The utility of the nitrotyrosine chromophore as a spectroscopic probe in troponin C and modulator protein

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Abstract
Rabbit skeletal and bovine cardiac troponin C (TN-C) and bovine brain protein modulator were nitrated by treatment with tetranitromethane. The resulting derivatives were characterized by Ca titration employing circular dichroism and by absorption difference spectroscopy. The nitrated proteins did not differ significantly in secondary structure from their native unmodified counterparts. Their response to Ca2+, as measured by the binding parameters n and K, is also comparable. Introduction of the nitrotyrosyl chromophore at specific residues in the protein chain allowed the performance of additional Ca titrations by the circular dichroism method. For each of the 3 proteins the ellipticity at 275 nm, a point of maximum change for this chromophore, was monitored as a function of Ca addition. The binding parameters obtained indicated that in each case a high affinity site was being probed. This is consistent with the known locations of the tyrosine residues in these polypeptide chains. Ca-induced absorption difference spectra were also generated for the 3 proteins in the wavelength range 550-250 nm. In the 320-550 nm region all 3 spectra were quite similar, suggesting a comparable change in the environment of the chromophoric moiety upon Ca2+-binding in all cases. Below 320 nm the spectra differed considerably. For skeletal TN-C, the large contribution from phenylalanine residues which has been noted previously for the native protein was still evident. For fully nitrated cardiac TN-C the contribution from nitrotyrosine at 280 nm is comparable to that for skeletal TN-C. For the partially nitrated sample the spectrum more closely resembles that of native cardiac TN-C, the major contribution coming from the 2.6 mol of unreacted tyrosine. The particularly large value of .DELTA..epsilon.290 nm of 600 M-1 cm-1 for the protein modulator was interpreted in terms of subtle interactions between the nitrotyrosine residues located at the 3rd and 4th Ca2+-binding sites producing an electronic enhancing effect. The 2 techniques demonstrate the sensitivity of the nitrotyrosyl chromophore to subtle changes in its microenvironment, making it a useful conformational probe in the continuing study of the molecular changes elicited when Ca binds to TN-C and homologous molecules.