The long term goal of this research is to understand the biochemical mechanism of amyloid disease. Amyloid fibril formation refers to the abnormal self-assembly of a normally soluble protein into an insoluble cross beta-sheet quaternary structural form and is the causative agent in amyloid disease. Understanding how a normally soluble protein is transformed into amyloid fibrils is a critical part of understanding the mechanism of this disease and is the focus of this proposal. The protein transthyretin composes the amyloid fibrils in two different types of human amyloid disease. The mechanism of transthyretin amyloid fibril formation will be studied using an in vitro system that simulates the low pH environment of the organelle where amyloid fibril formation is thought to occur in vivo. The advantage of this in vitro system is its simplicity, which will enable us to make careful measurements. The acid-mediated partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. A structured acidic-form of transthyretin appears to be the precursor that assembles into amyloid fibrils. Transthyretin denaturation and amyloid fibril formation are competitive processes that can now be studied individually due to the discovery of an inhibitor which prevents amyloid fibril formation without noticeably interfering with the denaturation pathway. Thus, in the presence of amyloid inhibitor, transthyretin denaturation can be studied and in the absence of inhibitor, fibril formation can be studied. It is our priority to identify the nature of the transthyretin denaturation intermediate competent to form fibrils. The denaturation pathway and the ability of transthyretin variants to form fibrils will also be examined in the course of these studies. Structurally based amyloid inhibitors will be tested with regard to their ability to curb amyloid fibril formation. Successful inhibitors in combination with kinetic and thermodynamic denaturation and fibril formation studies should allow a mechanism of fibril formation to be formulated. Long term structural studies on certain acid induced conformations of transthyretin will be initiated during these studies with the ultimate goal of characterizing the denaturation intermediate that is the precursor to amyloid fibrils.