Unraveling the mysteries of chaperone interactions of the myosin head.

Myosin is a ubiquitous protein that produces forces in the cell by converting the chemical energy from ATP hydrolysis into mechanical work. It is responsible for many fundamental biological processes from the small scale (cell motility, cellular trafficking) all the way up to the level of organisms (neuron growth, muscle contraction). Myosin, like most proteins, has a function defined by its folded native structure. It would be logical to assume that proteins evolved to fold to their native structure very efficiently so that the important processes under their control would continue to perform optimally. Surprisingly, for myosin and many other large proteins, this is not the case. The nascent myosin polypeptide chain requires assistance of other proteins, namely the UNC-45 chaperone, to obtain its final, functional structure; however, the details of the interaction between these proteins have remained obscure. In this issue of Biophysical Journal, Bujalowski et al. (1) use both traditional biochemical and biophysical techniques and a novel single-molecule method that indirectly measures chaperone-assisted folding to differentiate the roles of individual domains of the common muscle-specific chaperone UNC-45B in facilitating the folding of type II myosin (hereafter referred to as “UNC-45” and “Myosin”). Their results give more insight into the unique mode of operation of the UNC-45 chaperone and also pave the way for using single-molecule force-spectroscopy techniques to probe chaperone-assisted folding. The chaperone-assisted folding of myosin is absolutely crucial to human health because the phenotypes for the loss of the UNC-45 gene include inhibition of cell proliferation, embryonic lethality, and can cause severe problems to the sarcomere (2). UNC-45 seems to be a specialized chaperone for myosin, although it has been found to recruit generic chaperone proteins like Hsp90 and Hsp70, which may help to facilitate its functionality. The UNC-45 protein consists of three main domains—an N-terminal TPR domain, a central domain, and a C-terminal UCS domain. The TPR domain consists of TPR repeats that mediate binding to the general chaperones Hsp70 and Hsp90. The UCS domain (named because of highly conserved sequences among proteins UNC-45, CROI, and She4P) is known to contain the binding site to myosin. The central domain is important for forming oligomers (3) and is thought to regulate the activity of UNC-45 (2). However, there has been no definitive confirmation and no definitive information about its interactions with myosin and its chaperoning capabilities. Bujalowski et al. now confirms the critical role of the UCS domain in UNC-45 chaperone activity and show unequivocally that the central domain has no chaperoning activity on its own, although it binds with high affinity to the myosin motor domain at a site distinct from that of UCS. It seems that the central domain plays an important role in modulating UNC-45:myosin complex formation through an allosteric effect, but more work is warranted to identify the nature of this allosteric interaction. One of the most exciting results of this report is the use of AFM-based single-molecule force-spectroscopy (4–9) to probe chaperone-assisted mechanical refolding to determine the roles of the UCS and central domains of the UNC-45 chaperone. Kaiser et al. (10) previously developed this approach for the full UNC-45 protein, and here they have adapted it to examine the roles of individual domains of UNC-45. The method uses traditional single-molecule force-spectroscopy—where a protein of interest is covalently attached to a reporter protein (often the I27 immunoglobulin domain), which, in turn, is tethered to a cantilever. An extension pulse is applied to the tethered protein, during which the deflection of the cantilever is measured to determine the force versus extension. This technique can be used to capture refolding—i.e., when the molecule is retracted after extension, allowed to refold, and then another extension pulse is applied to determine whether it has refolded (5). Kaiser et al. (10) cleverly determined that they could use a protein attached to myosin that would be an indirect reporter of refolding. They attached three I27 domains to myosin and discovered that I27 refolding is interrupted when myosin is unfolded (I27 would normally fold by itself with ∼100% probability). In their assay, I27 refolding was only recovered when the UNC-45 chaperone was added to refold myosin, so that myosin’s unfolded residues could no longer interfere with refolding of I27. In this new report, Bujalowski et al. (1) found that they could use specifically the UCS domain of UNC-45 to refold myosin, but not the central domain, providing indirect evidence of the functions of each specific domain. To further clarify the role of UNC-45 in chaperoning the refolding of myosin, Bujalowski et al. (1) used dynamic light scattering, a bulk biophysical technique to look at thermal aggregation of myosin over time. They found that the UCS domain alone is capable of inhibiting aggregation but the central domain by itself cannot. These bulk observations confirmed the AFM single-molecule results and provided the additional information as to the possible mechanism by which UNC-45 helps to fold myosin—by preventing its aggregation. These complementary approaches, namely the combination of bulk and single-molecule methodologies, may be very useful in the future for studies like these. The single-molecule methods have the capability to provide detailed information about the misfolded state of monomeric polypeptides (if probed directly instead of indirectly), and also may be able to give information about the kinetics of chaperone-assisted folding, while bulk methods will help to capture the global aggregation outcome of failed folding events in protein populations. This report opens up a new avenue of interesting possibilities for study that has largely been neglected—the folding of large proteins and their interactions with chaperones. This avenue of research is notoriously difficult, because of technical limitations encountered using traditional techniques. However, these difficulties can be circumvented using single-molecule approaches as shown in this report.