title: Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis-Finnish type.
abstract: Mutations D187N and D187Y in domain 2 of the actin-regulating protein gelsolin cause familial amyloidosis-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 curvature 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 D187N and D187Y 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 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.
authors.affiliation: Cambridge University Chemical Laboratory, MRC Centre, Hills Road, Cambridge CB2 2QH, United Kingdom.
authors.forename: R L
authors.initials: RL
authors.lastname: Isaacson
authors.forename: A G
authors.initials: AG
authors.lastname: Weeds
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000686:Amyloidosis / Q000235:genetics*; D002352:Carrier Proteins / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D018260:Gelsolin / Q000737:chemistry*; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D002352:Carrier Proteins; D004220:Disulfides; D018260:Gelsolin; D008840:Microfilament Proteins; C026615:villin