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Biomedical subjects

L L Randall

Publications and source records attributed to L L Randall.

At least 19 recordsLinked to original sources
PubMed1992

Peptide binding by chaperone SecB: implications for recognition of nonnative structure.

The molecular basis for recognition of nonnative proteins by the molecular chaperone SecB was investigated with an in vitro assay based on the protection of SecB from proteolysis when a ligand is bound. The SecB tetramer has multiple binding sites for positively charged peptides. When the peptide binding sites are occupied, the complex undergoes a conformational change to expose hydrophobic sites that bind the fluorescent probe 1-anilinonaphthalene-8-sulfonate. A model is proposed for interaction of nonnative polypeptides with both hydrophilic and hydrophobic sites on SecB.

Amino Acid Sequence↗
PubMed1991

Mutations that affect the folding of ribose-binding protein selected as suppressors of a defect in export in Escherichia coli.

It has been proposed (Randall, L. L., and Hardy, S. J. S. (1986) Cell 46, 921-928) that export of protein involves a kinetic partitioning between the pathway that leads to productive export and the pathway that leads to the folding of polypeptides into a stable conformation that is incompatible with export. As predicted from this model, a decrease in the rate of export of maltose-binding protein to the periplasmic space in Escherichia coli resulting from a defect in the leader sequence was able to be partially overcome by a mutation that slowed the folding of the precursor, thereby increasing the time in which the polypeptide was competent for export. (Liu, G., Topping, T. B., Cover, W. H., and Randall, L. L. (1988) J. Biol. Chem. 263, 14790-14793). Here we describe mutations of the gene encoding ribose-binding protein that were selected as suppressors of a defect in export of that protein and that alter the folding pathway. We propose that selection of such suppressors may provide a general method to obtain mutations that affect the folding properties of any protein that can be expressed and exported in E. coli.

Biological Transport↗
PubMed1991

A kinetic partitioning model of selective binding of nonnative proteins by the bacterial chaperone SecB.

An in vitro assay for the interaction of SecB, a molecular chaperone from Escherichia coli, with polypeptide ligands was established based on the ability of SecB to block the refolding of denatured maltose-binding protein. Competition experiments show that SecB binds selectively to nonnative proteins with high affinity and without specificity for a particular sequence of amino acids. It is proposed that selectivity in binding is due to a kinetic partitioning of polypeptides between folding and association with SecB.

ATP-Binding Cassette Transporters↗
PubMed1990

No specific recognition of leader peptide by SecB, a chaperone involved in protein export.

Most proteins destined for export from Escherichia coli are made as precursors containing amino-terminal leader sequences that are essential for export and that are removed during the process. The initial step in export of a subset of proteins, which includes maltose-binding protein, is binding of the precursor by the molecular chaperone SecB. This work shows directly that SecB binds with high affinity to unfolded maltose-binding protein but does not specifically recognize and bind the leader. Rather, the leader modulates folding to expose elements in the remainder of the polypeptide that are recognized by SecB.

ATP-Binding Cassette Transporters↗
PubMed1989

Unity in function in the absence of consensus in sequence: role of leader peptides in export.

Passage of proteins across membranes during export from their site of synthesis to their final destination is mediated by leader peptides that paradoxically exhibit a unity of function in spite of a diversity of sequence. These leader peptides act in at least two stages of the export process: at entry into the pathway and subsequently during translocation across the membrane. How selectivity is imposed on the system in the absence of a consensus among the sequences of leader peptides is the main issue discussed here.

Amino Acid Sequence↗
PubMed1989

Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein.

It has been shown that folding of precursor maltose-binding protein of Escherichia coli in vitro is retarded by the leader peptide. We now present evidence that this modulation of folding plays a role during the export of maltose-binding protein in vivo. Maltose-binding protein synthesized in vivo without a leader sequence did not engage the cellular export apparatus. However, the requirement for the leader in at least one step, that of binding the export factor SecB, could be overcome by an amino acid substitution in the mature portion of maltose-binding protein. This substitution retarded the folding of the polypeptide even in the absence of a leader. Investigations using purified proteins in vitro demonstrated that SecB would stably bind to species of maltose-binding protein devoid of a leader when the folding of the binding proteins was sufficiently slow. Thus, we conclude that one of the roles of the leader is to retard folding and expose the binding site for SecB.

ATP-Binding Cassette Transporters↗
PubMed1989

Biochemical investigation of protein export in Escherichia coli.

Export of proteins from the bacterial cytoplasm to a final destination in the periplasm and outer membrane is one example of the fundamental process occurring in all cells whereby polypeptides are transferred across biological membranes. Investigations on a variety of different systems have indicated similarities in the mechanism of this process. In the cases of bacterial protein export and the transfer of polypeptides across the endoplasmic reticulum in eukaryotic cells the processes are so similar that understanding gleaned from studies of the one is usually directly applicable to the other. The study of protein export in E. coli has two advantages over that of eukaryotic secretion. Not only is there the possibility of doing sophisticated genetic experiments, but also one can carry out biochemical investigations in vivo, a facility not so readily available with eukaryotic organisms. Such studies have, for example, shown that membrane translocation can occur both cotranslationally and post-translationally, that export requires protonmotive force, that some component of the export apparatus prevents the exported protein from assuming its native structure in the cytosol, and that there are probably at least two functions for the leader sequence, one in targeting the protein to the export pathway and one in translocation across the membrane.

Bacterial Proteins↗
PubMed1988

Retardation of folding as a possible means of suppression of a mutation in the leader sequence of an exported protein.

We have proposed (Randall, L. L., and Hardy, S. J. S. (1986) Cell 46, 921-928) that during export of protein from Escherichia coli, there is a kinetic partitioning between the pathway that leads to productive translocation and the pathway that leads to folding of precursors into a stable conformation that is incompatible with export. This model predicts that a decrease in rate along the productive pathway resulting from a defect in the leader sequence could be partially overcome by slowing the folding of the precursor and thereby increasing the time during which that polypeptide would be competent to enter the export pathway. Here it is shown that a change in the mature portion of maltose-binding protein that is known to suppress a mutation in the leader sequence (Cover, W. H., Ryan, J. P., Bassford, P. J., Jr., Walsh, K. A., Bollinger, J., and Randall, L. L. (1987) J. Bacteriol. 169, 1794-1800) also decreases the rate of folding of the precursor.

ATP-Binding Cassette Transporters↗
PubMed1988

Role of the leader peptide of maltose-binding protein in two steps of the export process.

During the process of export of maltose-binding protein to the periplasm of Escherichia coli, the leader peptide is involved in at least two steps. The presence of the leader portion of maltose-binding protein was shown to be necessary to mediate initial binding of the precursor to the membrane. However, the presence of a mutationally altered leader which does not sustain export in vivo was sufficient to allow this interaction. Thus, the defect in export which is manifested in vivo by this mutational substitution occurs at a step that follows membrane association, most likely the translocation step. Translocation occurs at discrete sites that are not uniformly distributed over the cytoplasmic membrane. A large proportion of the membrane involved in translocation has a higher density than that of bulk cytoplasmic membrane.

ATP-Binding Cassette Transporters↗
PubMed1985

Export of alpha-amylase by Bacillus amyloliquefaciens requires proton motive force.

The secretion of protein directly into the extracellular medium by Bacillus amyloliquefaciens, a gram-positive bacterium, was shown to be dependent on proton motive force. When the electrochemical membrane potential gradient of protons was dissipated either by uncouplers or by valinomycin in combination with K+, a precursor form of alpha-amylase accumulated on the cellular membrane. The proton motive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition of export was not the result of decreased ATP concentration.

Adenosine Triphosphate↗
PubMed1978

Precursors of three exported proteins in Escherichia coli.

Arabinose-binding protein, maltose-binding protein, and lambda receptor are synthesized in vitro on membrane-bound polysomes from Escherichia coli. All three proteins are exported from the cytoplasm of E. coli and all three are made in vitro in a form a few thousand daltons larger than the authentic protein. The larger form of arabinose-binding protein is also detected in vivo by pulse labeling. It is concluded that the larger forms of the exported proteins are precursors containing an extra sequence. In contrast to the above, when the intracellular protein elongation factor Tu is synthesized in vitro on free polysomes, it is not detectably larger than the authentic form.

Arabinose↗
PubMed1978

Arrangement of protein I in Escherichia coli outer membrane: cross-linking study.

The arrangement of protein I in the outer membrane of Escherichia coli was investigated by cross-linking whole cells, isolated cell wall, protein-peptidoglycan complexes, and protein I released from peptidoglycan with NaCl. Both cleavable azide cross-linkers and imidoester reagents were used. The data presented suggest that protein I exists in the outer membrane as a trimer.

Azides↗
PubMed1977

Synthesis of exported proteins by membrane-bound polysomes from Escherichia coli.

A membrane-bound fraction of polysomes of Escherichia coli has been isolated after lysis of cells without the use of lysozyme. Protein-synthesis studies in vitro show that membrane-bound and free polysomes are different in the following respects. 1. Membrane-bound polysomes synthesize proteins which are exported from the cell. The products include proteins of the outer membrane and a secreted periplasmic protein, the maltose-binding protein. 2. The major product synthesized by free polysomes is elongation factor Tu, a soluble cytoplasmic protein. 3. The activity of membrane-bound polysomes in vitro is more resistant to puromycin than is the activity of free polysomes. In addition, the mRNA associated with membrane-bound polysomes is more stable than the bulk of cellular mRNA as revealed by studies with rifampicin.

Binding Sites↗