Fluorescence lifetime probe of biomolecular conformations |
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Authors: | Xiangguo Shi Joel H Parks |
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Institution: | 1.The Rowland Institute at Harvard,Cambridge,USA |
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Abstract: | 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, kq, were found to be stereoselective for these different chiralities with relative magnitudes kq(L-LDD)>kq(D-LDD)>kq(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 kq+≫kq2+≈kq3+. 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. |
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Keywords: | |
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