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Journal Issue - Volume 1 Issue 8 (August 1992)

Abstract Two isoforms of yeast cyclophilins, yCyPA and yCyPB, have been subcloned, expressed in Escherichia coli, and purified to homogeneity. The full‐length (163‐amino acid) yeast CyPA was easily expressed and purified; however, only a genetically truncated, 186‐residue form of yCyPB lacking a putative 20‐amino acid signal sequence could be purified. Each yeast cyclophilin isoform is a peptidyl‐prolyl isomerase, inhibitable by the...

Abstract A Crithidia fasciculata 83‐kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min−1 and a Km of 60 μM, whereas the homologous mammalian hsp90 binds ATP...

Abstract Several of the major heat shock proteins (HSPs) function normally as molecular chaperones to prevent aggregation of immature polypeptides and thereby facilitate folding and oligomerization. To determine their effect on nascent polypeptides, we added purified preparations of different isoforms of HSP70 to in vitro translation reactions primed by the 26S mRNA of Sindbis virus, which encodes an autoprotease that functions...

Abstract The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558–1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the...

Abstract Human and bovine α‐thrombin cleaved at the B‐chain by chymotrypsin generates catalytically competent ζ‐thrombins, which are comprised of two noncovalently linked fragments: a 36‐ (human) or 49‐ (bovine) residue A‐chain linked by a disulfide to B‐chain residues B1–148 (ζ1‐thrombin) and B‐chain residues B149–259 (ζ2‐thrombin). Human and bovine d‐Phe‐Pro‐Arg‐CH2‐ζ‐ and PhMeSO2‐ζ‐thrombins were prepared by reaction of the active‐site...

Abstract BM 06.022 is a t‐PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process. Until now, no X‐ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine‐derived inhibitors was carried out in order to assess that the active site...

Abstract Recent publication of the atomic structure of G‐actin (Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes, K.C., 1990, Nature 347, 37–44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno‐nucleotides bound to G‐ and F‐actin were examined in order to assess polymerization‐related changes in the nucleotide phosphate site....

Abstract Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low...

Abstract The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X‐ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman,...

Abstract A detailed analysis of the composition and properties of hydrophobic nuclei and microclusters in pancreatic ribonuclease A (RNase A) has been carried out. Distance calculations for all noncovalently bonded atoms revealed that the average number of nonpolar contacts between a side chain of an amino acid and its neighbors is substantially larger if it involves hydrophobic residues rather than nonhydrophobic ones. However, the...

Abstract The structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV‐1) protease with a peptide‐mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been determined. The inhibitor is Noa‐His‐Hch[CH(OH)CH(OH)]Vam‐Ile‐Amp (U‐75875), and its Ki for inhibition of the HIV‐1 protease is <1.0 nM (Noa = 1‐naphthoxyacetyl, Hch = a hydroxy‐modified form of cyclohexylalanine, ...

Abstract Crystals have been grown of two similar peptides that form ion‐conducting channels in diphytanoyl phosphatidylcholine bilayers. These crystals were grown by slow evaporation of the organic solvent, 2,2,2‐trifluoroethanol. Crystals of one of the peptides have been characterized by X‐ray diffraction, and X‐ray data have been measured to 2.3 Å resolution. Earlier it was proposed that the ion‐conducting channels formed by these...