Mechanical Unfolding of Acylphosphatase Studied by Single-Molecule Force Spectroscopy and MD Simulations

Arad-Haase, Gali; Chuartzman, Silvia; Dagan, Shlomi; Nevo, Reinat; Kouza, Maksim; Khanh, Binh; Nguyên, Hoàng; Li, Mai Suan; Reich, Ziv

doi:10.1016/j.bpj.2010.04.004

Cited by 28 publications

(20 citation statements)

References 68 publications

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“…Our single-molecule experiments on the mechanical unfolding of MBP and preMBP give further evidence to this kinetic partitioning mechanism. In previous studies, ligand binding has been shown to affect the mechanical properties of proteins, for example, dihydrofolate reductase (46) and protein G (47), and acylphosphatase (48). In all of them, the force required to unfold a protein is enhanced upon ligand binding, and this could be explained by an increase in the barrier height.…”

Section: Resultsmentioning

confidence: 99%

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins

Aggarwal

Kulothungan

Balamurali

et al. 2011

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 99%

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins

Aggarwal

Kulothungan

Balamurali

et al. 2011

Journal of Biological Chemistry

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“…The dependence of the shift in the forces on IFN binding confirms that the effects observed are indeed the result of ligand binding. Previous forced unfolding of multi-domain proteins, with and without their ligand, showed either an increase in the force of unfolding [ 43 ] or no change [ 44 , 45 ]. To the best of our knowledge , this is the first experimental demonstration that ligand binding lends a protein more easily mechanically unfolded .…”

Section: Resultsmentioning

confidence: 99%

Binding of interferon reduces the force of unfolding for interferon receptor 1

Chuartzman¹,

Nevo²,

Waichman³

et al. 2017

PLoS ONE

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Differential signaling of the type I interferon receptor (IFNAR) has been correlated with the ability of its subunit, IFNAR1, to differentially recognize a large spectrum of different ligands, which involves intricate conformational re-arrangements of multiple interacting domains. To shed light onto the structural determinants governing ligand recognition, we compared the force-induced unfolding of the IFNAR1 ectodomain when bound to interferon and when free, using the atomic force microscope and steered molecular dynamics simulations. Unexpectedly, we find that IFNAR1 is easier to mechanically unfold when bound to interferon than when free. Analysis of the structures indicated that the origin of the reduction in unfolding forces is a conformational change in IFNAR1 induced by ligand binding.

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“…The results of our simulations demonstrated that regardless of the structural type of protein, there is a switch from thermal to force-driven pathways and the crossover between different force regimes which corresponds to the emergence of an additional energy barrier, analogously to what we observed in our previous studies on mechanical unfolding. 26,27 These results could delve into certain deep and unanswered questions of unfolding and refolding studies. For example, one can use the force dependencies on time to estimate f switch .…”

Section: Introductionmentioning

confidence: 90%

Switch from thermal to force-driven pathways of protein refolding

Kouza

Lan

Gabovich

et al. 2017

The Journal of Chemical Physics

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The impact of the quenched force on protein folding pathways and free energy landscape was studied in detail. Using the coarse-grain Go model, we have obtained the low, middle, and high force regimes for protein refolding under the quenched force. The folding pathways in the low force regime coincide with the thermal ones. A clear switch from thermal folding pathways to force-driven pathways in the middle force regime was observed. The distance between the denatured state and transition state x in the temperature-driven regime is smaller than in the force-driven one. The distance x obtained in the middle force regime is consistent with the available experimental data suggesting that atomic force microscopy experiments deal with the force-regime which is just above the thermal one.

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Mechanical Unfolding of Acylphosphatase Studied by Single-Molecule Force Spectroscopy and MD Simulations

Cited by 28 publications

References 68 publications

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins

Binding of interferon reduces the force of unfolding for interferon receptor 1

Switch from thermal to force-driven pathways of protein refolding

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