Role of the mature protein sequence of maltose-binding protein in its secretion across the E. coli cytoplasmic membrane

Cell ◽  
1981 ◽  
Vol 25 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Koreaki Ito ◽  
Jonathan R. Beckwith
Keyword(s):  
Protein Sequence ◽  
Cytoplasmic Membrane ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Mature Protein ◽  
E Coli

scholarly journals Exploring the Role of Integral Membrane Proteins in ATP-Binding Cassette Transporters: Analysis of a Collection of MalG Insertion Mutants

1998 ◽  
Vol 180 (9) ◽  
pp. 2507-2514 ◽  
Author(s):  
Bryn D. Nelson ◽  
Beth Traxler
Keyword(s):  
Membrane Proteins ◽  
Cytoplasmic Membrane ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Integral Membrane Proteins ◽  
Atp Binding ◽  
Mutant Proteins ◽  
Transport Complex ◽  
Atp Binding Cassette

ABSTRACT The maltose transport complex of Escherichia coli is a well-studied example of an ATP-binding cassette transporter. The complex, containing one copy each of the integral membrane proteins MalG and MalF and two copies of the peripheral cytoplasmic membrane protein MalK, interacts with the periplasmic maltose-binding protein to efficiently translocate maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK2 assembly interactions and in subsequent transport interactions, we isolated and characterized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regions of MalG permitted either assembly or both assembly and transport interactions to occur. In particular, we isolated two insertions mapping to extracytoplasmic (periplasmic) regions of MalG which preserved both assembly and transport abilities, suggesting that these are permissive sites in the protein. Another periplasmic insertion seems to affect only transport-specific interactions between MalG and maltose-binding protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble into the MalFGK2 complex. These mutants contain insertions in only two different hydrophilic regions of MalG, consistent with the notion that a restricted number of domains in this protein are critical complex assembly determinants. These MalG mutants will allow us to further explore the intermolecular interactions of this model transporter.

Progress in the identification of interaction sites on the periplasmic maltose binding protein from E coli

Biochimie ◽  
1990 ◽  
Vol 72 (6-7) ◽  
pp. 397-402 ◽  
Author(s):  
P. Martineau ◽  
W. Saurin ◽  
M. Hofnung ◽  
J.C. Spurlino ◽  
F.A. Quiochio
Keyword(s):  
Binding Protein ◽  
Maltose Binding Protein ◽  
E Coli ◽  
Interaction Sites

Linker mutagenesis in the gene encoding the periplasmic maltose-binding protein of E. coli.

Biochimie ◽  
1985 ◽  
Vol 67 (7-8) ◽  
pp. 849-851 ◽  
Author(s):  
P DUPLAY ◽  
H BEDOUELLE ◽  
S SZMELCMAN ◽  
M HOFNUNG
Keyword(s):  
Binding Protein ◽  
Maltose Binding Protein ◽  
Gene Encoding ◽  
E Coli

scholarly journals Characterization of the Klebsiella aerogenes Urease Accessory Protein UreD in Fusion with the Maltose Binding Protein

2010 ◽  
Vol 192 (9) ◽  
pp. 2294-2304 ◽  
Author(s):  
Eric L. Carter ◽  
Robert P. Hausinger
Keyword(s):  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Maltose Binding Protein ◽  
Accessory Protein ◽  
Klebsiella Aerogenes ◽  
E Coli ◽  
Cell Extracts ◽  
Recombinant Cells

ABSTRACT Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a ΔureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (>670 kDa) that bound approximately 2.5 Ni2+ ions (Kd of ∼50 μM, where Kd is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (∼4 Zn2+ ions per protomer; Kd of 5 μM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.

The role of gene A protein and E. coli rep protein in the replication of ɸX 174 replicative form DNA

1980 ◽  
Vol 210 (1180) ◽  
pp. 337-349 ◽  
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Origin Of Replication ◽  
Electron Microscopic ◽  
Single Stranded Dna ◽  
Rep Protein ◽  
E Coli

The gene A protein of bacteriophage ɸX 174 initiates replication of super-twisted RFI DNA by cleaving the viral (+) strand at the origin of replication and binding to the 5' end. Upon addition of E. coli rep protein (single-stranded DNA dependent ATPase), E. coli single-stranded DNA binding protein and ATP, complete unwinding of the two strands occurs. Electron microscopic analyses of intermediates in the reaction reveal that the unwinding occurs by movement of the 5' end into the duplex, displacing the viral strand in the form of a single-stranded loop. Since unwinding will not occur in the absence of either gene A protein or rep protein, it is presumed that the rep protein interacts to form a complex with the bound gene A protein. Single-stranded DNA binding protein facilitates the unwinding by binding to the exposed single-stranded DNA. Further addition of the four deoxyribotriphosphates and DNA polymerase III holoenzyme to the reaction results in synthesis of viral (+) single-stranded circles in amounts exceeding that of the input template. A model describing the role of gene A protein and rep protein in duplex DNA replication is presented and other properties of gene A protein discussed.

Sign in / Sign up

Export Citation Format

Share Document