Global configuration of single titin molecules observed through chain-associated rhodamine dimers

The global configuration of individual, surface-adsorbed molecules of the g iant muscle protein titin, labeled with rhodamine conjugates, was followed with confocal microscopy. Fluorescence-emission intensity was reduced becau se of self-quenching caused by the close spacing between rhodamine dye mole cules that formed dinners. In the presence of chemical denaturants, fluores cence intensity increased, reversibly, up to 5-fold in a fast reaction; the kinetics were followed at the single-molecule level. We show that dinners formed in a concentrated rhodamine solution dissociate when exposed to chem ical denaturants. Furthermore, titin denaturation, followed by means of try ptophan fluorescence, is dominated by a slow reaction. Therefore, the rapid fluorescence change of the single molecules reflects the direct action of the denaturants on rhodamine dimers rather than the unfolding/refolding of the protein. Upon acidic denaturation, which we have shown not to dissociat e rhodamine dinners, fluorescence intensity change was minimal, suggesting that dinners persist because the unfolded molecule has contracted into a sm all volume. The highly contractile nature of the acid-unfolded protein mole cule derives from a significant increase in chain flexibility. We discuss t he potential implications this finding could have for the passive mechanica l behavior of striated muscle.