Intramolecular Interactions

SMFS has been used to study the multiple intra- and intermolecular forces that determine the structure and dynamics and, therefore, the function of protein molecules. In this section we will review the studies on mechanical unfolding and folding. We also include here proteins that occur naturally in complexes and whose mechanical properties have been studied as a whole. Subsequently, we will devote the a) © © © © ©

Fig.8.7. Mechanical refolding of polyubiquitin monitored by force-clamp SMFS. a Protein length vs. time. After unfolding a polyprotein stretch at 122 pN (first staircase pattern), the force is lowered to 15pN and the changes in the polyprotein folds are observed. Afterwards, the force is increased to unfold the polyprotein stretch to check that the protein was completely refolded, b Force-time relationship. Note the fluctuation of the force as the feedback system adjusts the force back to the set value. This follows and marks the unfolding of each module (after Fernandez and Li 2004, with permission, copyright 2004 American Association forthe Advancement of Science)

following section to studies that specifically probe intermolecular forces, and which typically use different methodologies.

Both mechanical and nonmechanical proteins have been studied by SMFS. Figure 8.8 shows their modular and atomic structures. We have summarized the findings of these mechanical analyses in Table 8.1, which includes the values of the WLC adjusting parameters used to fit the family of experimental curves (p and ALc) as well as representative values of mechanical parameters such as Fu (at a particular pulling speed), fc°, and Axu.

Fig. 8.8. Structure of proteins studied by SMFS.a Native proteins: modular structure, b Atomic structures of proteins and protein modules (displayed using the MOLMOL program, Koradi et al. 1996). The application point of the force is indicated by either N or C when the protein was pulled from its termini, or by the name and number of the specific amino acid residue in the one-letter code. 7 Modules from mechanical proteins. 2 Modules from nonmechanical proteins. T4 lysozyme polyprotein was synthesized by forming disulfide bonds between two exogenous cysteine residues. 3 We have classified ubiquitin in a distinct category since its mechanical role as a handle forthe proteasome unfoldase is still hypothetical

Fig. 8.8. Structure of proteins studied by SMFS.a Native proteins: modular structure, b Atomic structures of proteins and protein modules (displayed using the MOLMOL program, Koradi et al. 1996). The application point of the force is indicated by either N or C when the protein was pulled from its termini, or by the name and number of the specific amino acid residue in the one-letter code. 7 Modules from mechanical proteins. 2 Modules from nonmechanical proteins. T4 lysozyme polyprotein was synthesized by forming disulfide bonds between two exogenous cysteine residues. 3 We have classified ubiquitin in a distinct category since its mechanical role as a handle forthe proteasome unfoldase is still hypothetical

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