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...

Individual molecules of the giant protein titin span the A-bands and I-bands that make up striated muscle. The I-band region of titin is responsible for passive elasticity in such muscle, and contains tandem arrays of immunoglobulin domains. One such domain (I27) has been investigated extensively, using dynamic force spectroscopy and simulation. However, the relevance of these studies to the behaviour of the protein under physiological conditions was not established. Force studies reveal a lengthening of I27 without complete unfolding, forming a stable intermediate that has been suggested to be an important component of titin elasticity. To develop a more complete picture of the forced unfolding pathway, we use mutant titins--certain mutations allow the role of the partly unfolded intermediate to be investigated in more depth. Here we show that, under physiological forces, the partly unfolded intermediate does not contribute to mechanical strength. We also propose a unified forced unfolding model of all I27 analogues studied, and conclude that I27 can withstand higher forces in muscle than was predicted previously.

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. Awide variety of proteins are placed under mechanical stress during cell adhesive interactions (1-4) and in muscle contraction (5-9). A remarkable feature of these proteins is their tandem modular construction. For example, the giant muscle protein titin is composed of several hundred Ig and fibronectin type III (FnIII) domains placed in tandem (10). These modules show low sequence homology among themselves [20-30% identity, 30-40% similarity between Ig modules in human skeletal titin (11)] and widely different thermodynamic stability (12) (2.55 to 7.36 kcal⅐mol Ϫ1). The key residues of individual Ig repeat across different species, and the superrepeat patterns of avian and mammalian titins are highly conserved through evolution (11) (82.5% similarity between human and the reptile sequences), suggesting that their particular ordering is important for the elasticity of the protein. The ordering of modules is particularly striking in the protein projectin, a titin-like protein found in the muscles of invertebrates (13). Most of the projectin protein is arranged in a repeating pattern of FnIII-FnIII-Ig domains. Although the significance of these modular arrangements is unknown, it is likely that these patterns form mechanical units that determine the elasticity of the whole protein. If this view is true, it implies that, in contrast to most other proteins, the functional characteristics of elastic proteins are obtained by the summation of the mechanical strength of its modular units. To examine this hypothesis, we use protein engineering and single molecule atomic force microscopy (AFM) techniques (3, 6, 9) to construct simple tandem modular proteins and study their mechanical properties. For our studies, we have chosen to use the I27 and I28 Ig modules of human cardiac titin. These modules offer the advantage that they have been studied in detail by using NMR, steered molecular dynamics, and AFM techniques, and their thermodynamic properties are well established (9,12,(14)(15)(16)(17). Furthermore, because the I28 module is significantly less stable than I27 (⌬G DϪN ϭ 3.0 kcal⅐mol Ϫ1 for I28 vs. 7.6 kcal⅐mol Ϫ1 for I27), we expected that these modules would show very different mechanical properties, and that these could be readily identified by AF...

No abstract