Tryptophan fluorescence assay

The effect of CMP on the intrinsic tryptophan fluorescence of wild-type and mutant mCST was measured using a Photon Technology International dual-monochromator fluorometer. Since cytidine’s absorption spectrum (λ_max=270) partially overlaps that of tryptophan’s (λ_max=280), this leads to an apparent fluorescence quenching termed the inner-filter effect (Lakowicz, 2006). To account for this, we first minimized this effect by using a slightly higher excitation wavelength (300 nm) that is not as well absorbed by cytosine. What little inner-filter effect remained could be measured and corrected for by performing the CMP titration in the presence of mCST denatured with sodium dodecyl sulfate (SDS). A typical experiment involved putting 400 μl of 6 μM mCST into a quartz cuvette and then recording the emission spectrum from 315 to 500 nm. Subsequent spectra would then be recorded after adding 0.4–2.5 μl of a concentrated stock of CMP to achieve the desired concentration. An identical titration was performed in the presence of 2.5% SDS. A spectrum collected from a buffer-only sample was used for subtracting the background fluorescence from the spectra of protein samples. The λ_max for the tryptophan fluorescence of mCST was around 328 nm and did not change as a function of CMP concentration. Peak height was determined by averaging the values from 321 to 335 nm. To correct for the inner-filter effect, a correction factor for each CMP concentration was first calculated by dividing the peak height of the SDS-treated sample without CMP by the peak heights of each SDS-treated sample with various concentrations of CMP. The peak heights for the native protein samples were then multiplied by this correction factor. Correction values were typically very small up to 700 μM CMP (ranging between 1–1.03) and increased slightly to ~1.2 for 7 mM CMP. The corrected peak heights were then used to calculate fractional quenching as a function of CMP concentration. The quenching data were fit to a version of Equation 1 that lacked the S’ terms to determine the K_d’s for CMP.