Site-Resolved and Quantitative Characterization of Very Weak Protein-Ligand Interactions.

Very weak interactions between small organic molecules and proteins have long been predicted and are expected to have dissociation constants of hundreds of millimolar and above. Unfortunately, quantitative evaluation of binding in a high-resolution structural context for this affinity regime is particularly difficult and often impossible using existing experimental approaches. Here, we show that nanoscale encapsulation of single protein molecules within the water core of reverse micelles enables the detection and evaluation of weak binding interactions at atomic resolution using solution NMR spectroscopy. This strategy is used to survey the interactions of a set of small molecules with the cytokine interleukin-1β (IL-1β). The interaction of IL-1β with these molecules is found to vary from more diffuse and weak binding modes to more specific and with a relatively higher affinity. The interactions detected here cover a large portion of the protein surface and have dissociation constants mostly in the low molar range. These results illustrate the ability of a protein to interact productively with a variety of small molecule functional groups and point to a broader potential to target even relatively featureless protein surfaces for applications in chemical biology and drug discovery.