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 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.

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.

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.

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