Abstract
The tryptophan that is highly conserved among repeating structural units
of spectrin is reported to promote the conformational stability of one
such unit of chicken brain a-spectrin. Four constructs were inserted
into pET vectors for overexpression in Escherichia coil of the following
spectrin peptides: (i) two adjacent but separately expressed
''conformationally phased'' repeating units, R16 and R17, one of which
(R17) contains a single tryptophan; (ii) a mutant, M17, of the single
tryptophan-containing unit with alanine substituted for the tryptophan;
and (iii) a conformationally unphased unit, 1617, composed of half of
each of the phased units. Both the mutant unit and the unphased unit
were much more readily digested by chymotrypsin and by elastase than the
phased units and exhibited only 38% and 54% as much alpha-helical
structure, respectively, as the phased units by their far UV Co spectra;
90 degrees light scattering measurements revealed the folded peptides to
be predominantly monomeric in solution, whereas the unfolded,
protease-sensitive peptides consisted of dimers and/or trimers. This
trend was corroborated by their dynamic light scattering. Both the
blue-shifted wavelength of maximal emission and the relative
inaccessibility to acrylamide of the single tryptophan in the folded
unit indicate that the invariant tryptophan occupies a site that is
shielded from the aqueous phase.
