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Latent analysis of unmodified biomolecules and their complexes in solution

Authors: Emma V. Yates, Thomas Müller, Luke Rajah, Erwin J. De Genst, Paolo Arosio, Sara Linse, Michele Vendruscolo, Christopher M. Dobson, and Tuomas P. J. Knowles.

Nature Chemistry, 2015, 7, 802-9. DOI: 10.1038/nchem.2344 

 

Abstract

The study of biomolecular interactions is central to an understanding of function, malfunction and therapeutic modulation of biological systems, yet often involves a compromise between sensitivity and accuracy. Many conventional analytical steps and the procedures required to facilitate sensitive detection, such as the incorporation of chemical labels, are prone to perturb the complexes under observation.

Here they present a ‘latent’ analysis approach that uses chemical and microfluidic tools to reveal, through highly sensitive detection of a labelled system, the behaviour of the physiologically relevant unlabelled system. They implement this strategy in a native microfluidic diffusional sizing platform, allowing us to achieve detection sensitivity at the attomole level, determine the hydrodynamic radii of biomolecules that vary by over three orders of magnitude in molecular weight, and study heterogeneous mixtures. they illustrate these key advantages by characterizing a complex of an antibody domain in the solution phase and under physiologically relevant conditions.

In this paper, Yates et al. show that the technology behind Fluidic Analytics instruments—microfluidic diffusional sizing—can be used to accurately size a range of proteins. Results were compared to those obtained using Pulsed Field Gradient NMR and Analytical Ultracentrifugation and found to agree closely (Figure 1).

Sizing proteins, heterogeneous mixtures and protein complexes.
Figure 1: MDS is used for size determination of various proteins Rh values determined in this manner are compared to those obtained with PFG-NMR (lilac) and AUC (aqua). The studied molecules vary by over three orders of magnitude in molecular weight and include intrinsically disordered proteins and heterogeneous mixtures.

Finally, they demonstrate that Microfluidic Diffusional Sizing can be used to characterise protein interactions without the use of tags, using the platform to explore the binding of α-synuclein to a nanobody, NbSyn1 (Figure 2).

novel Parkinson's-related immune complex
Figure 2: MDS is utilised to characterise protein interactions without the use of tags. Characterisation of a novel Parkinson’s-related immune complex between a Nanobody and equimolar (5 μM) α-synuclein.

Find the paper here

 


Instrument: Fluidity One
Therapeutic area: basic research

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