Time Resolved Fluorescence Spectroscopy

时间分辨荧光光谱

• 批准号: 8149481
• 负责人: JAY R KNUTSON
• 金额: $ 28.27万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149481
• 关键词: Binding; Biochemical; Cardiac Myocytes; Crystalline Lens; Crystallins; Dipeptides; Dyes; Fluorescence; Fluorescence Spectroscopy; G-Quartets; Heterogeneity; Image; Learning; Light; Process; Protein Dynamics; Time; base; cofactor; insight; prevent; protein function

Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure and assembly of macromolecular complexes. This year, we pursued protein-protein association , ultrafast protein solvation, and probes of mitochondrial energetics. We also continued studies of DNA using fluorescent nucleotide analogs that reveal disruptions in DNA structures, publishing accounts of analog base pairing effects. We continued and expanded our femtosecond upconversion studies of Trp in proteins and peptides to quantify early "quasistatic self-quenching" processes. We found extremely rapid (10-100ps) decays are important in several proteins (crystallins, thioredoxin, GB1,etc.), as they detect previously silent conformers engaged in ultrafast charge transfer. Our earlier study of protein *solvation* on the 330fs-200ps time scale, using proteins such as Monellin, found QSSQ that others attributed to a class of unique water molecules that desorb from protein in 20ps. Local quenching is the dominant mechanism in all but a few cases we have studied. The QSSQ was found even in simple dipeptides (just published)(suggesting a general process underlies all protein QSSQ) and we suggest that in crystallins QSSQ is an important factor in preventing eye lens cataract formation. We contined collaborative studies with LCE into the status of a primary fuel of heart muscle mitochondria- NADH. Our efforts distinguish free and bound populations of NADH by their different fluorescence lifetimes, and in collaboration with Light Microscopy Core and LCE, we are refining 'Decay-Associated Images' software to more rapidly extract profiles of NADH binding within isolated cardiac myocytes. We studied the structural determinants of lifetime for pteridine-based fluorophores that mimic (to the point of base-pairing) the behavior of natural DNA bases, learning which neighbors provide a rigid base "sandwich" and which permit "flip-out". We employed the same probes to look at the G-quadruplex formation within DNA (important in teleomeres , division, and regulation).

时间分辨荧光光谱是生物化学的有力工具;它可以为大分子复合物的结构和组装提供独特的见解。今年，我们追求蛋白质-蛋白质关联，超快蛋白质溶剂化，和线粒体能量学的探针。 我们还继续使用荧光核苷酸类似物研究DNA，揭示DNA结构的破坏，发表模拟碱基配对效应的帐户。 我们继续并扩展了我们对蛋白质和肽中色氨酸的飞秒上转换研究，以量化早期的“准静态自淬灭”过程。 我们发现极快速（10- 100 ps）衰变在几种蛋白质（晶体蛋白、硫氧还蛋白、GB 1等）中是重要的，因为它们检测到以前沉默的构象参与超快电荷转移。 我们早期在330 fs-200 ps时间尺度上对蛋白质 * 溶剂化 * 的研究，使用莫内林等蛋白质，发现了QSSQ，其他人将其归因于一类独特的水分子，这些水分子在20 ps内从蛋白质中解吸。 除了我们研究过的少数情况外，局部猝灭是所有情况下的主要机制。 甚至在简单的二肽中也发现了QSSQ（刚刚发表）（表明所有蛋白质QSSQ的一般过程），我们认为在晶体蛋白中QSSQ是预防眼透镜白内障形成的重要因素。 我们继续与LCE合作研究心肌线粒体的主要燃料- NADH的状态。我们的努力通过不同的荧光寿命来区分游离和结合的NADH群体，并与光学显微镜核心和LCE合作，我们正在改进“衰变相关图像”软件，以更快地提取分离的心肌细胞内的NADH结合谱。 我们研究了结构决定因素的寿命为蝶啶为基础的荧光团，模仿（点的碱基配对）的行为的天然DNA碱基，学习哪些邻居提供了一个刚性的基地“三明治”，并允许“翻转”。 我们使用相同的探针来观察DNA中G-四链体的形成（在端粒、分裂和调控中很重要）。