Fluorescence lifetime study of ECFP/EYFP labeled MBP in Forster resonance energy transfer

Abstract

This paper studies the conformational change of the binding protein by a fluorescence lifetime method As a model protein maltose binding protein (MBP) where enhanced cyan protein (ECFP) and enhanced yellow fluorescent protein (EYFP) were genetically fused to act as a donor and an acceptor in Forster resonance energy transfer (FRET) was used The ECFP and the EYFP were linked to the C-terminal and N-terminal regions of the MBP respectively In order to investigate the conformational change of the MBP the lifetime of the ECFP which acts as a donor in the ECFP MBP EYFP fusion protein was analyzed during the FRET process We observed that two lifetime components exist when the ECFP is linked to the MBP and that the lifetime of the ECFP is shortened when ECFP MBP EYFP protein undergoes a conformational change as a result of the maltose binding In addition we observed that the lifetime of the donor is gradually shorter in the ECFP MBP EYFP fusion protein as the maltose concentration increases By a lifetime analysis and simulation study we found that the participant rate of the ECFP MBP EYFP protein in FRET is the main cause of the donor lifetime shortening in relation to the increase of the maltose concentration (C) 2010 Elsevier B V All rights reserved