Conformational stabilities of the structural repeats of erythroid spectrin and their functional implications.
The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T(m)) ranging from 21 to 72 degrees C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol(-1). Constructs comprising five successive repeats from the beta-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.
An
Xiuli
X
Red Cell Physiology Laboratory, New York Blood Center, 310 E. 67th Street, New York, NY 10021, USA. xan@nybloodcenter.org
Guo
Xinhua
X
Zhang
Xihui
X
Baines
Anthony J
AJ
Debnath
Gargi
G
Moyo
Damali
D
Salomao
Marcela
M
Bhasin
Nishant
N
Johnson
Colin
C
Discher
Dennis
D
Gratzer
Walter B
WB
Mohandas
Narla
N
D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D013058:Mass Spectrometry; D018625:Microscopy, Atomic Force; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013049:Spectrin / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
D010455:Peptides; D011994:Recombinant Proteins; D013049:Spectrin