH- replacing the scissile peptide bond) based on one ot the CA/NC cleavage junctions in the gag region of HIV-1 has been prepared and shown to provide effective inhibitors of HIV-1 PR. Furthermore, these compounds retain much of their potency when tested against mutant forms of HIV-1 PR in which residues 45, 48, and 82 have been changed. In contrast, when Ile 84 is changed to Val, binding is reduced and when Asp 30 is changed to Phe, binding is weakened substantially. To provide an independent measure of interaction, the binding of a fluorescent form of the methyleneamino-substituted inhibitors has been studied by kinetics and spectroscopy. A series of substrates of analogous structures was also prepared and evaluated by determining kcat, Km and kcat/Km values for cleavage by HIV-1 PR.
[Back to top] The Effect of Truncation on SHK Toxin: Elimination of the Amino-Carboxyl Terminal (3-35) Disulfide Linkage Stabilizing the Amino and Carboxyl Terminal Segments. M.W. Pennington, V.M. Mahnir, P. Baur, C. T. McVaugh, D. Behm and W.R. Kem.
We have synthesized two truncated analogs ot ShK toxin which contain the main residues comprising the binding surface of the toxin. In both of these analogs, the disulfide bond which links the N and C terminal Cys residues has been eliminated. The remaining two disulfide bonds (Cys12-Cys28, Cys17-Cys32) have been retained in one analog and the other analog contains only the Cys17-Cys32 linkage. The CD spectrum of the bicyclic peptide appears to have a small amount of the helical structure whereas the monocyclic peptide spectrum appears to be idisordered. The activity of the bicyclic and monocyclic peptides were respectively reduced by
1360- and > 3000-fold relative to wild type ShK toxin.
[Back to top] Human alpha-Fetoprotein is in the Molten Globule State Under Conditions Modelling Protein Environment Near the Membrane Surface. Natalya V. Narizhneva and Vladimir N. Uversky.
Conformational transitions induced in alpha-fetoprotein by such organic solvents as methanol and trifluoroethanol were studied using circular dichroism, tryptophan fluorescence spectroscopy and digital scanning calorimetry. The existence of well-populated intermediate state without rigid tertiary structure but with native-like secondary structure content and compactness (i.e., the molten globule-like intermediate state) was established. A possible implication of such structural rearrangement to function of this protein is discussed.
[Back to top] Sequence-Specific 1H-NMR Assignments and Secondary Structure of Neuropeptide Y Analogs in Aqueous Solution. S.D. Samarasinghe, L. Kar, A. Balasubramaniam and M.E. Johnson.
Solution structures of neuropeptide Y analogs NP158 and NP181 were studied by 1H NMR spectroscopy. Sequential assignments were made at pH 3.5 at 25ºC and 45ºC using COSY, HOHAHA and ROESY spectra. Modeling shows turn-type loop structures in aqueous solution that could be important for receptor binding and/or antagonistic potency. No significant conformational differences were observed between the two polypeptides despite vastly different antagonistic potency towards NPY YI receptors.
[Back to top] Use of TNBS Colorimetric Assay to Analyze Viral Protease Inhibitors. Q. May Wang and Robert B. Johnson.
Peptides derived from the rhinovirus-14 3C native cleavage sites have been synthesized and evaluated as substrates for this protease. Hydrolysis of the N-terminally acetylated peptides by 3C generates primary amino groups which react with 2,4,6-trinitrobenzenesulfonic acid at an alkaline pH. We have optimized the assay by selecting a sensitive monitoring wavelength to detect the resulting orange-colored trinitrophenyl products. Rhinovirus-14 3C cleaves the 3B/3C peptide with a Km value of 0.75 mM as determined by this method. Assay characterization and its use in protease inhibitor study have been performed.
[Back to top] Additivity of Lytic Activities for Mutant Lysozymes. Yoshio Hashimoto, Kazuhide Tokuyama, Yuji Ito and Taiji Imoto.
We constructed various lysozyme mutants and their addition mutants. Each mutation,
Asn27Asp (activity 84%), Lys33Asn (130%), Ser36Thr (175%), Ser5OThr (140%) and
Trp62Tyr (130%) was respectively added to the double mutant Asp101GIy/Gly102Pro
(225%). As the result of the additive mutations, activities of these triple mutants were 217%, 318%, 367%, 273% and 264% of wild type, respectively. These results show that the additivity is held in lytic activity whose mechanism is complex.
[Back to top] CNBR-Induced Formation of Tryptathionine in Proteins. L.G. Sparrow, C.P. Robinson, A. Kirkpatrick, D.E. Rivett and J.J. Gorman.
Cleavage of peptide bonds at methionine using cyanogen bromide under acid conditions is widely used in structural studies of proteins. Here we report a novel reaction in which a cross-link, tryptathionine, involving tryptophan and cysteine, may be introduced into certain proteins through the action of cyanogen bromide under acidic conditions. The reaction has been demonstrated both with free cysteine and with cysteine-containing peptides.
[Back to top] Protein Association and Stability of Thermophilic Enzymes. Raffaele Ragone.
The hypothesis is made that association could play an important evolutionary role in thermophilic enzymes. A few mesophilic model systems are examined, showing how association processes lead to assemblies that are thermodynamically stable over a wide temperature interval.
[Back to top] Aspartic Protease Inhibitors: Synthesis of a Radiolabelled Fatty Acid Linked Pepstatin. Michel Bessodes, Kostas Antonakis, Christian Rougeot, Françoise Capony, Marcel Garcia and Henri Rochefort.
To improve the bioavailability of the inhibitor of aspartic proteases, pepstatin A, the synthesis of a tritium labelled fatty acid derivative and its condensation with the C-terminal of pepstatine was performed. A comparative study of the inhibition properties of the title compound with peptatin A was made.
[Back to top] Preliminary X-Ray Crystallographic Analysis and Molecular Replacement Studies with 2,5-Diketo-D-Gluconate Reductase. Sumit Khurana, David B. Powers, Stephen Anderson and Michael Blaber.
2,5 diketo-D-gluconate reductase (2,5-DKG reductase) is an enzyme with considerable commercial interest because it can be used to manufacture 2-keto-L-gulonic acid, a key intermediate in the industrial synthesis of L-ascorbic acid. This protein was crystallized in the monoclinic space group P2 1 with one molecule in the asymmetric unit. The crystals diffract to a maximum resolution of 1.9 A. An x-ray intensity data set was collected from these crystals and is 85% complete between 60 to 2.1 A. An initial structure solution has been attempted using the molecular replacement method with human aldose reductase (38% sequence identity) as a search model. The rotation and translation frinction searches yield a single clear peak above the background noise level with a corresponding R factor of 49.2% and a correlation coefficient of 34.0%.