Single-Molecule Studies on PolySUMO Proteins Reveal Their Mechanical Flexibility

Abstract

Proteins with β-sandwich and β-grasp topologies are resistant to mechanical unfolding as shown by single-molecule force spectroscopy studies. Their high mechanical stability has generally been associated with the mechanical clamp geometry present at the termini. However, there is also evidence for the importance of interactions other than the mechanical clamp in providing mechanical stability, which needs to be tested thoroughly. Here, we report the mechanical unfolding properties of ubiquitin-like proteins (SUMO1 and SUMO2) and their comparison with those of ubiquitin. Although ubiquitin and SUMOs have similar size and structural topology, they differ in their sequences and structural contacts, making them ideal candidates to understand the variations in the mechanical stability of a given protein topology. We observe a two-state unfolding pathway for SUMO1 and SUMO2, similar to that of ubiquitin. Nevertheless, the unfolding forces of SUMO1 (∼130 pN) and SUMO2 (∼120 pN) are lower than that of ubiquitin (∼190 pN) at a pulling speed of 400 nm/s, indicating their lower mechanical stability. The mechanical stabilities of SUMO proteins and ubiquitin are well correlated with the number of interresidue contacts present in their structures. From pulling speed-dependent mechanical unfolding experiments and Monte Carlo simulations, we find that the unfolding potential widths of SUMO1 (∼0.51 nm) and SUMO2 (∼0.33 nm) are much larger than that of ubiquitin (∼0.19 nm), indicating that SUMO1 is six times and SUMO2 is three times mechanically more flexible than ubiquitin. These findings might also be important in understanding the functional differences between ubiquitin and SUMOs.

Rapid and Robust Polyprotein Production Facilitates Single-Molecule Mechanical Characterization of β-Barrel Assembly Machinery Polypeptide Transport Associated Domains

“…An inter-residue contact was assumed to be present if the shortest distance between side-chain atoms of different residues was <5 Å, the same cutoff used in a similar study on SUMO proteins. 57 For the six identified R þ β proteins we find that the number of contacts increases from 92 contacts in protein G to 171 in the SUMO1 protein. Interestingly, we find a clear correlation between both Δx U and k U and the number of contacts ( Figure 6B), where the Δx U and k U from each study have been obtained using a similar method to that employed in the present study using EcPOTRA2.…”

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].…”

Direct Observation of Multimer Stabilization in the Mechanical Unfolding Pathway of a Protein Undergoing Oligomerization

“…Interestingly, there is also a high correlation between the number of contacts in a monomer/dimer/tetramer versus its unfolding force ( Figure 3B). This has previously been found to be true for the unfolding forces of very homologous proteins, 58 and in general, it is true for proteins when they are normalized against the number of amino acids (comparing contact density). 59 However, to our knowledge, it has never been demonstrated for multimerization.…”