|
Using time-resolved nanosecond fluorescence spectroscopy, we investigated the conformational changes of skeletal and cardiac troponin C. A thiol specific fluorescence probe N-(iodoacety1)-N'-(5-sulfo-1-naphthyl)- ethylenediamine (IAEDANS) was attached to cysteine 98 of skeletal troponin C (STnC) and 2-(4'-iodoacetamido-anilino)-naphthalene-6-sulfonic acid (IAANS) was linked to cysteine 35 and 84 of cardiac troponin C (CTnC). With excitation at 340 nm for STnC-IAEDANS and at 335 nm for CTnC-IAANS, apo-STnC and apo-CTnC exhibited biexponential decay kinetics. At 20°C and neutral pH, the following lifetimes were observed: (1) apo-STnC, 9 and 16 ns, and (2) apo-CTnC, 2.3 and 7 ns. The long lived component of the emission in STnC-IAEDANS comprised ~ 61% of observed intensity, however, the corresponding component in CTnC contributed only. A decrease of pH from 7.2 to 5.2 induced an increase of the lifetimes 20% (STnC) and 10% (CTnC). These results suggest that Cys-98 of STnC and Cys-35 and Cys-84 of CTnC became less quenched by their neighboring residues at low pH. Addition of guanidine hydrochloride to STnC resulted in a decrease of ~30% of both lifetimes. The lifetimes increased slightly when the temperature was lowered. Variation of solution viscosity by addition of sucrose did not affect the long component of the lifetimes of STnC. However, the short component did sense a viscosity effect. These results suggest that there was likely a chromophore heterogeneity which may arise from differences in conformation, environment, and or ionization of the excited state of the chromophore. At 20°C and neutral pH, two rotational correlation times were observed: (1) apo-STnC, - 1.2 ns, .2 1 11.3 ns, and (2) apo-CTnC, .~ 0.6 ns, .2 1 13.6 ns. The short rotational correlation times likely reflect rapid motions of the chromophores covalently attached to the side chain of the cysteine residues, and the long correlation times reflect the overall protein motions. The .2 values suggest that both proteins were not highly asymmetric at neutral pH. In the presence of Ca2+, at 20°C and neutral pH the rotational correlation times were (1) STnC, .1 ~ 2.1 ns, .2 1 13.9 ns, and (2) CTnC, .1 ~ 1.9 ns, .2 1 13.0 ns. A decrease of pH from 7.2 to 5.2 led to an increase of .2 ~ 20% for STnC and a reduction of .2 ~ 7% for CTnC. The two proteins appear to have different hydrodynamic properties in an acidic environment.
|
