Free energies of urea and of thermal unfolding show that two tandem repeats of spectrin are thermodynamically more stable than a single repeat

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.