title: Multiple unfolding pathways of leucine binding protein (LBP) probed by single-molecule force spectroscopy (SMFS).
abstract: Experimental studies on the folding and unfolding of large multi-domain proteins are challenging, given the proteins' complex energy landscapes with multiple intermediates. Here, we report a mechanical unfolding study of a 346-residue, two-domain leucine binding protein (LBP) from the bacterial periplasm. Forced unfolding of LBP is a prerequisite for its translocation across the cytoplasmic membrane into the bacterial periplasm. During the mechanical stretching of LBP, we observe that 38% of the unfolding flux followed a two-state pathway, giving rise to a single unfolding force peak at ~70 pN with an unfolding contour length of 120 nm in constant-velocity single-molecule pulling experiments. The remaining 62% of the unfolding flux followed multiple three-state pathways, with intermediates having unfolding contour lengths in the range ~20-85 nm. These results suggest that the energy landscape of LBP is complex, with multiple intermediate states, and a large fraction of molecules go through intermediate states during the unfolding process. Furthermore, the presence of the ligand leucine increased the unfolding flux through the two-state pathway from 38% to 65%, indicating the influence of ligand binding on the energy landscape. This study suggests that unfolding through parallel pathways might be a general mechanism for the large two-domain proteins that are translocated to the bacterial periplasmic space.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research , Homi Bhabha Road, Colaba, Mumbai 400005, India.
authors.forename: Hema Chandra
authors.initials: HC
authors.lastname: Kotamarthi
authors.forename: Riddhi
authors.initials: R
authors.lastname: Sharma
authors.forename: Satya
authors.initials: S
authors.lastname: Narayan
authors.forename: Sayoni
authors.initials: S
authors.lastname: Ray
authors.forename: Sri Rama Koti
authors.initials: SR
authors.lastname: Ainavarapu
mesh_terms: D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D033902:Periplasmic Binding Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013057:Spectrum Analysis
chemical_list: D029968:Escherichia coli Proteins; C024913:LivK protein, E coli; D033902:Periplasmic Binding Proteins