The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
Andrew J Doig - One of the best experts on this subject based on the ideXlab platform.
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Amyloidogenic sequences in Native Protein structures
Protein Science, 2010Co-Authors: Susan Tzotzos, Andrew J DoigAbstract:Numerous short peptides have been shown to form beta-sheet amyloid aggregates in vitro. Proteins that contain such sequences are likely to be problematic for a cell, due to their potential to aggregate into toxic structures. We investigated the structures of 30 Proteins containing 45 sequences known to form amyloid, to see how the Proteins cope with the presence of these potentially toxic sequences, studying secondary structure, hydrogen-bonding, solvent accessible surface area and hydrophobicity. We identified two mechanisms by which Proteins avoid aggregation: Firstly, amyloidogenic sequences are often found within helices, despite their inherent preference to form beta structure. Helices may offer a selective advantage, since in order to form amyloid the sequence will presumably have to first unfold and then refold into a beta structure. Secondly, amyloidogenic sequences that are found in beta structure are usually buried within the Protein. Surface exposed amyloidogenic sequences are not tolerated in strands, presumably because they lead to Protein aggregation via assembly of the amyloidogenic regions. The use of alpha-helices, where amyloidogenic sequences are forced into helix, despite their intrinsic preference for beta structure, is thus a widespread mechanism to avoid Protein aggregation.
Richard A Friesner - One of the best experts on this subject based on the ideXlab platform.
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computational prediction of Native Protein ligand binding and enzyme active site sequences
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Raj Chakrabarti, Alexander M. Klibanov, Richard A FriesnerAbstract:Recent studies reveal that the core sequences of many Proteins were nearly optimized for stability by natural evolution. Surface residues, by contrast, are not so optimized, presumably because Protein function is mediated through surface interactions with other molecules. Here, we sought to determine the extent to which the sequences of Protein ligand-binding and enzyme active sites could be predicted by optimization of scoring functions based on Protein ligand-binding affinity rather than structural stability. Optimization of binding affinity under constraints on the folding free energy correctly predicted 83% of amino acid residues (94% similar) in the binding sites of two model receptor-ligand complexes, streptavidin-biotin and glucose-binding Protein. To explore the applicability of this methodology to enzymes, we applied an identical algorithm to the active sites of diverse enzymes from the peptidase, β-gal, and nucleotide synthase families. Although simple optimization of binding affinity reproduced the sequences of some enzyme active sites with high precision, imposition of additional, geometric constraints on side-chain conformations based on the catalytic mechanism was required in other cases. With these modifications, our sequence optimization algorithm correctly predicted 78% of residues from all of the enzymes, with 83% similar to Native (90% correct, with 95% similar, excluding residues with high variability in multiple sequence alignments). Furthermore, the conformations of the selected side chains were often correctly predicted within crystallographic error. These findings suggest that simple selection pressures may have played a predominant role in determining the sequences of ligand-binding and active sites in Proteins.
David Baker - One of the best experts on this subject based on the ideXlab platform.
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Native Protein sequences are close to optimal for their structures
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Brian Kuhlman, David BakerAbstract:How large is the volume of sequence space that is compatible with a given Protein structure? Starting from random sequences, low free energy sequences were generated for 108 Protein backbone structures by using a Monte Carlo optimization procedure and a free energy function based primarily on Lennard–Jones packing interactions and the Lazaridis–Karplus implicit solvation model. Remarkably, in the designed sequences 51% of the core residues and 27% of all residues were identical to the amino acids in the corresponding positions in the Native sequences. The lowest free energy sequences obtained for ensembles of Native-like backbone structures were also similar to the Native sequence. Furthermore, both the individual residue frequencies and the covariances between pairs of positions observed in the very large SH3 domain family were recapitulated in core sequences designed for SH3 domain structures. Taken together, these results suggest that the volume of sequence space optimal for a Protein structure is surprisingly restricted to a region around the Native sequence.
Susan Tzotzos - One of the best experts on this subject based on the ideXlab platform.
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Amyloidogenic sequences in Native Protein structures
Protein Science, 2010Co-Authors: Susan Tzotzos, Andrew J DoigAbstract:Numerous short peptides have been shown to form beta-sheet amyloid aggregates in vitro. Proteins that contain such sequences are likely to be problematic for a cell, due to their potential to aggregate into toxic structures. We investigated the structures of 30 Proteins containing 45 sequences known to form amyloid, to see how the Proteins cope with the presence of these potentially toxic sequences, studying secondary structure, hydrogen-bonding, solvent accessible surface area and hydrophobicity. We identified two mechanisms by which Proteins avoid aggregation: Firstly, amyloidogenic sequences are often found within helices, despite their inherent preference to form beta structure. Helices may offer a selective advantage, since in order to form amyloid the sequence will presumably have to first unfold and then refold into a beta structure. Secondly, amyloidogenic sequences that are found in beta structure are usually buried within the Protein. Surface exposed amyloidogenic sequences are not tolerated in strands, presumably because they lead to Protein aggregation via assembly of the amyloidogenic regions. The use of alpha-helices, where amyloidogenic sequences are forced into helix, despite their intrinsic preference for beta structure, is thus a widespread mechanism to avoid Protein aggregation.
Fred E. Regnier - One of the best experts on this subject based on the ideXlab platform.
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Native Protein proteolysis in an immobilized enzyme reactor as a function of temperature.
Analytical Chemistry, 2012Co-Authors: Dinelia Rivera-burgos, Fred E. RegnierAbstract:Trypsin concentration and the unmasking of cleavage sites in Proteins play important roles in the stoichiometry of peptide production and the number of limit peptides generated during proteolysis. The hypothesis explored in this work was that Native Proteins could be digested and identified without disulfide reduction by (i) enhancing the unmasking of cleavage sites through elevated reaction temperatures and (ii) increasing trypsin concentration by use of an immobilized enzyme reactor (IMER). Transferrin was chosen as a model Protein for these studies on the basis of its resistance to trypsin digestion. Results from this study showed greater than 70% sequence coverage in the peptides identified when nonreduced transferrin was digested at 60 °C. Large numbers of missed cleavages were observed from specific regions in Proteins. Proteolysis appeared to start at a small number of high frequency cleavage sites in the cases of both reduced and nonreduced transferrin. Although approximately the same number of pe...
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Eliminating disulfide exchange during glutamyl endopeptidase digestion of Native Protein.
Journal of Chromatography A, 1999Co-Authors: Shelly J Dormady, Fred E. RegnierAbstract:Abstract Numerous advantages of using immobilized enzymes over free-solution Protein digests have been cited in the literature. This investigation examines both the rate of hydrolysis and the extent of disulfide bond exchange in disulfide bridged dipeptide fragments formed during proteolysis of Native Protein. Gutamyl endopeptidase as both an immobilized enzyme and in free solution was used in these studies. It was found that extensive hydrolysis of insulin was achieved in 2 min with immobilized enzyme cartridges operated in the stopped-flow mode orders. This is orders of magnitude faster than was seen in free solution. Other advantages ranging from ease of use and reduction in sample size to the potential for automation were also noted with the immobilized enzyme cartridge. Normal free-solution proteolysis generally requires 12–24 h, based on the lower enzyme-to-substrate ratio in solution. A disturbing feature noted in these lengthy free-solution reactions was the tendency to form disulfide bridged peptide artifacts. This could lead to the erroneous conclusion that disulfide bonding in a sample was not that of the Native Protein. It is concluded that the advantage of immobilized enzymes over free-solution reactions will be most important in the pharmaceutical industry where proteolytic fragment “fingerprinting” of recombinant Proteins is being used to confirm structure.
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Native Protein separations and enzyme microassays by capillary zone and gel electrophoresis
Analytical Chemistry, 1993Co-Authors: Dan Wu, Fred E. RegnierAbstract:Native Protein separations by capillary gel electrophoresis are achieved using linear acrylamide gel matrices. Polyacrylamide gels with a concentration range of 3.5-5% did not exhibit size separations for Native Proteins with molecular weights from 20 000 to 47 000. The separation of Native Proteins in gel-filled capillaries is based solely on the charge of the Protein as in normal zonal electrophoresis. Retention of Protein activity in the acrylamide matrix was demonstrated by performing enzymatic assays in the gel matrix
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Native Protein separations and enzyme microassays by capillary zone and gel electrophoresis.
Analytical Chemistry, 1993Co-Authors: Dan Wu, Fred E. RegnierAbstract:: Native Protein separations by capillary gel electrophoresis are achieved using linear acrylamide gel matrices. Polyacrylamide gels with a concentration range of 3.5-5% did not exhibit size separations for Native Proteins with molecular weights from 20,000 to 47,000. The separation of Native Proteins in gel-filled capillaries is based solely on the charge of the Protein as in normal zonal electrophoresis. Retention of Protein activity in the acrylamide matrix was demonstrated by performing enzymatic assays in the gel matrix. Alkaline phosphatase (ALP) and beta-galactosidase assays were conducted in both C18-PF108-modified and polyacrylamide gel-filled capillaries. Enzyme assays were achieved by filling the capillary with an appropriate substrate dissolved in the electrophoresis buffer. The product formed by the reaction of enzyme with substrate was monitored using a standard UV-visible detector. Both constant potential and zero potential modes of analysis were demonstrated. The polyacrylamide gel columns provide the advantages of minimized diffusion and limited band spreading due to the high viscosity of the gel matrix. The lowest detection limit achieved was 5.2 x 10(-20) mol (7.6 x 10(-12) M sample injected) of ALP. The dual enzyme assay of ALP and beta-galactosidase was achieved in gel-filled capillaries simultaneously.