HEXIX-TURN-HELIX PEPTIDES THAT FORM ALPHA-HELICAL FIBRILS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major determinant of High Density Lipoprotein size is its main protein, Apolipoprotein A-I (apo A-I). Apo A-I contains 10 amphiphilic a-helical segments connected by turns. We synthesized four peptides with two 18-residue, amphiphilic a-helical segments (designed as apo A-I models, Anantharamaiah, G. M. et al.) connected by various putative turn sequences from apo A-I (1). 1. Ac-DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF-NH2 2. Ac-DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF-NH2 3. Ac-DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF-NH2 4. Ac-DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF-NH2 All of the peptides formed insoluble aggregates after incubating at 37?C in 10 mM phosphate buffer, pH 7.6. We were intrigued by this observation because variants of apo A-I have been implicated in systemic amyloidosis of the liver, heart, and kidney. The amyloidoses consist of a large number of diseases in which misfolding of extracellular protein plays a prominent role. The protein aggregates are deposited in tissues as b-sheet fibrils. 3 and 4 formed typical amyloid fibrils, as shown by thioflavin T and Congo red binding. In contrast to amyloid fibrils, however, peptides 1 and 2 formed a-helical fibrils by CD and FTIR, and were highly a-helical in solution and in fibril films. 1 and 2 did not bind thioflavin T, and induced a blue shift in the spectrum of Congo red. Synchrotron x-ray fiber diffraction on a magnetically aligned sample of 1 has confirmed the a-helical character of the fibrils. The diffraction data suggest that the a-helices are perpendicular to the main fibril axis, in contrast to previously described coiled-coil fibrils (2-6). To the best of our knowledge, the fibrils formed from peptide 1 represent a novel structure. Given our recent success with these model systems, further investigations of peptide 1 and the other (b-sheet forming) peptides may significantly improve our understanding of protein folding / misfolding in nature.