Single-molecule atomic force microscopy (AFM) was used to investigate the mechanical properties of titin, the giant sarcomeric protein of striated muscle. Individual titin molecules were repeatedly stretched, and the applied force was recorded as a function of the elongation. At large extensions, the restoring force exhibited a sawtoothlike pattern, with a periodicity that varied between 25 and 28 nanometers. Measurements of recombinant titin immunoglobulin segments of two different lengths exhibited the same pattern and allowed attribution of the discontinuities to the unfolding of individual immunoglobulin domains. The forces required to unfold individual domains ranged from 150 to 300 piconewtons and depended on the pulling speed. Upon relaxation, refolding of immunoglobulin domains was observed.

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. Individually folded domains are common building blocks of proteins (1, 2). The native state of proteins is the most stable, and therefore, proteins rarely unfold spontaneously. For example, the unfolding of isolated Ig and fibronectin type III domains is a rare event estimated to occur at a rate of 10, whereas refolding is typically much faster, at rates of Ϸ1 to 100 s Ϫ1 (3-6). Hence, unfolding is typically studied by using chemical denaturation, which forces the domains into various degrees of unfolding. By using protein engineering combined with a variety of spectroscopic techniques such as NMR and fluorescence, it is possible to examine the folding of protein domains after chemical denaturation (3, 7-9). These experimental approaches are widely used and give information about the folding free energy, transition state, and folding landscape.The atomic force microscope (AFM) is a simple instrument capable of causing the unfolding of a single protein by controlling its length with Å-scale resolution (10, 11). AFM techniques trigger unfolding by applying force to a single protein, which increases the rate of unfolding exponentially, thus making it easily observable without requiring chemical denaturants (10-12). However, despite these developments, single protein recordings by using AFM have remained limited, because when stretching the whole or part of a multimodular protein, it has been impossible to assign experimental observables to individual domains because of their heterogeneity. Furthermore, it is not known whether mechanical unraveling events represent true unfolding events. This has been a point that investigators have tried to address in previous papers (10, 11). However, it has been possible only to say that the unfolding and refolding rates observed and the stability measured have been ''in the range'' of the results observed for isolated domains of this kind. However, the comparison between mechanical and chemical data has remained uncertain because the range of unfolding and refolding rates of these modules varies by 2 orders of magnitude, and the stability ranges from 2 to 10 kcal͞mol (1 cal ϭ 4.18 J) (3)(4)(5)13).In this work, we use protein engineering to construct tandem repeats of a single protein module and stretch it with AFM to examine its stability and folding kinetics. Tandem repeats are necessary because the mechanical properties of a single module cannot be directly studied by using AFM techniques. A single module will extend only for a short distance and fall into...

The molecular mechanism by which a mechanical stimulus is translated into a chemical response in biological systems is still unclear. We show that mechanical stretching of single cytoplasmic proteins can activate binding of other molecules. We used magnetic tweezers, total internal reflection fluorescence, and atomic force microscopy to investigate the effect of force on the interaction between talin, a protein that links liganded membrane integrins to the cytoskeleton, and vinculin, a focal adhesion protein that is activated by talin binding, leading to reorganization of the cytoskeleton. Application of physiologically relevant forces caused stretching of single talin rods that exposed cryptic binding sites for vinculin. Thus in the talin-vinculin system, molecular mechanotransduction can occur by protein binding after exposure of buried binding sites in the talin-vinculin system. Such protein stretching may be a more general mechanism for force transduction.