Analysis of the local conformation of proteins with two-dimensional fluorescence techniques

Two 2D fluorescence techniques are described which allow the study of confo rmational changes in proteins in their native form in muM solutions using a romatic amino acids (tryptophan, tyrosine) as intrinsic fluorescence marker s. Simultaneous time- and wavelength-resolved fluorescence spectra are meas ured using a 80 ps laser source in conjunction with streak detection in the exit plane of an astigmatism-corrected spectrometer. This approach allows identification of different photophysical processes by their associated lif etime and spectral intensity distribution; errors due to the more common in tegration over a wider spectral range are avoided. Time-resolved spectra ar e sensitive to changes in the collisional environment (dynamic quenching) a nd can thus be used to monitor local conformation changes close to the resp ective fluorophors. This is demonstrated for the Ras protein which undergoe s a drastic conformation change while binding to different nucleotides. Excitation-emission spectra are two-dimensional fluorescence images with on e axis corresponding to the excitation and the other to the emission wavele ngth. Thus, they contain all conventional excitation and fluorescence spect ra of a given substance. The 2D structure facilitates the interpretation of these spectra and allows the direct identification of resonance effects, s cattering and the isolation of the contribution of different fluorophors to the complete spectrum. This is demonstrated for mixtures of tyrosine and t ryptophan, In this case, both wavelength-resolved spectra and temporal deca ys are affected by energy transfer processes between the two amino acids. In a last example, both static and time-resolved spectral methods are combi ned to determine the respective contribution of static and dynamic quenchin g in calsequestrin. Evaluation of the fluorescence data is in good agreemen t with a recent crystallographic analysis which shows that all tryptophans are located in a conserved domain of the protein. Addition of Ca2+ ions lea ds to a more compact form of calse-questrin and to polymers. This informati on would not be obtainable from either of the two techniques alone.