INTRAMOLECULAR DYNAMICS IN THE ENVIRONMENT OF THE SINGLE TRYPTOPHAN RESIDUE IN STAPHYLOCOCCAL NUCLEASE

The dipole relaxational dynamics in the environment of a single trypto phan residue Trp-140 in staphylococcal nuclease was studied by time-re solved (multi-frequency phase-modulation) spectroscopy and selective r ed-edge excitation. The long-wavelength position of the fluorescence s pectrum (at 343 nm) and the absence of red-edge excitation effects at 0 and 20 degrees C indicate that this residue is surrounded by very mo bile protein groups which relax on the subnanosecond time scale. For t hese temperatures (0-20 degrees C) the steady-state emission spectra d id not show the excitation-wavelength dependent shifts (red-edge effec ts) for excitation wavelengths from 295 to 308 nm; however, the anisot ropy decay rate is slow (tens of nanoseconds). This suggests that the spectral relaxation is due to mobility of the surrounding groups rathe r than :he motion of the tryptophan itself. The motions of the tryptop han surrounding are substantially retarded at reduced temperatures in viscous solvent (60% glycerol). The temperature dependence of the diff erence in position of fluorescence spectra at excitation wavelengths 2 95 and 305 nm demonstrate the existence of red-edge effect at sub-zero temperatures, reaching a maximum value at - 50 degrees C, where the s teady-state emission spectrum is shifted to 332 nm. The excitation and emission wavelength dependence of multi-frequency phase-modulation da ta at the half-transition point (-40 degrees C) demonstrates the exist ence of the nanosecond dipolar relaxations. At - 40 degrees C the time -dependent spectral shift is close to monoexponential with the relaxat ion time of 1.4 ns.