Conformation and size variants

Conformation and size variants illustration

In addition to self-association behavior (e.g. desired oligomerization), the conformational status of proteins and other biomolecules (e.g. denaturation, misfolded conformers) is crucial for understanding molecular functions and evaluate stability/safety/efficacy issues of drug products. Therefore, discriminating between size and conformation variants is an ubiquitous task in characterizing the size distribution of biomolecules in solution.

Nanolytics offers the most advanced instruments combined with an extensive experience in data evaluation, in order to differentiate conformation- and size variants using various AUC techniques. As alteration in conformation (i.e. the shape) of the analyte results in distinct frictional properties, application of specific data algorithms for evaluating sedimentation velocity experiments is an essential tool. Of particular relevance are data fits generating 2D sedimentation coefficient distributions assigning a pair of distinct sedimentation coefficient and frictional ratio to each sedimenting population.

CASE STUDY: Two size-variants (monomer and dimer) with a citrate-dependent change dimer’s conformation (Kaschner et al. (2017)).

Exemplary sedimentation velocity analysis of 0.5 mg/mL CitAPBsLA solutions with (first panel) and without citrate (second panel), respectively, using absorbance detection.
The conventional sedimentation coefficient distributions c(s) are shown as black solid lines. In a secondary analysis, the c(Pδ)(s) distributions based on the prior expectation that each protein sample exclusively features monodisperse species are shown as red (with citrate) and blue lines (without citrate), respectively.
The relative abundances of monomers and dimers are derived from the c(Pδ)(s) distributions.
A citrate-dependent change in conformation of dimers is confirmed via a smaller calculated frictional ratio f/f0 – indicative of a more globular conformation and/or lower hydration.
The frictional ratio reflects both the shape and hydration of the protein molecule and can be considered as an approximate measure of the molecules’ globularity.