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Journal Issue - Volume 9 Issue 10 (2000)

Abstract By using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase‐1, that was 15‐26 °C more thermostable than the parent fungal phytases used in its design (Lehmann et al., 2000). We now sought to use the backbone of consensus phytase‐1 and to modify its catalytic properties. This was done by replacing a considerable part of the active site (i.e., all the divergent...

Abstract A direct conflict between the stabilization free energy parameters of cytochrome c determined by optical methods and by hydrogen exchange (HX) is quantitatively explained when the partially folded intermediates seen by HX are taken into account. The results support the previous HX measurements of intermediate populations, show how intermediates can elude the standard melting analysis, and illustrate how they confuse the analysis when...

Abstract Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J, Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203–224). Both near‐ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid‐ and ...

Abstract α‐Macroglobulin inhibits a broad spectrum of proteinases by forming macromolecular cages inside which proteinases are cross‐linked and trapped. Upon formation of a complex with proteinase, α‐macroglobulin undergoes a large conformational change that results in the exposure of its receptor‐binding domain (RBD). Engagement of this domain by α‐macroglobulin receptor permits clearance of the α‐macroglobulin: proteinase complex...

Abstract Saquinavir is a widely used HIV‐1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV‐1 viral enzymes. Three HIV‐1 protease mutant species, G48V, L90M, and G48V/L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme (Jacobsen et al., 1996). Kinetic studies on these...

Abstract CAP‐23/NAP‐22, a neuron‐specific protein kinase C substrate, is Nα‐myristoylated and interacts with calmodulin (CaM) in the presence of Ca2+ ions. Takasaki et al. (1999, J Biol Chem 274:11848‐11853) have recently found that the myristoylated N‐terminal nonapeptide of CAP‐23/NAP‐22 (mC/N9) binds to Ca2+‐bound CaM (Ca2+/CaM). In the present study, small‐angle X‐ray scattering was used to investigate structural changes of Ca2+/CaM induced by its...

Abstract Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy, we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal...

Abstract Biotin and lipoic acid moieties are the covalently attached coenzyme cofactors of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of these moieties to the biotinyl‐ and lipoyl‐dependent enzymes is post‐translationally catalyzed by specific biotinylating and lipoylating protein enzymes. In Escherichia coli, two different enzymes, LplA and LipB, catalyze independent pathways for the lipoylation...

Abstract We have identified a similarity between the apical domain of the human transferrin receptor and several other protein families. This domain is found associated with two different families of peptidases. Therefore, we term it the PA domain for protease‐associated domain. The PA domain is found inserted within a loop of the peptidase domain of family M8/M33 zinc peptidases. The PA domain is also found in a vacuolar sorting...

Abstract The substrate specificity of porcine pepsin has been altered by site‐directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys‐Pro‐Xaa‐Yaa‐Phe*Nph‐Arg‐Leu (where Xaa is Ile or Pro, Yaa is Glu, Leu, Gln or Lys, Nph is...

Abstract Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature‐induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native‐like α‐helical structure. On heating to 70°C, partial unfolding occurs and...

Abstract The fluorescence time decay parameters of the β‐lactoglobulin‐1‐anilinonaphthalene‐8‐sulfonate complex have been investigated under physical and chemical perturbations (2 < pH < 8 and added electrolyte 0 < NaCl < 0.5 M) to obtain new insight on the nature of the protein binding interactions. A double exponential decay of the bound probe lifetime has been confirmed by the presence of a longer component, 11 to...

Abstract The prediction of binding energies from the three‐dimensional (3D) structure of a protein–ligand complex is an important goal of biophysics and structural biology. Here, we critically assess the use of empirical, solvent‐accessible surface area‐based calculations for the prediction of the binding of Src‐SH2 domain with a series of tyrosyl phosphopeptides based on the high‐affinity ligand from the hamster middle T antigen...

Abstract α2‐Macroglobulin (α2M) is a major carrier of transforming growth factor‐β (TGF‐β) in vitro and in vivo. By screening glutathione S‐transferase (GST) fusion proteins with overlapping sequences, we localized the TGF‐β‐binding site to aa 700–738 of the mature human α2M subunit. In separate experiments, we screened overlapping synthetic peptides corresponding to aa 696–777 of α2M and identified a single 16‐mer (718–733) that binds TGF‐β1....

Abstract Escherichia coli CspA is a member of the cold shock protein family. All cold shock proteins studied to date fold rapidly by an apparent two‐state mechanism. CspA contains an unusual cluster of aromatic amino acids on its surface that is necessary for nucleic acid binding and also provides stability to CspA (Hillier et al., 1998). To elucidate the role this aromatic cluster plays in the determining the folding rate and...

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