The Experts below are selected from a list of 15 Experts worldwide ranked by ideXlab platform
Carol A. Kumamoto - One of the best experts on this subject based on the ideXlab platform.
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Mutations of the molecular chaperone Protein SecB which alter the interaction between SecB and maltose-binding Protein.
The Journal of biological chemistry, 1993Co-Authors: P M Gannon, Carol A. KumamotoAbstract:SecB is a 16-kDa cytosolic chaperone Protein that is required for efficient export of particular Proteins in Escherichia coli. To identify regions of SecB that contribute to efficient Protein export, we isolated SecB point mutants that are defective for Protein export in vivo. We obtained missense mutations at residues Leu75 (SecBL75Q), Cys76 (SecBC76Y), and Glu77 (SecBE77K) in the center of the SecB gene. In vivo, mutant SecBL75Q and SecBE77K Proteins are capable of binding to precursor maltose-binding Protein (MBP) and preventing the formation of export-incompetent precursor MBP; however, export of MBP is still defective. In vitro, purified SecBL75Q and SecBE77K Proteins bound to unfolded MBP and blocked its refolding. SecBL75Q and SecBE77K were more effective than wild-type SecB at blocking the refolding of unfolded MBP, suggesting that SecBL75Q and SecBE77K have a higher affinity for unfolded MBP.
David Eisenberg - One of the best experts on this subject based on the ideXlab platform.
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crystallization of the chaperone Protein SecB
Protein Science, 1995Co-Authors: Alice Vrielink, Lesa J Beamer, David EisenbergAbstract:The secretory Protein SecB found in Escherichia coli is a molecular chaperone that binds to precursor forms of a number of Proteins targeted for export to the periplasmic space. SecB maintains these Proteins in a translocation-competent conformation facilitating the translocation process. The material has been cloned and expressed in E. coli. Crystals have been grown from polyethylene glycol 8000 by vapor diffusion using the hanging drop technique. These crystals are monoclinic, belonging to space group C2 with unit cell dimensions a = 56.0 A, b = 111.1 A, c = 134.7 A, and beta = 104 degrees. The crystals diffract to 8 A resolution on a Rigaku imaging plate detector. Dynamic light scattering experiments suggest that SecB exhibits aggregation behavior with a number of different precipitating agents. These results may explain resistance of SecB to forming ordered crystals.
P M Gannon - One of the best experts on this subject based on the ideXlab platform.
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Mutations of the molecular chaperone Protein SecB which alter the interaction between SecB and maltose-binding Protein.
The Journal of biological chemistry, 1993Co-Authors: P M Gannon, Carol A. KumamotoAbstract:SecB is a 16-kDa cytosolic chaperone Protein that is required for efficient export of particular Proteins in Escherichia coli. To identify regions of SecB that contribute to efficient Protein export, we isolated SecB point mutants that are defective for Protein export in vivo. We obtained missense mutations at residues Leu75 (SecBL75Q), Cys76 (SecBC76Y), and Glu77 (SecBE77K) in the center of the SecB gene. In vivo, mutant SecBL75Q and SecBE77K Proteins are capable of binding to precursor maltose-binding Protein (MBP) and preventing the formation of export-incompetent precursor MBP; however, export of MBP is still defective. In vitro, purified SecBL75Q and SecBE77K Proteins bound to unfolded MBP and blocked its refolding. SecBL75Q and SecBE77K were more effective than wild-type SecB at blocking the refolding of unfolded MBP, suggesting that SecBL75Q and SecBE77K have a higher affinity for unfolded MBP.
Gunter Blobel - One of the best experts on this subject based on the ideXlab platform.
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high affinity binding of escherichia coli SecB to the signal sequence region of a presecretory Protein
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Makoto Watanabe, Gunter BlobelAbstract:The Escherichia coli cytosolic homotetrameric Protein SecB is known to be involved in Protein export across the plasma membrane. A currently prevalent view holds that SecB functions exclusively as a chaperone interacting nonspecifically with unfolded Proteins, not necessarily exported Proteins, whereas a contrary view holds that SecB functions primarily as a specific signal-recognition factor--i.e., in binding to the signal sequence region of exported Proteins. To experimentally resolve these differences we assayed for binding between chemically pure SecB and chemically pure precursor (p) form (containing a signal sequence) and mature (m) form (lacking a signal sequence) of a model secretory Protein (maltose binding Protein, MBP) that was C-terminally truncated. Because of the C-terminal truncation, neither p nor m was able to fold. We found that SecB bound with 100-fold higher affinity to p (Kd 0.8 nM) than it bound to m (Kd 80 nM). As the presence of the signal sequence in p is the only feature that distinguished p from m, these data strongly suggest that the high-affinity binding of SecB is to the signal sequence region and not the mature region of p. Consistent with this conclusion, we found that a wild-type signal peptide, but not an export-incompetent mutant signal peptide of another exported Protein (LamB), competed for binding to p. Moreover, the high-affinity binding of SecB to p was resistant to 1 M salt, whereas the low-affinity binding of SecB to m was not. These qualitative differences suggested that SecB binding to m was primarily by electrostatic interactions, whereas SecB binding to p was primarily via hydrophobic interactions, presumably with the hydrophobic core of the signal sequence. Taken together our data strongly support the notion that SecB is primarily a specific signal-recognition factor.
Alice Vrielink - One of the best experts on this subject based on the ideXlab platform.
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crystallization of the chaperone Protein SecB
Protein Science, 1995Co-Authors: Alice Vrielink, Lesa J Beamer, David EisenbergAbstract:The secretory Protein SecB found in Escherichia coli is a molecular chaperone that binds to precursor forms of a number of Proteins targeted for export to the periplasmic space. SecB maintains these Proteins in a translocation-competent conformation facilitating the translocation process. The material has been cloned and expressed in E. coli. Crystals have been grown from polyethylene glycol 8000 by vapor diffusion using the hanging drop technique. These crystals are monoclinic, belonging to space group C2 with unit cell dimensions a = 56.0 A, b = 111.1 A, c = 134.7 A, and beta = 104 degrees. The crystals diffract to 8 A resolution on a Rigaku imaging plate detector. Dynamic light scattering experiments suggest that SecB exhibits aggregation behavior with a number of different precipitating agents. These results may explain resistance of SecB to forming ordered crystals.
