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Journal Issue - Volume 2 Issue 3 (March 1993)

Abstract A new method has been developed to compute the probability that each amino acid in a protein sequence is in a particular secondary structural element. Each of these probabilities is computed using the entire sequence and a set of predefined structural class models. This set of structural classes is patterned after Jane Richardson's taxonomy for the domains of globular proteins. For each structural class considered, a...

Abstract Protein Cα coordinates are used to accurately reconstruct complete protein backbones and side‐chain directions. This work employs potentials of mean force to align semirigid peptide groups around the axes that connect successive Cα atoms. The algorithm works well for all residue types and secondary structure classes and is stable for imprecise Cα coordinates. Tests on known protein structures show that root mean square errors in predicted...

Abstract The conformation of bovine Hsc70, a 70‐kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222–245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem....

Abstract We have investigated the spontaneous degradation of aspartate and asparagine residues via succinimide intermediates in model peptides in organic co‐solvents. We find that the rate of deamidation at asparagine residues is markedly reduced in solvents of low dielectric strength. Theoretical considerations suggest that this decrease in rate is due to the destabilization of the deprotonated peptide bond nitrogen anion that is...

Abstract The membrane‐intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca2+‐ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high‐performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform–methanol mixtures....

Abstract S‐peptide (residues 1–20) and S‐protein (residues 21–124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S‐peptide binds S‐protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of...

Abstract Cytochrome P450cam (P450CIA1) catalyzes the hydroxylation of camphor and several substrate analogues such as norcamphor and 1‐methyl‐norcamphor. Hydroxylation was found experimentally at the 3, 5, and 6 positions of norcamphor, but only at the 5 and 6 positions of 1‐methyl‐norcamphor. In the catalytic cycle, the hydroxylation of substrate is coupled to the consumption of NADH. For camphor, the degree of coupling is 100%,...

Abstract Based on the recently determined X‐ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three‐dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49...

Abstract The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three‐dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA‐binding proteins (leucine zippers), Leu residues are conserved at positions “d,” and β‐branched amino acids, Ile and Val, often occur at positions “a” in the hydrophobic core. In order to discern what effect ...

Abstract We have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesis/chemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides ...

Abstract Thioflavine T (ThT) associates rapidly with aggregated fibrils of the synthetic β/A4‐derived peptides β(1–28) and β(1–40), giving rise to a new excitation (ex) (absorption) maximum at 450 nm and enhanced emission (em) at 482 nm, as opposed to the 385 nm (ex) and 445 nm (em) of the free dye. This change is dependent on the aggregated state as monomeric or dimeric peptides do not react, and guanidine dissociation of aggregates destroys...

Abstract The IR absorption frequencies as derived from second derivatives of the Fourier transform IR spectra of the amide I' bands of globular proteins in D2O are compared to those obtained from band fitting of the vibrational circular dichroism (VCD) spectra. The two sets of frequencies are in very good agreement, yielding consistent ranges where amide I' VCD and IR features occur. Use of VCD to complement the IR allows one to add...

Abstract The first high‐level production of a binding‐active odorant binding protein is described. The expression cassette polymerase chain reaction was used to generate a DNA fragment encoding the pheromone binding protein (PBP) of the male moth Antheraea polyphemus. Transformation of Escherichia coli cells with a vector containing this construct generated clones which, when induced with isopropyl β‐d‐thiogalactopyranoside, produced the 14‐kDa PBP in both...

Abstract The plant cytotoxin ricin is a heterodimer with a cell surface binding (B) chain and an enzymatically active A chain (RTA) known to act as a specific N‐glycosidase. RTA must be separated from B chain to attack rRNA. The X‐ray structure of ricin has been solved recently; here we report the structure of the isolated A chain expressed from a clone in Escherichia coli. This structure of wild‐type rRTA has and will continue to serve as the...

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