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Journal Issue - Volume 2 Issue 4 (April 1993)

Abstract Peptide α‐amidation is a widespread, often essential posttranslational modification shared by many bioactive peptides and accomplished by the products of a single gene encoding a multifunctional protein, peptidylglycine α‐amidating monooxygenase (PAM). PAM has two catalytic domains that work sequentially to produce the final α‐amidated product peptide. Tissue‐specific alternative splicing can generate forms of PAM retaining or lacking a domain...

Abstract Escherichia coli cells permeabilized by treatment with low concentrations of toluene contain an adenylyl cyclase activity that can be stimulated 3.6–7.6‐fold by GTP. The stimulatory effect of GTP is maximal at concentrations of the nucleotide in the physiological range (above 0.7 mM). Studies of the dependence of velocity on substrate (ATP) concentration indicate that the velocity vs. substrate plots are sigmoid in the...

Abstract We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158–9169). We now report the sequencing of the ptsl gene of B. subtilis encoding Enzyme I (570 amino acids and 63, 076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject...

Abstract A new method based on protein fragmentation and directly coupled microbore high‐performance liquid chromatography–fast atom bombardment mass spectrometry (HPLC‐FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH...

Abstract Chemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high‐resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization. Both tensor element magnitudes and tensor orientations...

Abstract IIIGlc is an 18.1‐kDa signal‐transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two‐dimensional 1H‐15N and 1H‐13C heteronuclear multiple‐quantum coherence (HMQC) experiments and a two‐dimensional 13C‐13C‐1H correlation spectroscopy via JCC...

Abstract Computer‐based homology modeling techniques were used to construct a three‐dimensional model of the Escherichia coli serine receptor ligand‐binding domain based on the crystal structure of the Salmonella typhimurium aspartate receptor and the sequence homology between the two receptors. Residues that have been found in mutagenesis studies to be necessary for serine binding are located in a proposed serine‐binding site. Several other mutations that...

Abstract The effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the...

Abstract Crystal structures of four mutants of bovine pancreatic trypsin inhibitor (F22A, Y23A, N43G, and F45A), engineered to alter their stability properties, have been determined. The mutated residues, which are highly conserved among Kunitz‐type inhibitors, are located in the rigid core of the molecule. Replacement of the partially buried bulky residues of the wild‐type protein with smaller residues resulted in crevices open to...

Abstract Four mutants of bovine pancreatic trypsin inhibitor (BPTI) with replacements in the rigid core result in the creation of deep crevices on the surface of the protein. Other than crevices at the site of the mutation, few other differences are observed in the crystal structures of wild‐type BPTI and the mutants F22A, Y23A, N43G, and F45A. These mutants are highly destabilized relative to wild type (WT). The differences between...

Abstract The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 Å. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 Å between both...

Abstract In the crystal structure of troponin C, the holo C‐domain is bound in a head‐to‐tail fashion to the A‐helix of the apo N‐domain of a symmetry‐related molecule. Using this interaction, we have proposed a model for the calmodulin‐peptide complex. We find that the interaction of the C‐domain with the A‐helix is similar to that observed in the NMR structure of the calmodulin‐myosin light chain kinase (MLCK) peptide complex....

Abstract A new, automated, knowledge‐based method for the construction of three‐dimensional models of proteins is described. Geometric restraints on target structures are calculated from a consideration of homologous template structures and the wider knowledge base of unrelated protein structures. Three‐dimensional structures are calculated from initial partly folded states by high‐temperature molecular dynamics simulations followed...

Abstract The three‐dimensional structure of rubredoxin from the hyperthermophilic archaebacterium, Pyrococcus furiosus, has been modeled from the X‐ray crystal structures of three homologous proteins from Clostridium pasteurianum, Desulfovibrio gigas, and Desulfovibrio vulgaris. All three homology models are similar. When comparing the positions of all heavy atoms and essential hydrogen atoms to the recently solved crystal structure (Day, M.W., et al.,...

Abstract The affects of differences in amino acid sequence on the temperature stability of the three‐dimensional structure of the small β‐sheet protein, rubredoxin (Rd), was revealed when a set of homology models was subjected to molecular dynamics simulations at relatively high temperatures. Models of Rd from the hyperthermophile, Pyrococcus furiosus (Pf), an organism that grows optimally at 100°C, were compared to three mesophilic Rds of...

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