A large number of methods have been developed over the years to study protein-protein interactions. Many of these techniques are now available to the nonspecialist researcher thanks to new affordable instruments and/or resource centres. A typical protein-protein interaction study usually starts with an initial screen for novel binding partners. We start this review by describing three techniques that can be used for this purpose: (i) affinity-tagged proteins (ii) the two-hybrid system and (iii) some quantitative proteomic techniques that can be used in combination with, e.g., affinity chromatography and coimmunoprecipitation for screening of protein-protein interactions. We then describe some public protein-protein interaction databases that can be searched to identify previously reported interactions for a given bait protein. Four strategies for validation of protein-protein interactions are presented: confocal microscopy for intracellular colocalization of proteins, coimmunoprecipitation, surface plasmon resonance (SPR) and spectroscopic studies. Throughout the review we focus particularly on the advantages and limitations of each method.

The rapid removal of Ca2+ ions from the cytosol, necessary for the efficient relaxation of cardiac muscle cells, is performed by the Ca2+-pumping ATPase of the sarcoplasmic reticulum. The calcium pump is activated by cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban, an integral membrane protein of the sarcoplasmic reticulum. Using a heterobifunctional crosslinking agent which can be cleaved and photoactivated, we provide evidence for a direct interaction between the two proteins. Only the non-phosphorylated form of phospholamban interacts with the ATPase, demonstrating that phospholamban is an endogenous inhibitor that is removed from the ATPase by phosphorylation. Non-phosphorylated phospholamban interacts only with the calcium-free conformation of the ATPase and is released when it is converted to the calcium-bound state. We localized the site of interaction to a single peptide isolated after cyanogen bromide cleavage of the ATPase. The peptide derives from a domain just C-terminal to the aspartyl phosphate of the active site. This domain is unique to ATPases of the sarcoplasmic reticulum in that it has no homology with any other phosphorylation-type ion pump. The domain occurs in both slow- and fast-twitch isoforms of the ATPase, even though phospholamban is not expressed in fast-twitch muscles.

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