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Mutational and functional analysis o...

Chang, Zhiying.

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Mutational and functional analysis of substrate binding by the Escherichia coli DnaJ molecular chaperone.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Mutational and functional analysis of substrate binding by the Escherichia coli DnaJ molecular chaperone./
Author:	Chang, Zhiying.
Description:	161 p.
Notes:	Source: Dissertation Abstracts International, Volume: 66-04, Section: B, page: 2047.
Contained By:	Dissertation Abstracts International66-04B.
Subject:	Chemistry, Biochemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3172566
ISBN:	054210072X

Mutational and functional analysis of substrate binding by the Escherichia coli DnaJ molecular chaperone.
Chang, Zhiying.

Mutational and functional analysis of substrate binding by the Escherichia coli DnaJ molecular chaperone. - 161 p.

Source: Dissertation Abstracts International, Volume: 66-04, Section: B, page: 2047.

Thesis (Ph.D.)--The Johns Hopkins University, 2005.

The E. coli DnaJ is a member of a universally conserved molecular chaperone protein family. Acting in concert with DnaK and GrpE, DnaJ functions in various aspects of protein metabolism. This dissertation research was focused on identification of the sequence elements of DnaJ involved in the recognition of protein substrates. A series of internal deletion mutants of DnaJ was made; each had a potential motif or domain of DnaJ removed. Each deletion mutant protein was purified to homogeneity and the physical and functional properties of each were assessed. Circular dichroism spectroscopic analysis indicated that each mutant protein retained substantial secondary structure similar to that of native DnaJ. As assessed by native gel electrophoresis, the wild type and mutant proteins existed predominantly as dimers in solution. The tests for DnaJ function included assessments of each mutant protein for its capacity: (1) to stimulate ATP hydrolysis by DnaK; (2) to support initiation of phage lambda DNA replication in vitro; (3) to protect denatured luciferase from aggregation; and (4) to bind to peptides, partially folded proteins, or known native protein substrates. From these studies, we concluded that the cysteine-rich (CR) zinc binding domain plays some role in substrate binding, while the DnaJ regions comprised of residues 210 to 253 and 107 to 138 are essential for this function. An alanine scan across the region of 107 to 138 targeting conserved residues was performed. Either 2 or 4 consecutive amino acid residues were substituted with alanines at one time or a single residue was replaced by alanine. Each of these alanine-substitution-mutant proteins was purified and the functional properties of selected mutant proteins were examined. Surprisingly, most alanine-mutant proteins were found to be as active as wild type DnaJ. Mutations in a potential substrate-binding pocket caused small defects in DnaJ's interactions with substrates. It appears likely that some hydrophobic residues in this region contribute to the formation of a substrate binding pocket; however, our evidence indicates that single amino acid substitutions of these residues do not have a significant impact on DnaJ function.

ISBN: 054210072XSubjects--Topical Terms:

1017722
Chemistry, Biochemistry.

Mutational and functional analysis of substrate binding by the Escherichia coli DnaJ molecular chaperone.
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245 1 0 $a Mutational and functional analysis of substrate binding by the Escherichia coli DnaJ molecular chaperone.
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500 $a Source: Dissertation Abstracts International, Volume: 66-04, Section: B, page: 2047.
500 $a Adviser: Roger McMacken.
502 $a Thesis (Ph.D.)--The Johns Hopkins University, 2005.
520 $a The E. coli DnaJ is a member of a universally conserved molecular chaperone protein family. Acting in concert with DnaK and GrpE, DnaJ functions in various aspects of protein metabolism. This dissertation research was focused on identification of the sequence elements of DnaJ involved in the recognition of protein substrates. A series of internal deletion mutants of DnaJ was made; each had a potential motif or domain of DnaJ removed. Each deletion mutant protein was purified to homogeneity and the physical and functional properties of each were assessed. Circular dichroism spectroscopic analysis indicated that each mutant protein retained substantial secondary structure similar to that of native DnaJ. As assessed by native gel electrophoresis, the wild type and mutant proteins existed predominantly as dimers in solution. The tests for DnaJ function included assessments of each mutant protein for its capacity: (1) to stimulate ATP hydrolysis by DnaK; (2) to support initiation of phage lambda DNA replication in vitro; (3) to protect denatured luciferase from aggregation; and (4) to bind to peptides, partially folded proteins, or known native protein substrates. From these studies, we concluded that the cysteine-rich (CR) zinc binding domain plays some role in substrate binding, while the DnaJ regions comprised of residues 210 to 253 and 107 to 138 are essential for this function. An alanine scan across the region of 107 to 138 targeting conserved residues was performed. Either 2 or 4 consecutive amino acid residues were substituted with alanines at one time or a single residue was replaced by alanine. Each of these alanine-substitution-mutant proteins was purified and the functional properties of selected mutant proteins were examined. Surprisingly, most alanine-mutant proteins were found to be as active as wild type DnaJ. Mutations in a potential substrate-binding pocket caused small defects in DnaJ's interactions with substrates. It appears likely that some hydrophobic residues in this region contribute to the formation of a substrate binding pocket; however, our evidence indicates that single amino acid substitutions of these residues do not have a significant impact on DnaJ function.
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