The interaction between the Human Immunodeficiency Virus Nef protein (HIV-1 Nef) and the Src Homology Region 3 (SH3) domain of Hck was studied using scintillation proximity assay (SPA). SPA is a quick and sensitive method that does not require a separation step, thus allowing assays to be performed in a homogeneous environment. In contrast to most conventional techniques, SPA may also be used to detect low affinity protein-protein interactions. In this study, the assay was configured using biotinylated Hck SH3 domain expressed both as a GST fusion protein and synthesized chemically in its' native form. Biotinylated Hck protein was immobilized to streptavidin-coated fluoromicrosphere SPA beads and the binding of [3H]Nef was detected by scintillation counting. Analysis of binding yielded an average equilibrium dissociation constant (KD) of 183 ± 30 nM for the interaction in line with reported values by other methods. The data presented demonstrates that using SPA, protein-protein interactions of relatively low affinity can be detected with a high degree of sensitivity and screening studies of inhibitors of these associations could be facilitated by the high sample throughput achievable with SPA.
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