(570c) Understanding the Role of Glycine in Amyloid Protein Aggregation through Rationally Designed Protein Sequences

Vance, S. Z., University of South Carolina
Moss, M. A., University of South Carolina
Booth, G. L., University of Arkansas
Hestekin, C. N., University of Arkansas
Aggregation of amyloid proteins is associated with a myriad of medical conditions including Alzheimer’s disease (AD), Type II diabetes, and Parkinson’s disease. While attributable to different amyloidogenic proteins, these proteins all share a common feature: a periodic glycine motif (GxxxG). This glycine motif, associated with increased backbone flexibility, is extended in a number of familial mutations associated with early onset AD (i.e. Flemish, Arctic) that significantly increase disease severity. A better understanding of the role that the glycine motif, and by extension chain flexibility, plays in protein aggregation will allow for new and give insight into therapeutic approaches.

In this study, the glycine motif in amyloid-β (Aβ), the amyloid protein associated with AD, is targeted via either extension, by introduction of additional periodic glycine, or contraction, by replacement of glycine with a bulkier amino acid. Modifications that extend periodic glycine include those that align with familial AD mutations (L17G/A21G) as well as off sequence mutations (V18G/E22G). Modifications that attenuate the glycine motif target G25 via replacement with either a bulky (G25I) or more conservative (G25A) mutation.

To examine how these mutations impact aggregation kinetics, aggregation was monitored via thioflavin-T (ThT), a dye that fluoresces in the presence of β-sheet structure, and aggregation curves fit using an adapted sigmoidal kinetic model that accounts for unique features observed at late stages of aggregation. Kinetically, extensions of the glycine motif (L17G/A21G, V18G/E22G) correlate with faster nucleation and a reduction in lag time. Interestingly, the on sequence extension (L17G/A21G) results in an increased equilibrium plateau while the off sequence extension (V18G/E22G) closely resembles the WT at plateau. Both L17G/A21G and V18G/E22G resulted in aggregates with a greatly diminished fibril morphology as visualized by TEM.

L17G/A21G aggregate were fractionated by size exclusion chromatography revealing an enhancement of small, soluble aggregates. Aggregate hydrodynamic radii (RH), determined using dynamic light scattering, were smaller than WT; however, as observed with 8-anilino-1-naphthalenesulfonic acid (ANS), these aggregates have an unchanged surface hydrophobicity. Similarly, V18G/E22G soluble aggregates possessed a reduced RH but were very similar in hydrophobicity to the WT, suggesting L17G/A21G and V18G/E22G, increases in glycine motif, corresponds with formation of smaller aggregates with a conformation very similar to that of WT Aβ.

Broadly, the reduction of the glycine motif has the opposite effect. Both G25A and G25I result in aggregates resembling traditional fibrils via TEM, however, differences are apparent in aggregation kinetics. The more conservative mutation, G25A, resembles the WT with a very similar kinetic lag but an enhanced plateau. However, G25I completely inhibits the formation of ThT-binding aggregates. As a result, circular dichroism was used to assess secondary structure characteristics revealing the propensity of G25I alone in creating β-sheet negative aggregates. Soluble aggregate fractions of G25A show a reduction in hydrophobicity compared to the control, while G25I aggregates remain unchanged.

 Taken together with well-studied observations that smaller aggregates are more physiologically active, these results support the hypothesis that an extended glycine motif, including that associated with familial mutations, may contribute to disease progression and early onset of the disease.