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

TY - JOUR

T1 - Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis-Finnish type

AU - Isaacson, Rivka L.

AU - Weeds, Alan G.

AU - Fersht, Alan R.

PY - 1999/9/28

Y1 - 1999/9/28

N2 - 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.

AB - 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.

KW - Denaturation

KW - Hammond

KW - Protein

KW - Stability

KW - Two-state

UR - http://www.scopus.com/inward/record.url?scp=0033613236&partnerID=8YFLogxK

U2 - 10.1073/pnas.96.20.11247

DO - 10.1073/pnas.96.20.11247

M3 - Article

C2 - 10500162

AN - SCOPUS:0033613236

SN - 0027-8424

VL - 96

SP - 11247

EP - 11252

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 20

ER -