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Journal Issue - Volume 13 Issue 4 (April 2004)

Abstract We have introduced the mutation glycine 29 to alanine, designed to increase the rate of protein folding, into the B‐domain of protein A (BdpA). From NMR lineshape analysis, we find the G29A mutation increases the folding rate constant by threefold; the folding time is 3 μsec. Although wild‐type BdpA folds extremely fast, simple‐point mutations can still speed up the folding; thus, the folding rate is not evolutionarily...

Abstract Specific peptidases exist for nearly every amide linkage in peptidoglycan. In several cases, families of peptidoglycan hydrolases with different specificities turned out to be related. Here we show that lysostaphin‐type peptidases and D‐Ala‐D‐Ala metallopeptidases have similar active sites and share a core folding motif in otherwise highly divergent folds. The central Zn2+ is tetrahedrally coordinated by two histidines, an aspartate,...

Abstract We develop coarse‐grained, distance‐ and orientation‐dependent statistical potentials from the growing protein structural databases. For protein structural classes (α, β, and α/β), a substantial number of backbone–backbone and backbone–side‐chain contacts stabilize the native folds. By taking into account the importance of backbone interactions with a virtual backbone interaction center as the 21st anisotropic site, we...

Abstract We characterized the biochemical functions of the small nonessential (C101–C104) and the large essential (C173–C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. We also characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca2+ protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin,...

Abstract We present a method for prediction of functional sites in a set of aligned protein sequences. The method selects sites which are both well conserved and clustered together in space, as inferred from the 3D structures of proteins included in the alignment. We tested the method using 86 alignments from the NCBI CDD database, where the sites of experimentally determined ligand and/or macromolecular interactions are annotated....

Abstract The folding of collagen in vitro is very slow and presents difficulties in reaching equilibrium, a feature that may have implications for in vivo collagen function. Peptides serve as good model systems for examining equilibrium thermal transitions in the collagen triple helix. Investigations were carried out to ascertain whether a range of synthetic triple‐helical peptides of varying sequences can reach equilibrium, and...

Abstract An efficient enzyme kinetics assay using electrospray ionization mass spectrometry (ESI‐MS) was initially applied to the catalytic mechanism investigation of a carbohydrate sulfotransferase, NodST. Herein, the recombinant NodST was overexpressed with a His6‐tag and purified via Ni‐NTA metal‐affinity chromatography. In this bisubstrate enzymatic system, an internal standard similar in structure and ionization efficiency to...

Abstract The pyridoxal 5′‐phosphate‐dependent enzymes tyrosine phenol‐lyase and tryptophan indole‐lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the...

Abstract Electrostatics and solvation energies are important for defining protein stability, structural specificity, and molecular recognition. Because these energies are difficult to compute quickly and accurately, they are often ignored or modeled very crudely in computational protein design. To address this problem, we have developed a simple, fast, and accurate approximation for calculating Born radii in the context of protein...

Abstract Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's...

Abstract We report molecular dynamics calculations of neuraminidase in complex with an inhibitor, 4‐amino‐2‐deoxy‐2,3‐didehydro‐N‐acetylneuraminic acid (N‐DANA), with subsequent free energy analysis of binding by using a combined molecular mechanics/continuum solvent model approach. A dynamical model of the complex containing an ionized Glu119 amino acid residue is found to be consistent with experimental data. Computational analysis indicates...

Abstract We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G, which are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation‐condensation mechanism, each of which is described as helix‐assisted β‐1 and β‐2 hairpin...

Abstract Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55‐carbon long‐chain product. We previously reported the crystal structure of the apo‐enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg2+, and two Triton molecules (product‐like). In the...

Abstract Transglutaminases (TGases) catalyze the cross‐linking of peptides and proteins by the formation of γ‐glutamyl‐ε‐lysyl bonds. Given the implication of tissue TGase in various physiological disorders, development of specific tissue TGase inhibitors is of current interest. To aid in the design of peptide‐based inhibitors, a better understanding of the mode of binding of model peptide substrates to the enzyme is required. Using...

Abstract A novel method for the refinement of misfolded protein structures is proposed in which the properties of the solvent environment are oscillated in order to mimic some aspects of the role of molecular chaperones play in protein folding in vivo. Specifically, the hydrophobicity of the solvent is cycled by repetitively altering the partial charges on solvent molecules (water) during a molecular dynamics simulation. During...

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