Forced Unfolding of Periplasmic Binding Proteins (PBPS) Follows Kinetic Partitioning

Abstract

Periplasmic binding proteins (PBPs) are large two-domain proteins that are present in the periplasmic space of Gram-negative bacteria and they mediate the uptake of small ligands, for example, amino acids and sugars, from the surrounding environment. After their synthesis in the cytoplasm, forced unraveling of PBPs is essential for their translocation into the periplasm. Atomic force microscopy based single-molecule force spectroscopy (SMFS) is a versatile technique to study the mechanical unfolding mechanisms of PBPs such as maltose binding protein (MBP), leucine binding protein (LBP), and ribose binding protein (RBP). On mechanical stretching, MBP follows a kinetic partitioning between two-state and three-state unfolding pathways with 38% of molecules taking the two-state pathway. The flux through the two-state pathway further decreases to 21% upon binding to maltose (1). The unfolding mechanism of LBP is much more complex than MBP. LBP also follows two-state pathway and three-state pathways during the mechanical unfolding; the three-state pathways are more diverse in nature suggesting that LBP takes multiple three-state pathways during mechanical unfolding. Leucine binding influences the unfolding flux more towards a two-state pathway, increasing it from 38% to 65% (2). Similar studies on RBP show that it also follows kinetic partitioning during mechanical unfolding but the percentage of molecules taking the two-state pathway is very high (∼85%). These single-molecules studies on PBPs reveal that kinetic partitioning seems to be a general feature for their mechanical unfolding and ligand binding further modulates the unfolding propensity through these pathways.1. Aggarwal V, Kulothungan SR, Balamurali MM, Saranya SR, Varadarajan R, and Ainavarapu SRK, J. Biol. Chem. 2011, 286, pp. 28056-28065.2. Kotamarthi HC, Sharma R, Narayan S, Ray S, and Ainavarapu SRK, J. Am. Chem. Soc.2013, 135, pp. 14768-14774.