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.Small proteins (with fewer than 100 amino acids) are still under the scrutiny of having smooth versus the rough energy landscape; however, for proteins larger than 100 amino acid residues it is generally believed that they follow multiple pathways involving one or many intermediates during protein unfolding (1-4). Contrary to this notion, there exist few exceptions among large proteins, for example, Borrelia burgdorferi VlsE (5), Alteromonas haloplanctis ␣-amylase (6), and maltosebinding protein (MBP) 2 (7), which are shown to exhibit an apparent two-state equilibrium unfolding pathway in bulk solution. Very recent single-molecule pulling studies showed that out-of-equilibrium mechanical unfolding of MBP is very complex and consists of on-pathway sequential intermediates (8, 9). Our earlier studies on MBP have shown the existence of intermediates during the folding pathway of MBP (10, 11). Traditional bulk biophysical techniques like fluorescence, time-resolved unfolding and folding probe ensembles of molecules and hence suffer many drawbacks compared with methods that probe one molecule at a time. Therefore, single-molecule studies on protein folding/unfolding have proved vital in givin...

Identifying Sequential Substrate Binding at the Single-Molecule Level by Enzyme Mechanical Stabilization

“…The force required to unfold the maltose binding protein can be modulated by the introduction of maltose in the solution, 20 which also has consequences on the unfolding pathway. 45 A similar effect was reported in titin kinase, where the introduction of ATP into the solution modifies the number of mechanical intermediates present in force-extension curves. 46 A very sophisticated example of mechanical unfolding modulation is observed in protein G and the Fc fragment of IgG.…”

Single-Molecule Experiments Reveal the Flexibility of a Per-ARNT-Sim Domain and the Kinetic Partitioning in the Unfolding Pathway under Force

“…It allows force to be applied to a protein to trigger the conformational change and quantitatively measure the free-energy landscape along the force direction (23)(24)(25). Single-molecule AFM has revealed rich information on the conformational dynamics of proteins, such as the existence of mechanical unfolding intermediate states (26)(27)(28)(29)(30)(31)(32)(33), the presence of parallel unfolding pathways (34)(35)(36)(37)(38), the structural changes upon ligand binding or protein-protein interactions (39)(40)(41)(42)(43)(44)(45), and the capture of rare misfolding events (46,47), most of which are difficult or impossible to study at the ensemble level. Previous single-molecule AFM studies on photoactive yellow protein (48,49), a representative PAS domain, revealed that the PAS domain is extended by~3 nm and mechanically destabilized bỹ 30% in the light-activated state, indicating partial unfolding of photoactive yellow protein upon photoactivation (48).…”

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

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