Dataset Information

Effects of Macromolecular Cosolutes on the Kinetics of Huntingtin Aggregation Monitored by NMR Spectroscopy.

ABSTRACT: The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hht^ex1) with a 35 polyglutamine repeat, htt^ex1Q₃₅, are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis for quantitative analysis of htt^ex1Q₃₅ aggregation, monitored by measuring cross-peak intensities in a series of 2D ¹H-¹⁵N NMR correlation spectra acquired during the course of aggregation. The primary effects of the two cosolutes are associated with shifts in the prenucleation tetramerization equilibrium resulting in substantial changes in concentration of "preformed" htt^ex1Q₃₅ tetramers. Similar effects of the two cosolutes on the tetramerization equilibrium observed for a shorter, nonaggregating huntingtin variant with a 7-glutamine repeat, htt^ex1Q₇, lend confidence to the conclusions drawn from the fits to the htt^ex1Q₃₅ aggregation kinetics.

SUBMITTER: Torricella F

PROVIDER: S-EPMC11345868 | biostudies-literature | 2024 Jun

REPOSITORIES: biostudies-literature

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Effects of Macromolecular Cosolutes on the Kinetics of Huntingtin Aggregation Monitored by NMR Spectroscopy.

Torricella Francesco F Tugarinov Vitali V Clore G Marius GM

The journal of physical chemistry letters 20240610 24

The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hhtex1) with a 35 polyglutamine repeat, httex1Q35, are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis ...[more]

PMID: 38857530

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Project description:Product excipients are used to confer a number of desirable properties on the drug substance to maintain or improve stability and facilitate drug delivery. This is especially important for products where the active pharmaceutical ingredient (API) is a recombinant protein. In this study, we aimed to determine if excipients and formulation conditions affect the structure and/or modulate the dynamics of the protein API of filgrastim products. Samples of uniformly labeled 15N-Met-granulocyte-colony stimulating factor (GCSF) were prepared at 100 μM (near formulation concentration) with various concentrations of individual components (polysorbate-20 and -80, sorbitol) and three pH values. Nuclear magnetic resonance (NMR) spectroscopy techniques were applied to measure chemical shift perturbation (CSP) to detect structural changes, and relaxation parameters (T 1, T 2, and heteronuclear Overhauser effect) were measured to probe the effects on protein backbone motions. In parallel, the same solution conditions were subjected to protein thermal unfolding studies monitored by circular dichroism spectropolarimetry (CD). Detergents (polysorbate-20 and 80) do not induce any observable changes on the protein structure and do not modify its dynamics at formulation concentration. Lowering pH to 4.0, a condition known to stabilize the conformation of filgrastim, as well as the addition of sorbitol produced changes of the fast motion dynamics in the nanosecond and picosecond timescale. NMR-derived order parameters, which measure the local conformational entropy of the protein backbone, show that lowering pH leads to a compaction of the four-helix bundle while the addition of sorbitol relaxes helices B and C, thereby reducing the mobility of loop CD. CSPs and measurements of protein dynamics via NMR-derived order parameters provide a description in structural and motional terms at an atomic resolution on how formulation components contribute to the stabilization of filgrastim products.

Dataset Information

Effects of Macromolecular Cosolutes on the Kinetics of Huntingtin Aggregation Monitored by NMR Spectroscopy.

Publications

Effects of Macromolecular Cosolutes on the Kinetics of Huntingtin Aggregation Monitored by NMR Spectroscopy.

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