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

摘要

Mutations D187N and D187Y in domain 2 of the actin-regulating protein gelsolin cause familial amyloid- Osis-Finnish type (FAf). We have constructed and expressed a recombinant version of gelsolin domain 2 that is sufficiently stable for kinetic and equilibrium measurements. The wild- type domain and the two amyloidogenic mutants fold via simple two-state kinetics without the accumulation of an intermediate. Unfolding kinetics exhibits significant curva- ture with increasing urea concentration, indicating that the transition state for unfolding becomes more native-like under increasingly denaturing conditions in accordance with the Hammond postulate. Mutations DI87N and D187Y destabi- lize gelsolin domain 2 by 1.22 and2.16 kcalXmol~-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 disulfide linkage. The destabilization conferred on gelsolin domain 2 by the FAF mutations is sufficient to predict that an appreciable fraction is unfolded and, therefore, extremely susceptible to proteolysis at body temperature.