Abstract

As reported previously (MacDonald, R. I., Musacchio, A., Holmgren, R. A., and Saraste, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1299-1303), an unfolded peptide was obtained by site-directed mutagenesis of Trp-22 to Ala in the cloned, wild type 17th repeating unit (alpha17) of chicken brain alpha-spectrin. Trp occurs in position 22 of nearly all repeating units of spectrin. In the present study, Trp-22 was mutated to Phe or to Tyr to compare thermodynamic stabilities of urea-induced unfolding of alpha16 and mutants thereof. alpha16 was chosen for this study instead of alpha17, because alpha16 has two tryptophans, allowing urea-induced unfolding to be tracked by the fluorescence of the Trp remaining in each mutant peptide and by circular dichroism in the far UV. The free energies of unfolding of W22Y and W22F were 50% that of alpha16, showing that Trp-22 is crucial in stabilizing the triple helical bundle motif of the spectrin repeating unit. Mutation of the moderately conserved Trp-95 of alpha16 to Val, which occupies position 95 in alpha17, also yielded a peptide with 50% of the free energy of unfolding of alpha16. Thus, the thermodynamic stability of a given spectrin repeating unit may depend on both moderately and highly conserved tryptophans. Different structural roles of Trp-22 and Trp-95 in alpha16 are suggested by the slightly higher wavelength of maximum emission of Trp-22, the greater acrylamide quenching of Trp-95 than Trp-22, and the longer lifetime of Trp-95. For comparison with alpha16, urea-induced unfolding of spectrin dimer isolated from human red cells was monitored by far UV-CD and by tryptophan fluorescence. Thermodynamic parameters could not be rigorously derived for the stability of spectrin dimer because unfolding of spectrin dimer involved more than two states, unlike unfolding of cloned repeating units. However, the similar midpoints of CD-monitored denaturation curves of alpha16 and spectrin dimer, i. e. 2.7 and 3.2 M urea, respectively, indicate that investigation of cloned repeating units of spectrin can provide physiologically relevant information on these structures. Study holds ProTherm entries: 2969, 2970, 2971, 2972, 2973 Extra Details: additive : EDTA(0.1 mM), thermodynamic stability; site-directed mutagenesis;,alpha-spectrin; hydrophobic residue; structural role

Submission Details

ID: ZHWuvGsm

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:20 p.m.

Version: 1

Publication Details

Pantazatos DP;MacDonald RI,J. Biol. Chem. (1997) Site-directed mutagenesis of either the highly conserved Trp-22 or the moderately conserved Trp-95 to a large, hydrophobic residue reduces the thermodynamic stability of a spectrin repeating unit. PMID:9261107

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