A multidimensional spectrophotometer for monitoring thermal unfolding transitions of macromolecules.

We describe a multidimensional spectrometer that is capable of (nearly) simultaneous measurement of circular dichroism, steady-state fluorescence, and absorbance values on the same sample in a standard 1 x 1 cm cuvette. With a computer controlled thermoelectric cell holder, this instrument is capable of measuring the above types of spectral data at various wavelengths as a function of temperature. We have developed software to control the various acquisition functions and to convert the data files to a format appropriate for use with the nonlinear least squares program, NONLIN (Johnson and Frasier, 1985). We have tested various features of this instrument and we have applied this instrument and data analysis procedure to study the thermal unfolding of ribonuclease A, under conditions were the unfolding of this protein involves an intermediate state.