Equilibria and Kinetics of Folding of Gelsolin Domain 2 and Mutants Involved in Familial Amyloidosis-Finnish Type

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 96; no. 20; pp. 11247 - 11252
• Main Authors: Isaacson, R L, Weeds, A G, Fersht, A R
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 28-09-1999; National Acad Sciences; The National Academy of Sciences
• Subjects: Alzheimers disease; Amyloidosis - genetics; Amyloids; Biochemistry; Biological Sciences; Carrier Proteins - chemistry; Disulfides; Disulfides - chemistry; Fluorescence; gelsolin; Gelsolin - chemistry; Kinetics; Microfilament Proteins - chemistry; Mutation; Protein Conformation; Protein Folding; Protein precursors; Protein refolding; Proteins; Titration
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.96.20.11247

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 wildtype 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$\text{2.16 kcal}· \text{mol}^{-1}$(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.