Andres F. Oberhauser

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 137 pN. These peaks were ∼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,.

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 ∼7 Å 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 Å, 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 ∼15% of their slack length, and is therefore likely to be an important previously unrecognized component of titin elasticity.

Reverse engineering of the giant muscle protein titin

Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells. The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. Here we use protein engineering and single-molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behaviour of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.

The mechanical stability of ubiquitin is linkage dependent

Ubiquitin chains are formed through the action of a set of enzymes that covalently link ubiquitin either through peptide bonds or through isopeptide bonds between their C terminus and any of four lysine residues. These naturally occurring polyproteins allow one to study the mechanical stability of a protein, when force is applied through different linkages. Here we used single-molecule force spectroscopy techniques to examine the mechanical stability of N-C–linked and Lys48-C–linked ubiquitin chains. We combined these experiments with steered molecular dynamics (SMD) simulations and found that the mechanical stability and unfolding pathway of ubiquitin strongly depend on the linkage through which the mechanical force is applied to the protein. Hence, a protein that is otherwise very stable may be easily unfolded by a relatively weak mechanical force applied through the right linkage. This may be a widespread mechanism in biological systems.

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).

The mechanical hierarchies of fibronectin observed with single-molecule AFM.

Mechanically induced conformational changes in proteins such as fibronectin are thought to regulate the assembly of the extracellular matrix and underlie its elasticity and extensibility. Fibronectin contains a region of tandem repeats of up to 15 type III domains that play critical roles in cell binding and self-assembly. Here, we use single-molecule force spectroscopy to examine the mechanical properties of fibronectin (FN) and its individual FNIII domains. We found that fibronectin is highly extensible due to the unfolding of its FNIII domains. We found that the native FNIII region displays strong mechanical unfolding hierarchies requiring 80 pN of force to unfold the weakest domain and 200 pN for the most stable domain. In an effort to determine the identity of the weakest/strongest domain, we engineered polyproteins composed of an individual domain and measured their mechanical stability by single-protein atomic force microscopy (AFM) techniques. In contrast to chemical and thermal measurements of stability, we found that the tenth FNIII domain is mechanically the weakest and that the first and second FNIII domains are the strongest. Moreover, we found that the first FNIII domain can acquire multiple, partially folded conformations, and that their incidence is modulated strongly by its neighbor FNIII domain. The mechanical hierarchies of fibronectin demonstrated here may be important for the activation of fibrillogenesis and matrix assembly.

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 molecules 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.

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.

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells.

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.

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 β-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.