Stability and folding rates of domains spanning the large A-band super-repeat of titin

Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension. A prominent fea ture in the A-band portion of titin is the presence of an 11-domain super-r epeat of immunoglobulin superfamily and fibronectin-type-III-like domains. Seven overlapping constructs from human cardiac titin, each consisting of t wo or three domains and together spanning the entire 11-domain super-repeat , have been expressed in Escherichia coli. Fluorescence unfolding experimen ts and circular dichroism spectroscopy have been used to measure folding st abilities for each of the constructs and to assign unfolding rates for each super-repeat domain. Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II dom ain, suggesting close and possibly rigid association between these units. T he domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiolo gical conditions, correlate with previously reported mechanical forces requ ired to unfold titin domains. Individual domains vary greatly in their rate s of unfolding, with a range of unfolding rate constants between 2.6 X 10(- 6) and 1.2 s(-1). This variation in folding behavior is likely to be an imp ortant determinant in ensuring independent folding of domains in multi-doma in proteins such as titin.