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

As reported previously (MacDonald, a. I., Musacchio, ih,, Holmgren, R. a., and Saraste, M. (1994) PI oc. Natl. Acad Sci. U. S. A. 91, 1299-1 303), an unfolded peptide was obtained by site-directed mutagenesis of Trp-22 to Ala in the cloned, wild type 17th repeating unit (alpha 17) of chicken brain alpha-spectrin, Trp occurs in position 22 of nearly all repeating units of spectrin, In the present study, Trp-22 was muta ted to Phe or to Tyr to compare thermodynamic stabilities of urea-indu ced unfolding of alpha 16 and mutants thereof, alpha 16 was chosen for this study instead of alpha 17, because alpha 16 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% t hat of alpha 16, showing that Trp-22 if crucial in stabilizing the tri ple helical bundle motif of the spectrin repeating unit, Mutation of t he moderately conserved Trp-95 of alpha 16 to Val, which occupies posi tion 95 in alpha 17, also yielded a peptide? with 50% of the free ener gy of unfolding of alpha 16, Thus, the thermodynamic stability of a gi ven spectrin repeating unit may depend on both moderately and highly c onserved tryptophans. Different structural roles: of Trp-22 and Trp-95 in alpha 16 are suggested by the slightly higher wavelength of maximu m emission of Trp-22, the greater acrylamide quenching of Trp-95 than Trp-22, and the longer lifetime of Trp-95. For comparison with alpha 1 6, urea-induced unfolding of spectrin dimer isolated from human red ce lls was monitored by far W-CD and by tryptophan fluorescence, Thermody namic 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 s imilar midpoints of CD-monitored denaturation curves of alpha 16 and s pectrin dimer, i.e, 2.7 and 3.2 Rr urea, respectively, indicate that i nvestigation of cloned repeating units of spectrin can provide physiol ogically relevant information an these structures.