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May 1, 2011 (Vol. 31, No. 9)

Characterizing Protein-Protein Interactions

Concentration-Gradient Dynamic Light Scattering Finds Uses in High-Throughput Setting

  • Protein-protein interactions are intensively studied, not only to gain understanding of cellular function, but also for pharmaceutical development. In vitro, such complexes may be difficult to separate and analyze in pure form, as they equilibrate rapidly with their component monomers and partially formed complexes.

    Accordingly, methods that evaluate reversible interactions at true equilibrium are indispensable. Since any modification of the protein, either by immobilization or by labeling, can influence the interaction, free-solution, label-free methods are optimal. However, methods meeting these criteria—sedimentation equilibrium analytical ultracentrifugation, isothermal titration calorimetry, and more recently, concentration-gradient static light scattering—all require a relatively large quantity of sample when used in their standard configurations and are not ideally suited for high-throughput measurement.

    In contrast, there is a dynamic light scattering (DLS) method, concentration-gradient DLS, which enables accurate and quantitative characterization of protein-protein interactions in a high-throughput manner, using only picomoles of sample. DLS, also known as quasielastic light scattering or photon correlation spectroscopy, processes the time-dependent fluctuations in scattered light to yield the hydrodynamic radius, rh, of particles in solution.

    This method gives the ability to detect and characterize multiple binding stoichiometries, small-molecule inhibition of protein-protein interactions, and changes of interactions with temperature or solution conditions. Recent innovations in DLS instrumentation allow measurements from microtiter plates, enabling high-throughput measurements using only a few µL of sample per condition. These measurement volumes reduce protein requirements by several orders of magnitude over the methods listed above, and coupled with high-throughput measurements make DLS characterization of protein-protein interactions practical.

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