Single-Molecule Force Spectroscopy Identifies a Small Cold Shock Protein as Being Mechanically Robust Toni Hoffmann, †,‡,§ Katarzyna M. Tych, †,‡,§ David J. Brockwell, † and Lorna Dougan* ,†,‡ † Astbury Centre for Structural Molecular Biology and ‡ School of Physics and Astronomy, University of Leeds, Leeds, LS2 9JT, United Kingdom ABSTRACT: Single-molecule force spectroscopy has emerged as a powerful approach to examine the stability and dynamics of single proteins. We have completed force extension experiments on the small cold shock protein B from Thermotoga maritima, using a specially constructed chimeric polyprotein. The protein’s simple topology, which is distinct from the mechanically well- characterized β-grasp and immunoglobulin (Ig)-like folds, in addition to the wide range of structural homologues resulting from its ancient origin, provides an attractive model protein for single-molecule force spectroscopy studies. We have determined that the protein has mechanical stability, unfolding at greater than 70 pN at a pulling velocity of 100 nm s -1 . We reveal features of the unfolding energy landscape by measuring the dependence of the mechanical stability on pulling velocity, in combination with Monte Carlo simulations. We show that the cold shock protein has mechanically robust, yet malleable, features that may be important in providing the protein with stability and flexibility to function over a range of environmental conditions. These results provide insights into the relationship between the secondary structure and topology of a protein and its mechanical strength. This lays the foundation for the investigation of the effects of changes in environmental conditions on the mechanical and dynamic properties of cold shock proteins. ■ INTRODUCTION Single-molecule force spectroscopy is a powerful tool to study the conformational dynamics and stability of proteins. 1-5 By completing force-extension experiments using an atomic force microscope (AFM), a protein can be extended and unfolded at a constant velocity, yielding information on the mechanical stability of the protein in terms of the force (F U ) required to unfold it. Such studies have revealed rich information on the mechanical stability of proteins, the presence of intermediate states, and the importance of intramolecular interactions in determining protein mechanical stability. 6 This technique is rapidly advancing, and the number of natural and designed proteins studied in experiments, combined with those characterized by computational modeling, provide a growing data set for the detailed analysis of the mechanical stability of proteins. 7-9 With respect to their mechanical robustness, proteins can be ranked according to their secondary structure content and arrangement, where α-helical proteins generally exhibit lower mechanical stability than those with a high percentage of β- sheet content. The importance of the arrangement of the secondary structure in relation to the direction of the pulling force has also been demonstrated, where the shearing apart of two β-strands requires a greater force than a sequential “unzipping”. 10,11 Further studies have examined side chain packing and long-range interactions in topologically similar proteins, 12 hydrophobic packing in the hydrophobic core of a protein, 13 solvent accessibility of hydrogen bonds, 14 and bond patterns as well as the identification of “strong” and “weak” sequence motifs in protein families. 7,14-18 The family of cold shock proteins (Csps) belongs to a subset of the OB (oligonucleotide/oligosaccharide-binding) class of folds, a protein fold that is found in all three kingdoms of life. 19 The OB-fold is a β-barrel structure formed by five antiparallel β-strands arranged in two β-sheets that in most cases is capped by an α-helix. 19 This α-helix is missing in some cold shock protein domains, which simply contain a Greek-key β-barrel structure. To form the β-barrel, the β-sheets twist and coil to form closed structures in which the first strand is hydrogen- bonded to the last. The existence of Csps in a broad range of organisms, including the most primitive diverging lineages, suggests they may have ancient origin and were present in the earliest forms of life. 20-23 While the expression of many Csps is upregulated by a sudden decrease in temperature, as part of a cold shock response, most cold shock proteins are present under ambient environmental conditions and therefore may have an important role in response to other forms of stress. 21-24 In their proposed function as RNA chaperones, they are involved in the regulation of a number of biological functions by binding to single-stranded RNA and DNA to regulate ribosome trans- lation, mRNA decay, and termination of transcription. 24 Received: October 22, 2012 Revised: December 17, 2012 Published: January 8, 2013 Article pubs.acs.org/JPCB © 2013 American Chemical Society 1819 dx.doi.org/10.1021/jp310442s | J. Phys. Chem. B 2013, 117, 1819-1826