MultiSig: a new high-precision approach to the analysis of complex biomolecular systems

Richard B Gillis,Gary G Adams,Thomas Heinze,Arthur J Rowe,Stephen E Harding,Melanie Nikolajski

doi:10.1007/s00249-013-0924-y

Richard B Gillis, Gary G Adams + Show 4 more

Open Access

https://doi.org/10.1007/s00249-013-0924-y

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Abstract

MultiSig is a newly developed mode of analysis of sedimentation equilibrium (SE) experiments in the analytical ultracentrifuge, having the capability of taking advantage of the remarkable precision (~0.1 % of signal) of the principal optical (fringe) system employed, thus supplanting existing methods of analysis through reducing the ‘noise’ level of certain important parameter estimates by up to orders of magnitude. Long-known limitations of the SE method, arising from lack of knowledge of the true fringe number in fringe optics and from the use of unstable numerical algorithms such as numerical differentiation, have been transcended. An approach to data analysis, akin to ‘spatial filtering’, has been developed, and shown by both simulation and practical application to be a powerful aid to the precision with which near-monodisperse systems can be analysed, potentially yielding information on protein-solvent interaction. For oligo- and poly-disperse systems the information returned includes precise average mass distributions over both cell radial and concentration ranges and mass-frequency histograms at fixed radial positions. The application of MultiSig analysis to various complex heterogenous systems and potentially multiply-interacting carbohydrate oligomers is described.

Highlights

The analytical ultracentrifuge (AUC) is an instrument that subjects solutions of macromolecules to high centrifugal fields and explores, via a range of optical modes of analysis, the resultant re-distribution of solute particles
Moving on from and building upon the detailed knowledge that we have of the solution properties of many purified systems
It has long been realised that sedimentation equilibrium (SE) methods have very real potential for the definition of such complex systems, especially through their ability to define distribution parameters over a concentration range within a single experiment (Roark and Yphantis 1969; Teller 1973)

Summary

Introduction

The analytical ultracentrifuge (AUC) is an instrument that subjects solutions of macromolecules to high centrifugal fields (up to 300,0009g) and explores, via a range of optical modes of analysis, the resultant re-distribution of solute particles. Major advantages of AUC methods are that matrix-interaction effects (as with SEC or other columns) are not present, and the optical signal recorded for a given solute mass concentration is invariant with respect to solute particle size, unlike light-scattering systems. The two optical systems most widely used employ (1) absorption optics at wavelengths (200–600 nm) user specifiable or (2) interference fringe optics, based upon the refraction index increment with respect to solvent of the solute. For the former, the solutes under analysis there must possess a usable chromophore; for the latter it is convenient that all solutes have refraction increments that differ only

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