Spectrin is widely appreciated as an asymmetric highly flexible molecule that is an essential and central component of the cells cortical cytoskeleton. The flexibility of spectrin has been postulated in several models of the skeleton to be a feature important to its function. Yet, little is known about the structural basis of its flexibility. The only existing crystallographic study of a single spectrin repeat unit demonstrated an unusual dimeric structure that in allowed a reasonable model for a single repeat unit to be deduced. The only existing NMR study, also of a single repeat unit, was consistent with the triple helical repeat unit model. However, neither of these models provides any direct evidence for the structure of the intervening sequences that join each repeat unit together. It is these connecting regions that are thought to mediate spectrin's flexibility. The long-term objective of the present proposal is to understand the molecular mechanism of flexibility of the repeating spectrin unit domain, and that of related proteins. To provide this molecular characterization, a two-part investigation is proposed: 1) To obtain the first X-ray crystal structure of a spectrin peptide consisting of two linked repeating units; and 2) to build a dynamic model of this peptide in solution by endowing the X-ray structure with attributes of conformation and motions identified by fluorescence measurements. To carry out these studies, the crystallization of two closely related form of a two-unit spectrin peptide in two space groups has already been accomplished. One of these crystals diffracts to 3.2. Several mutants have also been cloned with cys introduced at different positions to facilitate the search for heavy atom derivatives. These mutant peptides will also be used to monitor by fluorescent energy transfer and anisotrophy decay experiments the 1) orientation in solution of the two linked units with respect ot each other ; 2) the degree of mobility of each of the helices of each of the bundle and of different regions of a single helix, and 3) the flexibility of the units about the linker region. The results of these studies will further our understanding of role of spectrin, and the role of the linker region in certain hemolytic disorders in which it is altered.
