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-four are associated with familial amyloidotic polyneuropathy. Normal TTR is the major protein found in the fibrillar deposits in the heart at time of autopsy of individuals with senile systemic amyloidosis. The mechanism by which normally soluble TTR deposits as organ-damaging, insoluble, pathological fibrils late in life is unknown. Understanding the mechanism of fibrillogenesis of normal TTR is critical to the design of clinical treatments aimed at retardation, prevention, or reversal of fibril deposition. We have employed a biophysical approach to explore the hypothesis that an instability in a particular secondary or tertiary structure plays a role in the ability of normal TTR to form fibrils at physiological pH. Using far UV circular dichroic (CD) spectroscopy as a function of temperature we have identified simultaneous, cooperative, reversible structural changes in the beta-sheet and alpha-helical regions. The flexible short, surface-located loops undergo an irreversible conformational change at a lower temperature. Spectra before and after heating are different, particularly in the wavelength region associated with these loops, strongly suggesting that the major portion of TTR returns to its initial conformation while the loops do not. Near UV CD reveals partially reversible and irreversible changes in tertiary structure. Using calorimetry to directly measure the enthalpy associated with these changes, two peaks are observed, with further analysis suggesting conformational intermediates. Precipitates from heated samples reveal pre-fibrillar morphology by negative stain electron microscopy. These biophysical studies suggest that heat-induced conformational rearrangements enable normal TTR to assemble into pre-fibrils at physiological pH.