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.

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

“…Leucine binding protein unfolds following a two-state pathway in the presence of leucine, but in the absence of substrate the unfolding become more complex and a multiple three-state pathway predominates. 30 Also, the phosphatidyl- myo -inositol mannosyltransferase A, shows a heterogeneous unfolding pathway with multiple steps. 48 In our case, TlGK is an enzyme of 467 residues with two domains, which include several β-strands, thus the presence of multiple intermediates is highly probable.…”

“…20-27 Substrates and ligands affect the energy landscape of different structural segments of a protein and in many cases increase the mechanical stability of the protein as a consequence of the favorable binding interactions. 28-30 …”

Characterization of Unfolding Mechanism of Human Lamin A Ig Fold by Single-Molecule Force Spectroscopy—Implications in EDMD

“…In singlemolecule force versus extension (F-X) traces I27 serves as a marker protein, whose mechanical properties have been characterized previously. 38,53,54 The unfolding force for the Ig domain was observed to be significantly lower (56 ± 29 pN) than that of I27 (200 ± 30 pN) at 1000 nm/s pulling velocity ( Figure 2B and 2C), although both belong to immunoglobulinlike family consisting of nine beta strands packed in betasandwich fold. This could possibly be ascribed to the favorable unzipping geometry of the terminal β-strands of lamin Ig domain in contrast to shearing geometry of I27 ( Figure 6).…”

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

“…Single-molecule force spectroscopy is a commonly used tool for the mechanical characterization of polymers and biological molecules, such as proteins and DNA [1][2][3][4][5][6][7][8][9][10], and ligand-protein interactions [11][12][13][14][15][16][17][18][19]. These experiments can be performed using a variety of experimental setups, including optical tweezers [3,20], the atomic force microscope (AFM) [1,4], magnetic tweezers [21,22], and the biomembrane force probe [23].…”

“…A recent review provides details of studies that have employed the Zhurkov-Bell model to extract parameters of the unfolding energy landscape of a protein from experimental data on polyproteins [43]. In addition, since that review, 12 additional studies have used the Zhurkov-Bell model to extract information from force spectroscopy experiments [15,[44][45][46][47][48][49][50][51][52][53][54]. Given the prevalence of the Zhurkov-Bell model in force spectroscopy experiments, it is interesting to examine its application and accuracy in more detail.…”