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Fig. 8.2. Protein mechanics in vivo, a The extracellular matrix protein elastin is an entropic elastomer. After pulling, elastin molecules stretch owing to their entropic elasticity (adapted, with permission, from Alberts et al. 2002, copyright 2002 Taylor & Francis Group), b Adhesion protein mechanics: modular cell adhesion receptors (2) may function as shock absorbers by increasing the range and lifetime of adhesion bonds, whereas nonextensible receptors (7) may display higher bond rupture forces (see text for details). The "protective" unfolding of sacrificial modules (weaker than the adhesive bond) of a modular protein results in an increase in the total work needed for the unbinding process. This mechanical shock-absorber effect increases the range and lifetime of bonds and offers an explanation for why adhesion proteins (whose function is often related to mechanical resistance) are typically modular (adapted from Fisher et al. 2000). c Changes in the elastic protein titin during sarcomere contraction, extension, and overextension. The regions from titin are represented by different colors: fibronectin and fibronectin/immu-noglobulin (Ig) domains anchored to the thick filament (red), PEVKand N2B unique sequences (orange), and Ig domains (proximal and distal to the Z disk; violet); the last two regions are the extensible part oftitin (modified, with permission, from Tskhovrebova and Trinick 2003, copyright 2003 Macmillan Publishers Ltd.).d) Mechanosensitive ion channels. These types of ion channels are present, for instance, in the cilia of the renal epithelial cells and in the streoreocilia of the hair cells in the auditory and vestibular systems. The bending of the cilia opens mechanically gated transduction channels allowing ion influx. The key proteins involved in the mechanics of these channels are still under debate (modified from Gillespie and Walker 2001, with permission, copyright 2001 Macmillan Publishers Ltd.). e Folding-unfolding cell machines: chaperonins. 1 In the bacterial chaperonin GroEL, prior to the folding process, conformational changes increase the relative distance between the substrate binding sites, suggesting a "racklike" model for forced protein unfolding (after Shtilerman et al. 1999, with permission, copyright 1999 American Association for the Advancement of Science). 2 Comparison between protein folding mediated by GroEL-GroES and CCT chaperonines. GroEL-GroES is assumed to perform a "free folding" of protein substrates, while CCT may induce conformational changes that mechanically force the substrate protein to fold in the correct way (after Valpuesta et al. 2002, with permission, copyright 2002 Elsevier Science), f Mitochondrial protein transport. 1 "Pulling model": mitochondrial chaperone mtHsp70 involved in protein translocation functions as a molecular motor that actively pulls to unfold the protein. 2 This chaperone may alternatively act as a Brownian motor taking advantage of spontaneous unfolding events to translocate the protein substrate into the mitochondrion ("trapping model"). It has recently been proposed that these two models could also coexist, each one being just an extreme case of the same process (reviewed by Oster and Wang 2003) (modified from Neupert and Brunner 2002, with permission, copyright 2002 Macmillan Publishers Ltd), g Compartmental proteases: the AAA+ (hexameric ring) ATPase from the proteasome and other related proteases unfold proteins, in an ATP-dependent manner, prior to their translocation to the catalytic chamber for degradation. Force attempt failures would result in the release of the substrate (dotted arrow) (modified from Sauer et al. 2004, with permission, copyright 2004 Elsevier Science), h The Haldane-Pauling model ("strain and distortion") for enzyme-mediated catalysis postulates that mechanical tension would break the links between molecules proteins are located within particular cellular machines such as the cytoskeleton (sarcomeric and cortical), the adhesion machinery located at the cell-cell interface (cell-cell and cell-substrate adhesion proteins), or the mechanosensitive ion channels. Examples of these proteins can be seen in Fig. 8.2a-d.

tte enthalpic group of mechanical proteins seems to have evolved to resist force such that mechanical unfolding is critical for their function. However, mechanical unfolding in a cell may not be limited to proteins with a mechanical function, ttere is increasing evidence to indicate that many "unfoldases" may operate mechanically, i.e., these proteins would actively unfold (or fold) their protein substrates by exerting mechanical forces on them using chemical energy from the hydrolysis of ATP (reviewed by Valpuesta et al. 2002; Matouschek 2003; Prakash and Matouschek 2004; Sauer et al. 2004). ttese unfoldases include molecular chaperones, compart-mental proteases such as proteasomes, and the protein translocases that constitute the protein import machinery of the mitochondria, chloroplasts, and endoplasmic reticulum (examples of these machines can be seen in Fig. 8.2e-g). If this model is confirmed, most proteins in the cell would be mechanically unfolded at some point or another during their lifespan. Ms means that the mechanical properties of many proteins may be critically important. In addition to being generated by these mech-anochemical enzymes, mechanical forces may also underlie the activity of other enzymes, according to the hypothesis of the tension-induced catalysis proposed by Haldane-Pauling (reviewed by Bustamante et al. 2004; Fig. 8.2h). Ms hypothesis, which remains to be demonstrated, postulates that enzyme catalysis may work by inducing mechanical tension on the enzyme-substrate complex.

tte nanomachinery involved in all these processes and the mechanochemistry behind them are still not well understood. Although current technology still does not allow us to directly measure the forces involved in vivo (except for the case of extracellular proteins), much can be learned in vitro by the study of well-defined model systems, while we await the exciting developments to come. In the meantime, a first generation of nanomanipulation techniques allows us to apply mechanical forces to single protein molecules and single interacting proteins and to analyze their responses.

Single-Molecule Techniques

Technical innovation has always been central to scientific progress. Classical biochemical techniques deal with large ensembles of molecules in the range of the Avogadro's number (Na~1023 molecules per mol) and can therefore only provide average parameters from them. In contrast, single-molecule techniques allow the direct study of individual molecules, eliminating the "population noise" and the need for the difficult-to-accomplish synchronization of reactions (a must when analyzing the dynamics of multistep processes in the ensemble). Using these techniques, we can obtain a histogram of the actual distribution of values for an experimental parameter (i.e., the probability distribution function), instead of the population average. One clear advantage of this approach is that it enables us to directly probe the existence of rare intermediates, ttus, single-molecule biophysics permits the dynamics of molecular processes to be analyzed in real time with an unprecedented resolution (reviewed in Leuba and Zlatanova 2001). A pioneer among these techniques was patch-clamping, which enabled the electrical activity of a single ion channel to be recorded directly (Neher and Sakmann 1976).

Single-Molecule Manipulation Techniques

With the recent advent of the so-called single-molecule manipulation techniques, we are now able to manipulate single proteins and study their mechanical properties as well as their function (i.e., the dynamics of protein structure, which typically involves structural changes in their intramolecular -"conformation" - or intermolecular - "interactions" - bonding), tte techniques for single-molecule manipulation include mechanical force transducers such as AFM, biomembrane force probe (also dubbed dynamic force spectroscopy, another form of SMFS), microneedles and optical fibers, as well as external field manipulators like optical tweezers, magnetic tweezers, and the flow-field apparatus, ttese are complementary techniques that cover overlapping ranges of force and that also differ in their spatial resolution and dynamic range (time window) (Fig. 8.1). ttree of these techniques have been used extensively to characterize the mechanical properties of intra- and intermolecular interactions of single proteins: optical tweezers (Ashkin 1970), biomembrane force probe (Evans et al. 1995), and AFM (Binning et al. 1986). While the first two are based on soft springs, the last one is based on stiff springs.

tte biomembrane force probe technique has essentially been used to measure the bond strength of intermolecular interactions in protein-biomolecule pairs (Evans and Ritchie 1999). Optical tweezers have been widely used to study the mechanics of motor proteins (reviewed by Howard 2001, Oster and Wang 2003 and Bustamante et al. 2004). Finally, AFM has been applied mostly to characterize the mechanical resistance of both individual polypeptides (intramolecular interactions) and protein-biomolecule bonds (intermolecular interactions) (Bustamante et al. 2001). With the exception of AFM (several companies now sell apparatuses designed to carry out this technique) most single-molecule manipulation instruments are currently custom-built, they are not commercially available and, therefore, remain inaccessible to the nonspecialist.

Compared to all the other nanomanipulation techniques, the remarkable advantages of AFM are the distance resolution and the precision of positioning, while its bottleneck is the limited resolution in the low-force regime. Furthermore, AFM is particularly suitable for single-molecule studies (whose goal is to address a single molecule or a single molecular pair), since the curvature radius of an AFM tip (see section 8.3.1.1) is much smaller than that of optical or magnetic beads and, therefore, the number of individual molecules (or molecule pairs) that it can hold is orders of magnitude smaller.

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