Effect of Crowding Agents, Signal Peptide, and Chaperone SecB on the Folding and Aggregation ofE. coliMaltose Binding Protein

Abstract

SecB, a soluble cytosolic chaperone component of the Sec export pathway, binds to newly synthesized precursor proteins and prevents their premature aggregation and folding and subsequently targets them to the translocation machinery on the membrane. PreMBP, the precursor form of maltose binding protein, has a 26-residue signal sequence attached to the N-terminus of MBP and is a physiological substrate of SecB. We examine the effect of macromolecular crowding and SecB on the stability and refolding of denatured preMBP and MBP. PreMBP was less stable than MBP (DeltaT(m )= 7 +/- 0.5 K) in both crowded and uncrowded solutions. Crowding did not cause any substantial changes in the thermal stability of MBP (DeltaT(m )= 1 +/- 0.4 K) or preMBP (DeltaT(m )= 0 +/- 0.6 K), as observed in spectroscopically monitored thermal unfolding experiments. However, both MBP and preMBP were prone to aggregation while refolding under crowded conditions. In contrast to MBP aggregates, which were amorphous, preMBP aggregates form amyloid fibrils. Under uncrowded conditions, a molar excess of SecB was able to completely prevent aggregation and promote disaggregation of preformed aggregates of MBP. When a complex of the denatured protein and SecB was preformed, SecB could completely prevent aggregation and promote folding of MBP and preMBP even in crowded solution. Thus, in addition to maintaining substrates in an unfolded, export-competent conformation, SecB also suppresses the aggregation of its substrates in the crowded intracellular environment. SecB is also able to promote passive disaggregation of macroscopic aggregates of MBP in the absence of an energy source such as ATP or additional cofactors. These experiments also demonstrate that signal peptide can greatly influence protein stability and aggregation propensity.

Chaperone addiction of toxin–antitoxin systems

“…Whether Tat signal peptide stimulates aggregation of Tat substrates and whether such aggregation is inhibited by dedicated REMPs is not known. In the case of the maltose-binding protein precursor (pre-MBP), a known SecB-dependent substrate in E. coli , it has been shown that the presence of the N-terminal signal peptide kinetically interfere with the folding to mature species, and that pre-MBPs were more prone to aggregation than mature MBPs, thus somehow mirroring the effect of ChAD on the folding of antitoxins, GFP and luciferase4243. Yet, despite the fact that pre-MBP is a bona fide E. coli SecB substrate, its signal peptide is not the primary chaperone-binding site, as it seems to be the case for ChAD/SecB TA .…”

Macromolecular crowding: Macromolecules friend or foe

“…These fragmented fibrils are generally considered more infectious than larger species [94,95]. Furthermore, E. coli preMaltose binding protein was observed to form amyloids during refolding in the presence of Ficoll 70 in contrast to the formation of amorphous aggregates under dilute conditions [97]. Similar to Ficoll, another polymer crowding agent, Dextran has also been reported to accelerate the rate of fibril formation of human apolipoprotein C-II [98] and reduced apo α-LA [99].…”

Effect of Signal Peptide on Stability and Folding of Escherichia coli Thioredoxin

“…We have previously shown that the maltose-binding protein containing its native the N-terminal 26-residue malE signal peptide is substantially less stable and more aggregation prone than the corresponding mature protein [14], [15]. We now explore the effects of two different signal peptides, pelB and malE on protein stability and aggregation in a smaller protein, E. coli thioredoxin.…”

“…Prior to translocation, pre-proteins must be maintained in an export competent conformation in the cytoplasm which is thought to be a loosely folded, protease-sensitive structure [9]. The export competent conformation is maintained by chaperone proteins SecB, GroEL, DnaK, and DnaJ, which also aid in preventing aggregation and improper intramolecular interactions of the exported proteins [10]–[15]. In addition, the presence of non-optimal codons in the signal sequence have been shown to play an important role in export for both SecB and SRP dependent export [16], [17].…”