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Growing evidence supports the confident association between distinct amyloid beta (Aβ) isoforms and Alzheimer's Disease (AD) pathogenesis. As such, critical investigations seeking to uncover the translational factors contributing to Aβ toxicity represent a venture of significant value. Herein, we comprehensively assess full-length Aβ42 stereochemistry, with a specific focus on models that consider naturally-occurring isomerization of Asp and Ser residues. We customize various forms of d -isomerized Aβ as natural mimics, ranging from fragments containing a single d residue to full length Aβ42 that includes multiple isomerized residues, systematically evaluating their cytotoxicity against a neuronal cell line. Combining multidimensional ion mobility-mass spectrometry experimental data with replica exchange molecular dynamics simulations, we confirm that co- d -epimerization at Asp and Ser residues within Aβ42 in both N-terminal and core regions effectively reduces its cytotoxicity. We provide evidence that this rescuing effect is associated with the differential and domain-specific compaction and remodeling of Aβ42 secondary structure.
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Developability Assessment of an Isolated CH2 Immunoglobulin Domain
The IgG CH2 domain continues to hold promise for the development of new therapeutic entities due to its bi-functional role as a biomarker and effector protein. The need for further understanding of molecular stability and aggregation in therapeutic proteins has led to the development of a breakthrough quantum cascade laser microscope to allow for real-time comparability assessment of an array of related proteins in solution upon thermal perturbation. Our objective was to perform a comprehensive developability assessment of three similar monoclonal antibody fragments: CH2, CH2s, and m01s. The CH2 construct consists of residues Pro238 to Lys340 of the IgG1 heavy chain sequence. CH2s has a 7-residue deletion at the N-terminus and a 16-residue C-terminal extension containing a histidine tag. The m01s construct is identical to CH2s except for two cysteines introduced at positions 242 and 334. A series of hyperspectral images was acquired during thermal perturbation from 28 to 60 oC for all three proteins in an array. Co-distribution and 2D IR correlation spectroscopies yielded the mechanism of aggregation and stability for these three proteins. The level of detail is unprecedented, identifying the regions within CH2 and CH2s that are prone to self-association and establishing the differences in stability. Furthermore, CH2 helical segments, beta-sheets, beta-turns, and random coil regions were less stable than in CH2s and m01s, due to the presence of the N-terminal 310-helix and beta-turn type III. The engineered disulfide bridge in m01s eliminated the self-association process and rendered this mAb fragment the most stable.
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- Award ID(s):
- 1632420
- PAR ID:
- 10337673
- Date Published:
- Journal Name:
- Analytical chemistry
- Volume:
- 93
- Issue:
- 3
- ISSN:
- 0003-2700
- Page Range / eLocation ID:
- 1342-1351
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acs.analchem.0c02663
