Biophysical analysis of normal transthyretin: implications for fibril formation in senile systemic amyloidosis

Transthyretin (TTR) is a plasma protein that transports thyroid hormone and retinol binding protein-vitamin A complex Eighty-four variants of TTR have been identified and seventy-Sour are associated with familial amyloidotic polyneuropathy. Normal TTR is the major protein found in the fibrillar depo sits in the heart at time of autopsy of individuals with senile systemic am yloidosis. The mechanism by which normally soluble TTR deposits as organ-da maging, insoluble, pathological fibrils late in life is unknown. Understand ing the mechanism of fibrillogenesis of normal TTR is critical to the desig n of clinical treatments aimed at retardation, prevention, or reversal of f ibril deposition. We have employed a biophysical approach to explore the hy pothesis that an instability in a particular secondary or tertiary structur e plays a role in the ability of normal TTR to form fibrils at physiologica l pH! Using far UV circular dichroic (CD) spectroscopy as a function of tem perature we have identified simultaneous, cooperative, reversible structura l changes in the P-sheet and ct-helical regions. The flexible short surface -located loops undergo an irreversible conformational change at a lower tem perature. Spectra before and after heating are different, particularly in t he wavelength region associated with these leaps, strongly suggesting that the major portion of TTR returns to ifs initial conformation while the loop s do not. Near UV CD reveals partially reversible and irreversible changes in tertiary structure. Using calorimetry to directly measure the enthalpy a ssociated with these changes, two peaks are observed with further analysis suggesting conformational intermediates. Precipitates from heated samples r eveal pre-fibrillar morphology by negative stain electron microscopy. These biophysical studies suggest that heat-induced conformational rearrangement s enable normal TTR to assemble into pre-fibrils at physiological pH.