Thermal stabilities of brain spectrin and the constituent repeats of subunits.
The different genes that encode mammalian spectrins give rise to proteins differing in their apparent stiffness. To explore this, we have compared the thermal stabilities of the structural repeats of brain spectrin subunits (alphaII and betaII) with those of erythrocyte spectrin (alphaI and betaI). The unfolding transition midpoints (T(m)) of the 36 alphaII- and betaII-spectrin repeats extend between 24 and 82 degrees C, with an average higher by some 10 degrees C than that of the alphaI- and betaI-spectrin repeats. This difference is reflected in the T(m) values of the intact brain and erythrocyte spectrins. Two of three tandem-repeat constructs from brain spectrin exhibited strong cooperative coupling, with elevation of the T(m) of the less stable partner corresponding to coupling free energies of approximately -4.4 and -3.5 kcal/mol. The third tandem-repeat construct, by contrast, exhibited negligible cooperativity. Tandem-repeat mutants, in which a part of the "linker" helix that connects the two domains was replaced with a corresponding helical segment from erythroid spectrin, showed only minor perturbation of the thermal melting profiles, without breakdown of cooperativity. Thus, the linker regions, which tolerate few point mutations without loss of cooperative function, have evidently evolved to permit conformational coupling in specified regions. The greater structural stability of the repeats in alphaII- and betaII-spectrin may account, at least in part, for the higher rigidity of brain compared to erythrocyte spectrin.
An
Xiuli
X
Red Cell Physiology Laboratory, New York Blood Center, New York, New York 10021, USA. xan@nybloodcenter.org
Zhang
Xihui
X
Salomao
Marcela
M
Guo
Xinhua
X
Yang
Yang
Y
Wu
Yu
Y
Gratzer
Walter
W
Baines
Anthony J
AJ
Mohandas
Narla
N
D000818:Animals; D004355:Drug Stability; D006358:Hot Temperature; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics; D013552:Swine
D011994:Recombinant Proteins; D013049:Spectrin