Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Aggarwal V

PROVIDER: S-EPMC3151051 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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

Aggarwal Vasudha V Kulothungan S Rajendra SR Balamurali M M MM Saranya S R SR Varadarajan Raghavan R Ainavarapu Sri Rama Koti SR

The Journal of biological chemistry 20110608 32

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 sin ...[more]

PMID: 21659518

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Project description:Two mechanisms, induced fit (IF) and conformational selection (CS), have been proposed to explain ligand recognition coupled conformational changes. The histidine binding protein (HisJ) adopts the CS mechanism, in which a pre-equilibrium is established between the open and the closed states with the ligand binding to the closed state. Despite being structurally similar to HisJ, the maltose binding protein (MBP) adopts the IF mechanism, in which the ligand binds the open state and induces a transition to the closed state. To understand the molecular determinants of this difference, we performed molecular dynamics (MD) simulations of coarse-grained dual structure based models. We find that intra-protein contacts unique to the closed state are sufficient to promote the conformational transition in HisJ, indicating a CS-like mechanism. In contrast, additional ligand-mimicking contacts are required to "induce" the conformational transition in MBP suggesting an IF-like mechanism. In agreement with experiments, destabilizing modifications to two structural features, the spine helix (SH) and the balancing interface (BI), present in MBP but absent in HisJ, reduce the need for ligand-mimicking contacts indicating that SH and BI act as structural restraints that keep MBP in the open state. We introduce an SH like element into HisJ and observe that this can impede the conformational transition increasing the importance of ligand-mimicking contacts. Similarly, simultaneous mutations to BI and SH in MBP reduce the barrier to conformational transitions significantly and promote a CS-like mechanism. Together, our results show that structural restraints present in the protein structure can determine the mechanism of conformational transitions and even simple models that correctly capture such structural features can predict their positions. MD simulations of such models can thus be used, in conjunction with mutational experiments, to regulate protein ligand interactions, and modulate ligand binding affinities.

Dataset Information

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

Publications

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

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