Ligand-modulated parallel mechanical unfolding pathways of maltose-binding proteins.

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.

Mimosine mitigates oxidative stress in selenium deficient seedlings of Vigna radiata--Part I: Restoration of mitochondrial function.

Mimosine, a non-protein plant amino acid found in Mimosa pudica and certain species of Leucaena, was beneficial for the growth of seedlings of Vigna radiata germinated under selenium-deficient stressed condition (-Se stressed) despite the recognized toxicity of the allelochemical. Exposure of mimosine at 0.1 mM (Mim-0.1) promoted the growth of the seedlings and significantly enhanced mitochondrial functional efficiency. Growth-related parameters including root and shoot lengths and dry weight were increased by 44-58% in the Mim-0.1 group compared to that of the -Se-stressed group. Oxygen uptake by mitochondria of Mim-0.1 group, studied with different substrates, revealed enhanced State 3 respiratory rates with regulated State 4 rates, resulting in high respiratory control ratio (RCR) of 3.4 to 3.9 indicative of a high degree of oxidative coupling. Specific activities of mitochondrial electron transport enzymes, nicotinamide adenine dinucleotide (reduced form) (NADH)-cytochrome (cyt) c oxidoreductase, succinate dehydrogenase, and cyt c oxidase in the Mim-0.1 group were enhanced by 53% to threefold over those of the Se-stressed group. Marked decreases in the extent of mitochondrial lipid peroxidation ensued upon mimosine exposure, indicative of its antioxidant function. Mitochondrial 45Ca2+ uptake was notably augmented twofold in the Mim-0.1 group, compared to the Se-stressed group. Detailed kinetic analyses of Ca2+ uptake revealed positive cooperative interactions in both -Se-stressed group and Mim-0.1 groups with Hill coefficient (nH) values of 1.7 and 2, respectively. The present study establishes the beneficial effects of mimosine exposure at 0.1 mM on the growth and mitochondrial function of the seedlings grown under selenium-deficient stressed condition and a significant physiological role can be ascribed to mimosine.

Effect of signal peptide on stability and folding of Escherichia coli thioredoxin.

The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

Effect of crowding agents, signal peptide, and chaperone SecB on the folding and aggregation of E. coli maltose binding protein.

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.

SecB-mediated protein export need not occur via kinetic partitioning.

In Escherichia coli, the cytosolic chaperone SecB is responsible for the selective entry of a subset of precursor proteins into the Sec pathway. In vitro, SecB binds to a variety of unfolded substrates without apparent sequence specificity, but not native proteins. Selectivity has therefore been suggested to occur by kinetic partitioning of substrates between protein folding and SecB association. Evidence for kinetic partitioning is based on earlier observations that SecB blocks the refolding of the precursor form of maltose-binding protein (preMBP)(5) and slow-folding maltose-binding protein (MBP) mutants, but not faster-folding mature wild-type MBP. In order to quantitatively validate the kinetic partitioning model, we have independently measured each of the rate constants involved in the interaction of SecB with refolding preMBP (a physiological substrate of SecB) and mature MBP. The measured rate constants correctly predict substrate folding kinetics over a wide range of SecB, MBP, and preMBP concentrations. Analysis of the data reveals that, for many substrates, kinetic partitioning is unlikely to be responsible for SecB-mediated protein export. Instead, the ability of SecB-bound substrates to continue folding while bound to SecB and their ability to interact with other components of the secretory machinery such as SecA may be key opposing determinants that inhibit and promote protein export, respectively.