Protein analysis by dynamic light scattering: Methods and techniques for students

Abstract

Dynamic light scattering (DLS) analyses are routinely used in biology laboratories to detect aggregates in macromolecular solutions, to determine the size of proteins, nucleic acids, and complexes or to monitor the binding of ligands. This article is written for graduate and undergraduate students with access to DLS and for faculty members who wish to incorporate DLS into a lab activity, a practical course or research. It reviews the basic concepts of light scattering measurements and addresses four critical aspects of the analysis and interpretation of DLS results. To ensure reproducible quantitative data, attention should be paid to controlling the preparation and handling of proteins or assemblies because variations in the state of aggregation, induced by minor changes in experimental condition or technique, might compromise DLS results and affect protein activity. Variables like temperature, solvent viscosity, and inter-particle interactions may also influence particle size determination. Every point is illustrated by case studies, including a commercially available albumin, a small RNA virus isolated from plants, as well as four soluble proteins and a ribonucleoprotein assembly purified and characterized by students in the frame of their master degree.Keywords: Light scattering, protein, temperature, viscosity.During the last two decades the panel of methods used to study the structure and function of proteins and nucleic acids has grown continuously. An increasing number of instruments based on physical methods have appeared in biology laboratories. Amongst them, noninvasive spectroscopic methods based on light scattering have become a popular tool in biomolecule characterization. Light scattering measurements have numerous applications in condensed matter physics [1][2][3], biology [4][5][6][7], and medicine [8]. For half a century they have been used to monitor aggregation phenomena in protein solutions. Examples include the study of the influence of various factors on their state of aggregation [9], the detection and monitoring of undesirable aggregates in biotherapeutics that lead to immunogenic reactions or have adverse effects during administration in patients [10], and the investigation of protein aggregation diseases [11], such as caused by interactions between human gD-crystallin molecules with point mutations whose low solubility is responsible for lens opacity during cataract [12].Owing to intense monochromatic laser light sources, dynamic light scattering (DLS, also known as photon correlation spectroscopy or quasi-elastic light scattering) measurements provide a quick and straightforward means to determine the mutual translational diffusion coefficient (or diffusivity) D t of macromolecules in solution. D t describes the ease with which a substance displaces inside another one by diffusion and is equal to the mean square displacement of the particles divided by two-fold time [13]. According to Fick's first law of diffusion, D t relates the concentration gradient of a solute || To whom correspo...

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Dynamic light scattering: a practical guide and applications in biomedical sciences

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