Under physiological conditions, protein oxidation and misfolding occur with very low probability and on long times scales. Single molecule techniques provide the ability to distinguish between properly folded and damaged proteins that are otherwise masked in ensemble measurements. However, at physiological conditions these rare events occur with a time constant of several hours, inaccessible to current single molecule approaches. Here we present a magnetic tweezers based technique that allows, for the first time, the study of folding of single proteins during week-long experiments. This technique combines HaloTag anchoring, sub-micron positioning of magnets, and an active correction of the focal drift. Using this technique and protein L as a molecular template we generate a magnet-law by correlating the distance between the magnet and the measuring paramagnetic bead with unfolding/folding steps. We demonstrate that using this magnet law, we can accurately measure the dynamics of proteins over a wide range of forces, with minimal dispersion from bead to bead. We also show that the force calibration remains invariant over week-long experiments applied to the same single proteins. The approach demonstrated in this article opens new exciting ways to examine proteins on the “human” time scale and establishes magnetic tweezers as a valuable technique to study low probability events that occur during protein folding under force.

Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker intermediates that recover strength with time or ligand binding. Based on the high force extensibility, CnaA-containing pili can dissipate ∼28-fold as much energy compared with their inextensible counterparts before reaching forces sufficient to cleave covalent bonds. We propose that efficient mechanical energy dissipation is key for sustained bacterial attachment against mechanical perturbations.bacterial adhesion | mechanical stability | single-molecule force spectroscopy | Gram-positive pili | isopeptide bond B acterial infections of solid tissues begin with the attachment of bacteria to target surfaces. In many instances, bacteria adhere against forces that oppose such attachment: micturition in the genitourinary tract (1) or mucociliary flow in the respiratory tract (2), for example. In such environments, a completely immobile adherent bacterium experiences a drag force that can be approximated by Stokes law, F = 6·π·r·η·v, where r is the Stokes radius of the bacterium (∼0.5 μm), η is the viscosity of the fluid (in the respiratory mucus, 1-100 Pa·s −1 ) (3), and v is the velocity of the fluid surrounding the bacterium (Fig. 1A). Under normal mucociliary flow (1-100 μm·s −1 ) (4), forces on a single bacterium can exceed several nanonewtons. Such high forces are sufficient to cleave covalent bonds within the initial adherence structures (5), which would terminate attachment. Understanding how bacteria manage to remain attached under such strong mechanical perturbations is of fundamental interest and could identify new targets for antibiotic development.The initial interaction between bacteria and the host is mediated by micrometer-long adhesive structures termed pili or fimbriae (Fig. 1A). Due to their adhesive role, pili are virulence factors that contribute to the development of infections (6). Structurally, pili are polymers of tens to hundreds of subunits, termed shaft pilins, that are assembled in series and are presented at the extracellular surface, often with inclusion of minor pilins that can have adhesive properties (6). Remar...

A hallmark of tissue ageing is the irreversible oxidative modifications of its constituent proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded loses completely its ability to contract by folding, becoming a labile polymer. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week-long experiments show greatly reduced rates of ageing. We demonstrate a novel assay where protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.

Bacteria must withstand large mechanical shear forces when adhering to and colonizing hosts. Recent structural studies on a class of Gram-positive bacterial adhesins have revealed an intramolecular Cys-Gln thioester bond that can react with surfaceassociated ligands to covalently anchor to host surfaces. Two other examples of such internal thioester bonds occur in certain anti-proteases and in the immune complement system, both of which react with the ligand only after the thioester bond is exposed by a proteolytic cleavage. We hypothesized that mechanical forces in bacterial adhesion could regulate thioester reactivity to ligand analogously to such proteolytic gating. Studying the pilus tip adhesin Spy0125 of Streptococcus pyogenes, we developed a single molecule assay to unambiguously resolve the state of the thioester bond. We found that when Spy0125 was in a folded state, its thioester bond could be cleaved with the small-molecule nucleophiles methylamine and histamine, but when Spy0125 was mechanically unfolded and subjected to forces of 50 -350 piconewtons, thioester cleavage was no longer observed. For folded Spy0125 without mechanical force exposure, thioester cleavage was in equilibrium with spontaneous thioester reformation, which occurred with a half-life of several minutes. Functionally, this equilibrium reactivity allows thioester-containing adhesins to sample potential substrates without irreversible cleavage and inactivation. We propose that such reversible thioester reactivity would circumvent potential soluble inhibitors, such as histamine released at sites of inflammation, and allow the bacterial adhesin to selectively associate with surface-bound ligands.Thioester bonds are ubiquitous in biology and are commonly employed as reactive intermediates in metabolic pathways, including ubiquitinylation (1), fatty acid synthesis (2), and nonribosomal peptide synthesis (3). In addition, there are two rare but notable examples of intramolecular thioester bonds that form between Cys and Gln/Glu side chains: in the ␣2-macroglobulin (A2M) 2 anti-proteases and in the C3 and C4 proteins of the immune complement system (4 -6). In both cases, the thioester bond functions as the electrophilic substrate to draw a nucleophilic ligand and create an intermolecular covalent bond with the target (7). Moreover, both A2Ms and the immune complement proteins utilize a proteolytic gating mechanism to regulate thioester reactivity; the thioester bond is protected and buried in the hydrophobic core until a specific proteolytic cleavage unmasks the bond to react with local nucleophiles (7). For example, the A2M anti-protease acts through a "Venus flytrap mechanism" whereby a bait region of A2M attracts a protease that subsequently cleaves A2M, exposing the thioester to irreversibly react with and inactivate the protease (4, 6). For C3, proteolysis occurs at target surfaces by upstream effectors of the immune complement pathway. Once cleaved, C3 readily reacts with ligands on the microbial cell with a half-life of microsecond...

Gram-positive bacteria colonize mucosal tissues against large mechanical perturbations, such as coughing, which generate shear forces that exceed the ability of non-covalent bonds to remain attached. To overcome these challenges, the pathogen Streptococcus pyogenes utilizes the protein Cpa, a pilus tip-end adhesin equipped with a Cys-Gln thioester bond. The reactivity of this bond towards host surface ligands enables covalent anchoring; however, colonization also requires cell migration and spreading over surfaces. The molecular mechanisms underlying these seemingly incompatible requirements remain unknown. Here, we demonstrate a magnetic tweezers force spectroscopy assay that resolves the dynamics of Cpa thioester bond under force. While folded at forces < 6 pN, Cpa thioester bond reacts reversibly with amine ligands, that are a common occurrence in inflammation sites; however, mechanical unfolding and exposure to forces > 6 pN block thioester reformation. We hypothesize that this folding-coupled reactivity switch—“smart covalent bond”—could allow the adhesin to undergo binding and unbinding to surface ligands under low force and remain covalently attached under mechanical stress.