Mechanical unfolding of spectrin reveals a super-exponential dependence of unfolding rate on force.

We investigated the mechanical unfolding of single spectrin molecules over a broad range of loading rates and thus unfolding forces by combining magnetic tweezers with atomic force microscopy. We find that the mean unfolding force increases logarithmically with loading rate at low loading rates, but the increase slows at loading rates above 1pN/s. This behavior indicates an unfolding rate that increases exponentially with the applied force at low forces, as expected on the basis of one-dimensional models of protein unfolding. At higher forces, however, the increase of the unfolding rate with the force becomes faster than exponential, which may indicate anti-Hammond behavior where the structures of the folded and transition states become more different as their free energies become more similar. Such behavior is rarely observed and can be explained by either a change in the unfolding pathway or as a reflection of a multidimensional energy landscape of proteins under force.

Multiple conformations of PEVK proteins detected by single-molecule techniques.

An important component of muscle elasticity is the PEVK region of titin, so named because of the preponderance of these amino acids. However, the PEVK region, similar to other elastomeric proteins, is thought to form a random coil and therefore its structure cannot be determined by standard techniques. Here we combine single-molecule electron microscopy and atomic force microscopy to examine the conformations of the human cardiac titin PEVK region. In contrast to a simple random coil, we have found that cardiac PEVK shows a wide range of elastic conformations with end-to-end distances ranging from 9 to 24 nm and persistence lengths from 0.4 to 2.5 nm. Individual PEVK molecules retained their distinctive elastic conformations through many stretch-relaxation cycles, consistent with the view that these PEVK conformers cannot be interconverted by force. The multiple elastic conformations of cardiac PEVK may result from varying degrees of proline isomerization. The single-molecule techniques demonstrated here may help elucidate the conformation of other proteins that lack a well-defined structure.

Stepwise unfolding of titin under force-clamp atomic force microscopy.

Here we demonstrate the implementation of a single-molecule force clamp adapted for use with an atomic force microscope. We show that under force-clamp conditions, an engineered titin protein elongates in steps because of the unfolding of its modules and that the waiting times to unfold are exponentially distributed. Force-clamp measurements directly measure the force dependence of the unfolding probability and readily captures the different mechanical stability of the I27 and I28 modules of human cardiac titin. Force-clamp spectroscopy promises to be a direct way to probe the mechanical stability of elastic proteins such as those found in muscle, the extracellular matrix, and cell adhesion.

Point mutations alter the mechanical stability of immunoglobulin modules.

Immunoglobulin-like modules are common components of proteins that play mechanical roles in cells such as muscle elasticity and cell adhesion. Mutations in these proteins may affect their mechanical stability and thus may compromise their function. Using single molecule atomic force microscopy (AFM) and protein engineering, we demonstrate that point mutations in two beta-strands of an immunoglobulin module in human cardiac titin alter the mechanical stability of the protein, resulting in mechanical phenotypes. Our results demonstrate a previously unrecognized class of phenotypes that may be common in cell adhesion and muscle proteins.

Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.

Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).

Atomic force microscopy captures quantized plastic deformation in gold nanowires.

Scanning probe microscopy has become a powerful tool to detect structural changes in small clusters of atoms. Herein, we use an atomic force microscope to measure the length of gold nanowire structures during extension and compression cycles. We have found that nanowires elongate under force in quantized steps of up to three integer multiples of 1.76 A and that they shorten spontaneously in steps of 1.52 A. Our results can be explained by the sliding of crystal planes within the gold nanowires creating stacking faults that change the local structure from face-centered cubic to hexagonal close packed. Our data also show that there can be up to three simultaneous slip events, in good agreement with the tetrahedral arrangement of slip planes in a gold crystal. These experiments provide direct evidence for the mechanism underlying the plastic deformation of a nanowire. A similar approach can be used to examine the atomic events underlying the plastic failure of other metals and their alloys.

Atomic force microscopy reveals the mechanical design of a modular protein.

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering.

Single protein misfolding events captured by atomic force microscopy.

Using single protein atomic force microscopy (AFM) techniques we demonstrate that after repeated mechanical extension/relaxation cycles, tandem modular proteins can misfold into a structure formed by two neighboring modules. The misfolding is fully reversible and alters the mechanical topology of the modules while it is about as stable as the original fold. Our results show that modular proteins can assume a novel misfolded state and demonstrate that AFM is able to capture, in real time, rare misfolding events at the level of a single protein.

Mechanical unfolding intermediates in titin modules.

The modular protein titin, which is responsible for the passive elasticity of muscle, is subjected to stretching forces. Previous work on the experimental elongation of single titin molecules has suggested that force causes consecutive unfolding of each domain in an all-or-none fashion. To avoid problems associated with the heterogeneity of the modular, naturally occurring titin, we engineered single proteins to have multiple copies of single immunoglobulin domains of human cardiac titin. Here we report the elongation of these molecules using the atomic force microscope. We find an abrupt extension of each domain by approximately 7 A before the first unfolding event. This fast initial extension before a full unfolding event produces a reversible 'unfolding intermediate' Steered molecular dynamics simulations show that the rupture of a pair of hydrogen bonds near the amino terminus of the protein domain causes an extension of about 6 A, which is in good agreement with our observations. Disruption of these hydrogen bonds by site-directed mutagenesis eliminates the unfolding intermediate. The unfolding intermediate extends titin domains by approximately 15% of their slack length, and is therefore likely to be an important previously unrecognized component of titin elasticity.

The micro-mechanics of single molecules studied with atomic force microscopy.

The atomic force microscope (AFM) in its force-measuring mode is capable of effecting displacements on an angstrom scale (10 A = 1 nm) and measuring forces of a few piconewtons. Recent experiments have applied AFM techniques to study the mechanical properties of single biological polymers. These properties contribute to the function of many proteins exposed to mechanical strain, including components of the extracellular matrix (ECM). The force-bearing proteins of the ECM typically contain multiple tandem repeats of independently folded domains, a common feature of proteins with structural and mechanical roles. Polysaccharide moieties of adhesion glycoproteins such as the selectins are also subject to strain. Force-induced extension of both types of molecules with the AFM results in conformational changes that could contribute to their mechanical function. The force-extension curve for amylose exhibits a transition in elasticity caused by the conversion of its glucopyranose rings from the chair to the boat conformation. Extension of multi-domain proteins causes sequential unraveling of domains, resulting in a force-extension curve displaying a saw tooth pattern of peaks. The engineering of multimeric proteins consisting of repeats of identical domains has allowed detailed analysis of the mechanical properties of single protein domains. Repetitive extension and relaxation has enabled direct measurement of rates of domain unfolding and refolding. The combination of site-directed mutagenesis with AFM can be used to elucidate the amino acid sequences that determine mechanical stability. The AFM thus offers a novel way to explore the mechanical functions of proteins and will be a useful tool for studying the micro-mechanics of exocytosis.

Atomic force microscopy captures length phenotypes in single proteins.

We use single-protein atomic force microscopy techniques to detect length phenotypes in an Ig module. To gain amino acid resolution, we amplify the mechanical features of a single module by engineering polyproteins composed of up to 12 identical repeats. We show that on mechanical unfolding, mutant polyproteins containing five extra glycine residues added to the folded core of the module extend 20 A per module farther than the wild-type polyproteins. By contrast, similar insertions near the N or C termini have no effect. Hence, our atomic force microscopy measurements readily discriminate the location of the insert and measure its size with a resolution similar to that of NMR and x-ray crystallography.

The study of protein mechanics with the atomic force microscope.

The unfolding and folding of single protein molecules can be studied with an atomic force microscope (AFM). Many proteins with mechanical functions contain multiple, individually folded domains with similar structures. Protein engineering techniques have enabled the construction and expression of recombinant proteins that contain multiple copies of identical domains. Thus, the AFM in combination with protein engineering has enabled the kinetic analysis of the force-induced unfolding and refolding of individual domains as well as the study of the determinants of mechanical stability.

Atomic levers control pyranose ring conformations.

Atomic force microscope manipulations of single polysaccharide molecules have recently expanded conformational chemistry to include force-driven transitions between the chair and boat conformers of the pyranose ring structure. We now expand these observations to include chair inversion, a common phenomenon in the conformational chemistry of six-membered ring molecules. We demonstrate that by stretching single pectin molecules (1 --> 4-linked alpha-D-galactouronic acid polymer), we could change the pyranose ring conformation from a chair to a boat and then to an inverted chair in a clearly resolved two-step conversion: 4C1 right arrow over left arrow boat right arrow over left arrow 1C4. The two-step extension of the distance between the glycosidic oxygen atoms O1 and O4 determined by atomic force microscope manipulations is corroborated by ab initio calculations of the increase in length of the residue vector O1O4 on chair inversion. We postulate that this conformational change results from the torque generated by the glycosidic bonds when a force is applied to the pectin molecule. Hence, the glycosidic bonds act as mechanical levers, driving the conformational transitions of the pyranose ring. When the glycosidic bonds are equatorial (e), the torque is zero, causing no conformational change. However, when the glycosidic bond is axial (a), torque is generated, causing a rotation around C---C bonds and a conformational change. This hypothesis readily predicts the number of transitions observed in pyranose monomers with 1a-4a linkages (two), 1a-4e (one), and 1e-4e (none). Our results demonstrate single-molecule mechanochemistry with the capability of resolving complex conformational transitions.

Mechanical and chemical unfolding of a single protein: a comparison.

Is the mechanical unraveling of protein domains by atomic force microscopy (AFM) just a technological feat or a true measurement of their unfolding? By engineering a protein made of tandem repeats of identical Ig modules, we were able to get explicit AFM data on the unfolding rate of a single protein domain that can be accurately extrapolated to zero force. We compare this with chemical unfolding rates for untethered modules extrapolated to 0 M denaturant. The unfolding rates obtained by the two methods are the same. Furthermore, the transition state for unfolding appears at the same position on the folding pathway when assessed by either method. These results indicate that mechanical unfolding of a single protein by AFM does indeed reflect the same event that is observed in traditional unfolding experiments. The way is now open for the extensive use of AFM to measure folding reactions at the single-molecule level. Single-molecule AFM recordings have the added advantage that they define the reaction coordinate and expose rare unfolding events that cannot be observed in the absence of chemical denaturants.

The molecular elasticity of the extracellular matrix protein tenascin.

Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were approximately 25 nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain.

Polysaccharide elasticity governed by chair-boat transitions of the glucopyranose ring.

Many common, biologically important polysaccharides contain pyranose rings made of five carbon atoms and one oxygen atom. They occur in a variety of cellular structures, where they are often subjected to considerable tensile stress. The polysaccharides are thought to respond to this stress by elastic deformation, but the underlying molecular rearrangements allowing such a response remain poorly understood. It is typically assumed, however, that the pyranose ring structure is inelastic and locked into a chair-like conformation. Here we describe single-molecule force measurements on individual polysaccharides that identify the pyranose rings as the structural unit controlling the molecule's elasticity. In particular, we find that the enthalpic component of the polymer elasticity of amylose, dextran and pullulan is eliminated once their pyranose rings are cleaved. We interpret these observations as indicating that the elasticity of the three polysaccharides results from a force-induced elongation of the ring structure and a final transition from a chair-like to a boat-like conformation. We expect that the force-induced deformation of pyranose rings reported here plays an important role in accommodating mechanical stresses and modulating ligand binding in biological systems.

A fusion pore phenotype in mast cells of the ruby-eye mouse.

Using patch-clamp capacitance and amperometric techniques, we have identified an exocytotic phenotype that affects the function of the fusion pore, the molecular structure that connects the lumen of a secretory vesicle with the extracellular environment during exocytosis. Direct observation of individual exocytotic events in mast cells from the ruby-eye mouse (ru/ru) showed a 3-fold increase in the fraction and duration of transient fusion events with respect to wild-type mice. The fraction of the total fusion events that were transient increased from 0.22 +/- 0.02 (wild type) to 0.65 +/- 0.02 (ru/ru), and the average duration of these events increased from 418 +/- 32 ms (wild type) to 1207 +/- 89 ms (ru/ru). We also show that this phenotype can reduce and delay an evoked secretory response by causing the fusion of vesicles that have been previously emptied by repeated cycles of transient fusion. The exocytotic phenotype that we describe here may be a cause of diseases like platelet storage pool deficiency and prolonged bleeding times for which the ruby-eye mouse serves as an animal model. Furthermore, the identification of the gene causing the fusion pore phenotype reported here will illuminate the molecular mechanisms regulating exocytotic fusion.

Simultaneous capacitance and amperometric measurements of exocytosis: a comparison.

We measured the exocytotic response induced by flash photolysis of caged compounds in isolated mast cells and chromaffin cells. Vesicle fusion was measured by monitoring the cell membrane capacitance. The release of vesicular contents was followed by amperometry. In response to a GTP gamma S stimulus we found that the time integral of the amperometric current could be superimposed on the capacitance trace. This shows that the integrated amperometric signal provides an alternative method of measuring the extent and kinetics of the secretory response. Very different results were obtained when photolysis of caged Ca2+ (DM-nitrophen) was used to stimulate secretion. In mast cells, there was an immediate, graded increase in membrane capacitance that was followed by step increases (indicative of granule fusion). During the initial phase of the capacitance increases, no release of oxidizable secretory products was detected. In chromaffin cells we also observed a considerable delay between increases in capacitance, triggered by uncaging Ca2+, and the release of oxidizable secretory products. Here we demonstrate that there can be large increases in the membrane capacitance of a secretory cell, triggered by flash photolysis of DM-nitrophen, which indicate events that are not due to the fusion of granules containing oxidizable substances. These results show that increases in capacitance that are not resolved as steps cannot be readily interpreted as secretory events unless they are confirmed independently.