Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis-Finnish type

Mutations D187N and D187Y in domain 2 of the actin-regulating protein gelso lin cause familial amyloidosis-Finnish type (FAF). We have constructed and expressed a recombinant version of gelsolin domain 2 that is sufficiently s table for kinetic and equilibrium measurements. The wildtype domain and the two amyloidogenic mutants fold via simple two-state kinetics without the a ccumulation of an intermediate. Unfolding kinetics exhibits significant cur vature with increasing urea concentration, indicating that the transition s tate for unfolding becomes more native-like under increasingly denaturing c onditions in accordance with the Hammond postulate. Mutations D187N and D18 7Y destabilize gelsolin domain 2 by 1.22 and 2.16 kcal.mol(-1) (1 kcal = 4. 18 kJ) respectively. The mutations do not prevent disulfide bond formation despite their direct contiguity with a cysteine residue involved in disulfi de linkage. The destabilization conferred on gelsolin domain 2 by the FAF m utations is sufficient to predict that an appreciable fraction is unfolded and, therefore, extremely susceptible to proteolysis at body temperature.