Merging fluorescence resonance energy transfer and expressed protein ligation to analyze protein-protein interactions.
Determination of protein oligomerization state can be technically challenging. We have combined the methods of expressed protein ligation (EPL) and fluorescence resonance energy transfer (FRET) for the analysis of protein homo-oligomerization states. We have attached fluorescein (donor) and rhodamine (acceptor) chromophores via dipeptide linkages to the C-termini of three recombinant proteins and examined the potential for FRET between mixtures of these semisynthetic proteins. The known protein dimer (glutathione S-transferase) showed evidence of FRET and the known protein monomer (SH2 domain phosphatase-1) did not display FRET. Using this method, the previously uncharacterized circadian rhythm enzyme, serotonin N-acetyltransferase, displayed significant FRET, indicating its likely propensity for dimerization or more complex oligomerization. These results establish the potential of the union of EPL and FRET in the analysis of protein-protein interactions and provide insight into the unusual enzymatic behavior of a key circadian rhythm enzyme.
Scheibner
Kara A
KA
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Rm. 316, Hunterian Bldg., Baltimore, MD 21205, USA.
Zhang
Zhongsen
Z
Cole
Philip A
PA
D000595:Amino Acid Sequence; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019793:Fluorescein / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D054645:Protein Phosphatase 1; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D013050:Spectrometry, Fluorescence
D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D001191:Arylamine N-Acetyltransferase; D005982:Glutathione Transferase; D054645:Protein Phosphatase 1; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D019793:Fluorescein