Determination of protein secondary structure and solvent accessibility using site-directed fluorescence labeling. Studies of T4 lysozyme using the fluorescent probe monobromobimane

We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. I n our experiments, we used 21 consecutive single-cysteine substitution muta nts in T4 lysozyme (residues T115-K135), located in a helix-turn-helix moti f. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measu rements show that introduction of the label is substantially perturbing onl y when it is located at buried residue sites. At buried sites (solvent surf ace accessibility of <40 Angstrom(2)), the destabilizations are between 3 a nd 5.5 kcal/mol, whereas at more exposed sites, Delta Delta G values of les s than or equal to 1.5 kcal/mol are obtained. Of all the-fluorescence param eters that were explored (excitation lambda(max), emission lambda(max), flu orescence lifetime, quantum yield, and steady-state anisotropy), the emissi on lambda(max) and the steady-state anisotropy values most accurately refle ct the solvent surface accessibility at each site as calculated from the cr ystal structure of cysteine-less T4 lysozyme. The parameters we identify al low the classification of each site as buried, partially buried, or exposed . We find that the variations in these parameters as a function of residue number reflect the sequence-specific secondary structure, the determination of which is a key step for modeling a protein of unknown structure.