Fluorescence lifetime probe of biomolecular conformations.

Methods have been developed to measure the fluorescence lifetime versus temperature of trapped biomolecular ions derivatized with a fluorescent dye. Previous measurements for different sequences of polyproline peptides demonstrated that quenching rates are related to conformations and their spatial fluctuations. This paper presents the results of extending these methods to study the conformational dynamics of larger biomolecules. Vancomycin-peptide noncovalent complexes in the 1+ charge state were studied as a function of temperature for different W-KAA peptide chiralities (L-LDD, D-LDD, L-DLL). Fluorescence-quenching rates, k(q), were found to be stereoselective for these different chiralities with relative magnitudes k(q)(L-LDD) > k(q)(D-LDD) > k(q)(L-DLL). The variation in fluorescent quenching resulting from switching the chirality of the single Trp residue was readily detectable. Molecular dynamics analysis of complexes formed by W-KAA (L-LDD) and W-KAA(L-DLL) indicates that increased flexibility in the (L-DLL) complex is correlated with reduced quenching rates. Fluorescence measurements were also performed for the Trp-cage protein comparing quenching rates in the 1+, 2+, and 3+ charge states for which k(q)(+) >> k(q)(2+) approximately k(q)(3+). Measurements of a sequence including a single-point mutation infer the presence of a salt-bridge structure in the 1+ charge state and its absence in both the 2+ and 3+ states. Molecular dynamics structures of Trp-cage indicate that a salt bridge in the 1+ charge state produces more compact conformations leading to larger quenching rates based on the quenching mechanism. In both these experimental studies the fluorescence-quenching rates were consistent with changes in structure induced by either intermolecular or intramolecular interactions.