Ri. Macdonald et Ev. Pozharski, Free energies of urea and of thermal unfolding show that two tandem repeats of spectrin are thermodynamically more stable than a single repeat, BIOCHEM, 40(13), 2001, pp. 3974-3984
Free energies of both urea and thermal denaturation have been measured for
three pairs of one- and two-repeat fragments, cloned in tandem from the cyt
oskeletal protein, alpha -spectrin, from chicken brain to ascertain whether
one- and two-repeat fragments are equally stable. One- and two-repeat frag
ments of each pair were designed with the same N-terminus, whereas the C-te
rminus of the two-repeat fragment was 106 residues or the length of one rep
eat downstream from that of the one-repeat fragment. The averaged free ener
gies of urea and thermal denaturation of the paired fragments, (R16)(00) an
d (R16R17)(00), (R16)(0+3) and (R16R17)(0+3), and (R16)(+8-4) and (R16R17)(
+8-4) [subscripts represent the N- and C-terminal positions with "00" refer
ring to the N- and C-termini defining a repeat according to X-ray crystal s
tructures of two repeat fragments [Grum, V. L., Li, D., MacDonald, R. I., a
nd Mondragon, A. (1999) Cell 98, 523-535] and "+" and "-" referring to posi
tions upstream and downstream therefrom, respectively], increased from 3.7
+/- 0.4 kcal/mol for (R16)(00), 3.7 +/- 0.5 kcal/mol for (R16)(0+3), 4.4 +/
- 0.4 kcal/mol for (R16)(+8-4), 6.2 +/- 0.6 kcal/mol for (R16R17)(+8-4), 8.
3 +/- 0.4 kcal/mol for (R16R17)(00) to 9.9 +/- 1.0 kcal/mol for (R16R17)(03). Thus, the two-repeat fragment of each pair was significantly more therm
odynamically stable than the single repeat by both urea and thermal denatur
ation. Differences in phasing among single repeats did not have the same ef
fect as the same differences in phasing among two-repeat fragments. Additio
n of nine residues to the C-terminus of (R16R17)(00) yielded a free energy
of unfolding of 7.9 +/- 0.8 kcal/mol, whereas addition of seven residues to
the C-terminus of (R16)(+8-4) yielded a free energy of unfolding of 5.9 +/
- 0.3 kcal/mol.