Experimental and Simulation Studies on the Mechanical Properties of Sumo Proteins

Abstract

Protein structure plays an important role in determining its mechanical stability. In order to understand the role of amino acid sequence on the mechanical stability, mechanical unfolding experiments were performed using atomic force microscope based single-molecule force spectroscopy (SMFS) on structurally homologous proteins - ubiquitin, SUMO1, and SUMO2. SUMOs are found to be mechanically weaker than ubiquitin, in spite of their high structural homology, when pulled along N-C direction [1]. The unfolding forces of these proteins follow the same trend as the number of sidechain contacts within them. The energy landscape parameters reveal that SUMOs are more flexible than ubiquitin. We have performed steered molecular dynamics simulations on these proteins to gain atomistic insights into their unfolding pathways. The hydrogen bonds in the mechanical clamp between the terminal β-strands of these proteins are ruptured at the transtion state and proteins unfolded in an all-or-none fashion. The interactions between the β2 strand and the α-helix also weaken at the transition state. We examine the different types of inter-resiude interactions in these proteins to rationalize the lower mechanical stability of SUMOs relative to ubiquitin. Role of water penetration during mechanical unfolding was also investigated.We have further studied the effect of ligand binding on the mechanical stability of SUMO proteins using SMFS in presence of short peptides derived from the binding motifs of SUMO targets. The unfolding force of SUMO1 increased from ∼130pN to ∼170pN upon ligand binding. Dependence of the unfolding force on the pulling speed was higher for SUMO1 bound to the peptides than the unbound protein. The flexibility of SUMOs decreased upon ligand binding suggesting a possible role of SUMO flexibility in SUMOylation.[1] Kotamarthi HC, Sharma R, Ainavarapu SRK, Biophys. J (2013), 104, p2273.