Single and Two-Photon Sensitized Acceptor Emission and Anisotropy Studies of Protein-Protein Interactions

Donor lifetime modification by Forster Resonance Energy Transfer (FRET) is widely used to detect protein-protein interactions and protein conformation change. Such measurements can be compromised by the presence of a significant non-interacting fraction of molecules. We have recently shown that FRET arising from homodimerisation in 3-phosphoinositide dependent protein kinase 1 (PDK1) can be measured for a small fraction of the total protein population from the rise-time in sensitized acceptor emission [1]. We present single and two-photon lifetime and anisotropy measurements of homodimerisation in EGFP and mCherry labelled PDK1 constructs. A computational model was employed to determine the limits and relative probabilities of the FRET orientation parameter using intrinsic donor and acceptor anisotropy decays together with that of sensitized acceptor emission. A consideration of the physically allowable minimum separation of the donor and acceptor fluorescent proteins allows physically unrealistic values of the orientation parameter to be removed. This was seen to lead to a reduction in the uncertainty of the mean donor-acceptor distance. Combined wavelength-resolved lifetime and anisotropy measurements show promise as a valuable tool for obtaining accurate angular and distance information from FRET in samples containing a small proportion of interacting molecules.[1] Masters, T. J., V. Calleja, D. A. Armoogum, R. J. Marsh, C. J. Applebee, M. Laguerre, A. J. Bain and B. Larijani, 2010. “In vivo regulation of 3-phosphoinositide dependent protein kinase 1 (PDK1) activity by homodimerisation”. Science Signalling (in press).