Single Molecule Mechanics vs Bulk Biochemical Assays

Until recently, the only way to measure the stability of a protein was to change its physical (temperature or pressure) or chemical environment (using concentrated solutions of guanidinium chloride or urea, acid or alkaline pH) and monitor the loss of protein conformation by spectroscopic techniques in order to determine the change in the Gibbs free energy. Most of these folding studies have been performed using chemical dénaturants acting on untethered proteins (i.e., in solution). However, a considerable number of proteins from the cytoskeleton and the extracellular matrix, as well as unfoldase substrates from chaperone, translocation, and degradation machines, are likely to be subjected to mechanical forces and are tethered, ttus, at least for those proteins, SMFS experiments may more closely mimic the real physiological conditions in which they function in the cell, i.e., typically immobilized and subjected to a mechanical force (in some cases acting as a denaturing agent) that imposes on them a "natural" reaction coordinate (i.e., the length of the protein in the direction of the force to which it is subjected). Protein length is also a well-defined reaction coordinate with a clear physical meaning, in contrast to the less well physically defined kinetic "m values" used in chemical folding experiments (defined as the derivative of the natural logarithm of the folding, or unfolding, rate constant with respect to the dénaturant concentration, they measure the sensitivity of the rate of the process to dénaturant concentration and are generally interpreted as a measure of the change in solvent exposure of the lateral chains of amino acid residues).

ttermodynamic comparisons of the change in free energy (a function of state) between both methods should in principle give identical results, provided that the entropie contribution of tethering the ends of the molecule is properly corrected. On the other hand, we should consider that the kinetics of the reaction depends on the pathway and that SMFS unfolding experiments impose a reaction coordinate to the molecule that is different from that of the bulk (chemical) experiments. Hence, the kinetic parameters of unfolding obtained by both methods may differ, ttus, force acts along a single dimension in specific regions of the protein, typically the N- and

C-termini, while conventional dénaturants have a more global effect. Interestingly, in this survey as a whole there is poor correlation between mechanical and chemical unfolding kinetic rates (a measure of kinetic stability) (Table 8.1). In contrast, a remarkable agreement has been found between the unfolding rate constants of the distal Ig modules of titin (Table 8.1, titin modules 127 and 128; Carrion-Vazquez et al. 1999a; Li et al. 2000b). ttis is noteworthy and raises interesting questions on the mechanical design of these particular domains, tte unfolding pathway of 127 has been analyzed in detail comparing chemical and mechanical methods and using protein engineering based on the O-value (Fowler et al. 2002) and hydrophobic core destabilization analyses (Brockwell et al. 2002; Fowler et al. 2002; Best et al. 2003b; Williams et al. 2003). In this way it was confirmed that the two unfolding pathways are different. It was concluded that the 127 mechanically unfolds through an intermediate which is not populated in the chemical pathway, and that the transition state presents a different structure in both pathways. However, the A'G region is the only region in the 127 structure that is critical in both pathways, being responsible for kinetic stability in both cases, ttis may explain why the two unfolding rates are so close in this module.

tte relationship between mechanical and chemical refolding for the two proteins that have been studied so far by SMFS, i.e., ubiquitin and filamin, has not yet been explored (Fernandez and Li 2004; Schwaiger et al. 2005). Still, both refolding pathways may differ considerably, based on the entropie cost of tethering (Carrion-Vazquez et al. 1999a) and on the fact that the application of force imposes a different reaction coordinate from that of the chemical experiments.

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