TY - JOUR
T1 - Determination of protein secondary structure and solvent accessibility using site-directed fluorescence labeling. Studies of T4 lysozyme using the fluorescent probe monobromobimane
AU - Mansoor, Steven E.
AU - Mchaourab, Hassane S.
AU - Farrens, David L.
PY - 1999/12/7
Y1 - 1999/12/7
N2 - We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix- turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measurements show that introduction of the label is substantially perturbing only when it is located at buried residue sites. At buried sites (solvent surface accessibility of <40 Å2), the destabilizations are between 3 and 5.5 kcal/mol, whereas at more exposed sites, ΔΔG values of ≤ 1.5 kcal/mol are obtained. Of all the fluorescence parameters that were explored (excitation λ(max), emission λ(max), fluorescence lifetime, quantum yield, and steady-state anisotropy), the emission λ(max) and the steady-state anisotropy values most accurately reflect the solvent surface accessibility at each site as calculated from the crystal structure of cysteine-less T4 lysozyme. The parameters we identify allow 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.
AB - We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix- turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measurements show that introduction of the label is substantially perturbing only when it is located at buried residue sites. At buried sites (solvent surface accessibility of <40 Å2), the destabilizations are between 3 and 5.5 kcal/mol, whereas at more exposed sites, ΔΔG values of ≤ 1.5 kcal/mol are obtained. Of all the fluorescence parameters that were explored (excitation λ(max), emission λ(max), fluorescence lifetime, quantum yield, and steady-state anisotropy), the emission λ(max) and the steady-state anisotropy values most accurately reflect the solvent surface accessibility at each site as calculated from the crystal structure of cysteine-less T4 lysozyme. The parameters we identify allow 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.
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U2 - 10.1021/bi991331v
DO - 10.1021/bi991331v
M3 - Article
C2 - 10587464
AN - SCOPUS:0033534145
SN - 0006-2960
VL - 38
SP - 16383
EP - 16393
JO - Biochemistry
JF - Biochemistry
IS - 49
ER -