Investigating Protein Aggregation using Segmental Isotope Labeling and 2D IR Spectroscopy

Abstract

Protein misfolding and aggregation are important processes in numerous human diseases, but the detailed structural changes that occur therein evade traditional crystallographic and NMR-based techniques. Rapid-scan 2D IR spectroscopy, combined with heavy isotope labeling, provides information on local secondary structure and coupling between IR chromophores, which can be used to study systems that aggregate in vitro on a timescale of minutes to hours. Recently, we have utilized single-position 1-13C=18O labels to develop a detailed kinetic model of the aggregation of synthetic hIAPP, a 37 AA peptide implicated in type II diabetes mellitus. Here we demonstrate a segmental, 13C labeling approach to the study the much larger human gammaD-crystallin, a 173 AA eye lens protein that aggregates via an unknown mechanism during cataract formation. Using bacterial expression and native chemical ligation, we have generated a gammaD-crystallin variant in which the highly similar N- and C-terminal domains are uniformly enriched in 12C and 13C, respectively. This results two well-resolved diagonal peaks in the 2D IR spectrum of the natively folded protein, with a separation of ∼40 cm−1. Aggregation under acidic conditions results in spectral changes consistent with amyloid fiber formation in the C-terminal domain and unfolding of the N-terminal domain, with evidence of interaction between the two based on the presence of cross-peaks. The structural model we propose, currently resolved at the level of individual domains, is nonetheless the most detailed available for a gammaD-crystallin aggregate to date. We anticipate that the method described will be broadly applicable to difficult systems including many aggregating, natively unstructured, or membrane-associated proteins.