Rowland Institute at Harvard, Cambridge, Massachusetts 02142, USA.
J Am Soc Mass Spectrom. 2010 May;21(5):707-18. doi: 10.1016/j.jasms.2010.01.009. Epub 2010 Jan 25.
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.
已经开发出了一些方法来测量被荧光染料衍生化的捕获生物分子离子的荧光寿命与温度的关系。以前对不同序列的多脯氨酸肽的测量表明，猝灭速率与构象及其空间波动有关。本文介绍了将这些方法扩展到研究更大生物分子构象动力学的结果。作为温度的函数，研究了万古霉素-肽非共价复合物在 1+电荷态下不同 W-KAA 肽手性（L-LDD、D-LDD、L-DLL）的构象动力学。发现这些不同手性的荧光猝灭速率 k(q)具有立体选择性，相对大小为 k(q)(L-LDD) > k(q)(D-LDD) > k(q)(L-DLL)。通过切换单个色氨酸残基的手性来检测到荧光猝灭的变化。W-KAA（L-LDD）和 W-KAA（L-DLL）形成的复合物的分子动力学分析表明，（L-DLL）复合物的灵活性增加与猝灭速率降低相关。还对 Trp-cage 蛋白进行了荧光测量，比较了 1+、2+和 3+电荷态下的猝灭速率，其中 k(q)(+)>>k(q)(2+)≈k(q)(3+)。对包含单点突变的序列的测量推断出在 1+电荷态下存在盐桥结构，而在 2+和 3+态下则不存在。Trp-cage 的分子动力学结构表明，在 1+电荷态下的盐桥会产生更紧凑的构象，从而根据猝灭机制导致更大的猝灭速率。在这两项实验研究中，荧光猝灭速率都与分子间或分子内相互作用引起的结构变化一致。
