Erythroid and non-erythroid alpha spectrin model system studies on stability and function.

摘要

Spectrin is one of the important structural proteins that line the inner surface of the membrane-associated (cyto)skeleton. The functional form of spectrin is tetrameric. High sequence homology is considered to imply similar structure and hence function in proteins. Intact erythroid and non-erythroid isoforms of alpha spectrin (Spalpha) exhibit ∼56% sequence homology. Yet, the affinity with which non-erythroid alpha spectrin (SpalphaII) binds to beta spectrin (SpbetaI) in order to form tetramers is higher than those exhibited by erythroid alpha spectrin (SpalphaI). In this study, recombinant, model functional peptides of SpalphaI and SpalphaII that mimick the tetramerization process have been used. The conformational stability of SpalphaI and SpalphaII fragments and their association with SpbetaI were investigated. Several SpalphaII mutants were also used to determine the effect of mutations at the functional site on stability and association with SpbetaI.;We used chemical and heat-induced denaturation methods to determine the stability of the native SpalphaI and SpalphaII. The recombinant peptides with the tetramerization region followed by one complete structural domain (SpalphaI-1 and SpalphaII-1) or three complete structural domains (SpalphaI-3 and SpalphaII-3) were used and their conformational stability is reported for the first time. SpalphaI-1 and SpalphaI-3 exhibited similar stabilities with urea concentration of 3.2 M and 3.8 M to achieve 50% unfolding ([U] m). Similarly, SpalphaII-1 and SpalphaII-3 exhibited similar stabilities with [U]m of 6.4 M and 6.6 M respectively. SpalphaII peptides exhibited two times higher conformational stability, in terms of denaturant concentration than the corresponding SpalphaI peptides. This result parallels the higher affinity exhibited by SpalphaII than SpalphaI to bind the same SpbetaI (mimicking the function of tetramerization).;We suggest that the higher stability and affinity exhibited by SpalphaII peptides is a coincidence. Though the few mutants of SpalphaII-3 (SpalphaII-3Delta) exhibited similar conformational stability as the native, they exhibited varied binding affinities. This proves that local conformation of the binding site (Helix C') is an important factor, which modulates tetramer formation in spectrin. Therefore, the function of Spalpha to form tetramers with Spbeta is determined by the local conformation of the binding site, rather than the overall conformational stability.