Labeling of the beta gamma subunit complex of transducin with an environmentally sensitive cysteine reagent. Use of fluorescence spectroscopy to monitor transducin subunit interactions

Phillips, W.J.; Cerione, R.A.

Journal of Biological Chemistry 266(17): 11017-11024


ISSN/ISBN: 0021-9258
PMID: 2040617
Accession: 007502389

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In this study, we have examined the interactions of the .beta.gamma. subunit complex of the retinal GTP-binding protein transducin (.beta.gamma.T) with its .alpha. subunit (.alpha.T) using fluorescence spectroscopic approaches. The .beta.gamma.T subunit complex was covalently labeled with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), an environmentally sensitive fluorescent cysteine reagent. The formation of the MIANS .beta.gamma.T complexes (two to five MIANS adducts per .beta.gamma.T) resulted in 2-3-fold enhancements in the MIANS fluorescence, and 20-25-nm blue shifts in the fluroescence emission maxima, relative to the emission for identical concentrations of MIANS-labeled MIANS complexes. The addition of .alpha.T.cntdot.GDP to these MIANS .beta.gamma.T complexes resulted in an additional enhancement in the MIANS fluorescence (typically ranging from 20 to 40%) and a 5-10-nm blue shift in the wavelength for maximum emission. These fluorescence changes were specifically elicited by the GDP-bound form of .alpha.T and were not observed upon the addition of purified .alpha.T.cntdot.guanosine 5'-O-(3-thiotriphosphate) (GTP.gamma.S) complexes to the MIANS .beta.gamma.T species. Conditions which resulted in the activation of the .alpha.T.cntdot.GDP subunit (i.e. the addition of AIF4- or the addition of rhodopsin-containing vesicles and GTP.gamma.S) resulted in a reversal of the .alpha.T.cntdot.GDP-induced enhancement of the MIANS .beta.gamma.T fluorescence. Thus, the MIANS .beta.gamma.T fluorescence provided a spectroscopic monitor for transducin-subunit association and transducin-activation. Based on the results from studies using this spectroscopic read-out, it appears that the association of the .alpha.T.cntdot.GDP species with the .beta.gamma.T subunit complex to form the holotransducin molecule is rapid and does not limit the rate of the rhodopsin-stimulated activation of holotransducin. However, either the dissociation of the activated .alpha.T subunit from the .beta.gamma.T complex, or a conformational change in .beta.gamma.T complex, or a conformational change in .beta.gamma.T which occurs as a result of the subunit dissociation event, appears to be slow relative to the G protein-subunit association event.