Abstract
Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein–peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kd values for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided. Finally, we show how a competition assay can be set up to perform focused library screens, so that compound labeling is not required anymore. These data demonstrate the utility of droplet compartments for the quantitative characterization of biomolecular interactions and establish fluorescence anisotropy imaging as a quantitative technique in a miniaturized droplet format, which is shown to be as reliable as its macroscopic test tube equivalent.
Highlights
Protein−ligand interactions interfere with, or promote, essential biological processes such as immune recognition, signal transduction or enzyme inhibition
We address the interactions of the BRC repeat 4 (BRC4) peptide, a reductionist model of the recombination mediator BRCA2, with mutants of an archaeal surrogate of the recombinase RAD51.10 The protein interaction pair BRCA2/RAD51 (Figure 1A) is a potential drug target: it is relevant for monitoring cancer progression, as RAD51 levels are often upregulated in cancerous cells, conferring resistance to chemotherapy.[11]
The two binding partners have been converted into analogues that maintain the relevant interactions, but for which it is easier to deconvolute the binding interactions and elucidate structure−activity relationships: (i) Eight BRC repeats have been identified in BRCA2 as interaction partners for RAD51 and a 30−35 amino acid peptide, corresponding to BRC repeat 4 (BRC4), has been shown to block RAD51 activity, suggesting a role as a cancer suppressor.[12] (ii) For a fragment-based drug discovery campaign a monomeric variant, RadA-ct, was derived from an archaeal homologue (RadA) and humanized, to facilitate measurement of small molecule binding constants.[13]
Summary
Protein−ligand interactions interfere with, or promote, essential biological processes such as immune recognition, signal transduction or enzyme inhibition. A wide range of assays exists to assess the strength of binding interactions, including techniques based on fluorescence probes (e.g., lifetime,[1] resonance energy transfer,[2] and anisotropy3), on surface immobilization (e.g., surface plasmon resonance, biolayer interferometry), or on calorimetry (isothermal titration calorimetry, ITC) Each of these approaches have shortcomings: surface immobilization may affect the properties of the molecular binding partners (e.g., as a consequence of conformational changes or molecular crowding);[4] ITC requires large volumes (typically hundreds of microliters) and highly concentrated reagents, precluding its use for precious and relatively insoluble samples.[5] An attractive choice is fluorescence anisotropy (FA) that enables measurements in homogeneous solution ( it still requires one of the binding partners to be fluorescently labeled). RadA-ct and its humanized variants (HumRadAs) are much more stable than RAD51, enabling biophysical analysis of interactions and development of small molecule inhibitors of this protein−
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