The Yin and Yang of Amyloids: Insights from alpha-Synuclein and the Pmel17 Repeat Domain

Abstract

While amyloidogenic proteins are commonly associated with human diseases such as Alzheimer's and Parkinson's disease, it is intriguing that amyloid fibrils also are utilized for essential biological processes. A key question then is why many amyloids are harmful whereas some serve essential functional roles. To begin to address this question, the environmental factors regulating the conformational changes in the Parkinson's disease-related protein, alpha-synuclein (alpha-syn), and a critical polypeptide fragment, the repeat domain (RPT) of Pmel17, the protein required for melanin formation are examined.

The role of membranes in modulating alpha-syn conformation is investigated because membranes are ubiquitous in vivo and affect alpha-syn aggregation in vitro. Using single tryptophan-containing alpha-syn variants (F4W, Y39W, F94W, Y125W) as site-specific fluorescent probes, distinct phospholipid vesicle and SDS micelle interactions have been identified and membrane binding equilibria measured. The role of specific N-terminal residues in membrane binding also has been assessed. Specifically, environments of the highly sensitive Trp4 probe in alpha-syn polypeptide fragments (residues: 1 - 4, 1 - 6, 1 - 10, and 1 - 15) upon membrane binding were characterized using steady-state fluorescence and time-resolved anisotropy. The penetration depths of alpha-syn and N-terminal peptides into the lipid bilayer also were determined using brominated lipids as heavy-atom quenchers. To simultaneously monitor alpha-syn and bilayer structure, neutron reflectometry (NR) and a sparsely-tethered bilayer lipid membrane (stBLM) were employed. Using NR and an stBLM, alpha-syn concentration dependent effects on both protein structure and membrane properties were measured.

To begin to address biophysical and biochemical differences between pathological and functional amyloid, a systematic investigation of the effects of solution pH (4→7) on RPT aggregation was performed since melanosomes, acidic organelles where Pmel17 fibrils are formed, change pH during maturation. Using intrinsic tryptophan fluorescence, circular dichroism spectroscopy, and transmission electron microscopy, local, secondary, and fibril morphological structure were monitored, respectively. Notably, RPT fibril morphology can be transformed directly by changing solution pH, suggesting that pH is a natural regulatory mechanism for Pmel17 amyloid formation and its subsequent dissolution in vivo.