Global configuration of single titin molecules observed through chain-associated rhodamine dimers.
The global configuration of individual, surface-adsorbed molecules of the giant muscle protein titin, labeled with rhodamine conjugates, was followed with confocal microscopy. Fluorescence-emission intensity was reduced because of self-quenching caused by the close spacing between rhodamine dye molecules that formed dimers. In the presence of chemical denaturants, fluorescence intensity increased, reversibly, up to 5-fold in a fast reaction; the kinetics were followed at the single-molecule level. We show that dimers formed in a concentrated rhodamine solution dissociate when exposed to chemical denaturants. Furthermore, titin denaturation, followed by means of tryptophan 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 dissociate rhodamine dimers, fluorescence intensity change was minimal, suggesting that dimers persist because the unfolded molecule has contracted into a small volume. The highly contractile nature of the acid-unfolded protein molecule derives from a significant increase in chain flexibility. We discuss the potential implications this finding could have for the passive mechanical behavior of striated muscle.
Grama
L
L
Department of Biophysics, Research Group of the Hungarian Academy of Sciences, Pécs University Medical School, Szigeti ut 12, Pécs, H-7624 Hungary.
Somogyi
B
B
Kellermayer
M S
MS
D000818:Animals; D064211:Connectin; D019281:Dimerization; D066298:In Vitro Techniques; D018613:Microscopy, Confocal; D009124:Muscle Proteins / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry; D011489:Protein Denaturation; D011494:Protein Kinases / Q000737:chemistry*; D011817:Rabbits; D012235:Rhodamines; D013053:Spectrophotometry
D064211:Connectin; D009124:Muscle Proteins; D012235:Rhodamines; D011494:Protein Kinases