Two-dimensional IR spectroscopy and segmental ¹³ C labeling reveals the domain structure of human γD-crystallin amyloid fibrils

Abstract

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly 13 C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins. C ataracts are a protein misfolding disease caused by the aggregation of lens crystallin proteins into insoluble deposits that blur vision (1, 2). Because these proteins are not regenerated, damage from UV radiation, oxidative stress, and other chemical modifications accumulates with time (1, 2). As a result, over 50% of the population over 55 develops age-related cataracts (2). Additionally, numerous mutations that destabilize crystallin protein folds are linked to inherited and juvenile-onset cataracts (1). Although the causative factors associated with this disease are known, the structures of the aggregates and the mechanisms by which they form are unknown.Like other protein aggregation diseases such as type II diabetes mellitus and Alzheimer's disease, the molecular structures of proteins in cataracts are difficult to determine. Atomic-level structures have been obtained for some amyloid aggregates of peptides using NMR spectroscopy (3, 4) and X-ray crystallography (5). However, the most widely used techniques for studying aggregate structures and aggregation mechanisms are circular dichroism spectroscopy, fluorescence spectroscopy, and transmission electron microscopy, which provide little detailed structural information. Two-dimensional (2D) IR spectroscopy is emerging as an important tool for studying protein aggregates such as amyloid fibrils (6-8) because it provides bond-by-bond structural resolution on kinetically evolving samples (6, 8-10). Two-dimensional IR spectroscopy probes secondary structure through cross peak couplings and solvent exposure through 2D lineshapes. Its bond-specific structural resolution comes from isotope labeling. Mech...

Structural motif of polyglutamine amyloid fibrils discerned with mixed-isotope infrared spectroscopy

“…This spectrum is nearly identical to that of the unlabeled peptide, except that the amide features are downshifted uniformly by 44 cm −1 . Such a frequency shift is consistent with the change in effective mass upon 13 C substitution of the amide carbon and has been observed in other fully labeled peptide samples (25). The similarity between the spectra indicates that uniform 13 C labeling does not disrupt the structure or coupling of the peptides.…”

Myeloperoxidase-mediated Methionine Oxidation Promotes an Amyloidogenic Outcome for Apolipoprotein A-I

“…The new intense and sharp peak at ϳ1620 cm Ϫ1 indicates formation of amyloid ␤-sheets. The line-width and anharmonic shift of this feature are also consistent with amyloid fiber formation and comparable to what observed in previously studied amyloid fibrils (45)(46)(47)(48). Notably, a similar enrichment in ␤-sheet structure was observed in MPO-2:1-H 2 O 2 -apoA-I incubated under the same conditions (Fig.…”

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

“…The only difference is the vastly disparate timescales probed by the two methods -picoseconds versus milliseconds -, which refers to the "direct time resolution" inherent to both techniques. In the context of protein dynamics, 2D IR spectroscopy has been applied to investigate the structure of membrane peptides [21,22], fibril formation [23,24], the structural flexibility of enzyme active sites [25], ligand migration in heme proteins [26], ligand binding [27,28], and the dynamics of the protein hydration shell [29,30], to list just a few examples. Recent reviews can be found in Refs.…”

“…For most cases, the -CO groups of the peptide backbone have been considered, either as single (-13 C 16 O) or as double (-13 C 18 O) substitution, where the latter is preferred since it completely separates the corresponding vibrational mode from the main amide I band. This approach has been used extensively to study structure and dynamics of small peptides [5, 7-9, 21, 22, 24, 34, 40-43], but unfortunately, it is essentially no longer feasible even for small proteins (elegant exceptions to that statement exist [23]). First, the synthesis becomes extremely tedious once the size of the protein exceeds that which can be handled on a peptide synthesizer ( > ∼ 50 amino acids), so the protein has to be expressed in E. coli.…”