TIME RESOLVED FLUORESCENCE SPECTROSCOPY

时间分辨荧光光谱

• 批准号: 6432667
• 负责人: JAY R KNUTSON
• 金额: --
• 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/6432667
• 关键词: biomedical equipment development; conformation; fluorescence spectrometry; human immunodeficiency virus; integrase; intermolecular interaction; laser spectrometry; neuronal transport; prazosin; protease inhibitor; protein folding; protein structure; spectrophosphorimetry; stop flow technique; time resolved data; virus DNA; virus infection mechanism; virus protein

Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry. Fluorometry can provide unique insights into the structure, assembly and flexibility of complex macromolecules. We continue to develop new laser-based technology for such studies.1) This year, we continued our collaborative studies into oligomerization and DNA binding of HIV-integrase, the enzyme used by the AIDS virus to incorporate itself into human DNA. We employed single-tryptophan containing mutant proteins to make site-specific observations of distance between the Trp and a fluorophore at the end of a 35bp oligonucleotide from the viral LTR (long terminal repeat) . We found, unexpectedly, that Trps in either the "DNA binding" domain or located near the center of the "catalytic core" domain were not significantly quenched by the Marina-blue labeled DNA, but Trp in the zinc-finger containing N domain (thought to mediate oligomerization) was. Thus the oligomerization domain, not the core, is within about 26A from the DNA end after "3prime processing", the first step in viral incorporation. These resonance energy transfer (distance measuring) experiments are intended to help us resolve the architecture of the entire DNA-protein complex. This task is impossible with alternative methods. Our current distance constraints will soon be supplanted by similar measurements using labeled single-cysteine versions of the protein (mutants were prepared and recently purified).One can imagine our task as if we were building a "scaffold" of distances that the complex fits in. Hopefully, this kind of structural insight can help us design appropriate drugs.2) We completed and published collaborative studies into the "molten globule" states of apomyoglobins and protein G, using DAS (decay-associated spectra) and time-resolved anisotropy to look for changes in tryptophan environments and motions. We found that the flexibility of apomyoglobin A helix side chains were restricted not only in native, but also in "molten globule" states. We found that detailed time-resolved data can resolve and identify the nature of several folding intermediates for GB1 (B1domain of protein G) that are unseen by other methods.3) We completed and published similar studies on the pH-dependent multimerization of R67 DHFR, an enzyme responsible for antibiotic (trimethoprim) resistance. We also prepared for "double kinetic" studies (fluorescence lifetime and time resolved anisotropy measured during folding reactions) on this protein.4) We built and calibrated two new new femtosecond laser-driven "upconversion"fluorometers ( a sort of laser strobe light for Trp fluorescence )with better than 100 femtosecond time resolution. We began measuring very early events (solvent relaxation, internal conversion, picosecond rotations) in tryptophan fluorescence that reflect on the polarity of its surroundings, to help us better interpret the behavior we observe for Trp in proteins. We found picosecond solvent relaxation (color shifts) due to the sudden increase in electric dipole strength of excited Trp. We also found that Trp measurements are a valid reflection of the environment after only a few tens of femtoseconds- so fluorescence will be a valid benchmark when it is used to crosscheck molecular dynamics simulations.

时间分辨荧光光谱是生物化学的有力工具。 荧光测定法可以提供对复杂大分子的结构、组装和柔性的独特见解。我们将继续为此类研究开发基于激光的新技术。1）今年，我们继续对HIV整合酶的寡聚化和DNA结合进行合作研究，HIV整合酶是艾滋病病毒用于将自身整合到人类DNA中的酶。我们采用含有单色氨酸的突变体蛋白质，对来自病毒LTR（长末端重复序列）的35 bp寡核苷酸末端的Trp和荧光团之间的距离进行位点特异性观察。 出乎意料的是，我们发现，在“DNA结合”结构域或位于“催化核心”结构域的中心附近的Trp不被Marina蓝标记的DNA显著淬灭，但在含有锌指的N结构域（被认为介导寡聚化）中的Trp被淬灭。因此，寡聚化结构域，而不是核心，在“3 prime加工”（病毒掺入的第一步）之后，在距离DNA末端约26 A内。这些共振能量转移（距离测量）实验旨在帮助我们解决整个DNA-蛋白质复合物的结构。 用其他方法是不可能完成这项任务的。我们目前的距离限制将很快被类似的测量所取代，这些测量使用的是标记的单半胱氨酸蛋白质（突变体已经制备好，最近才纯化出来）。希望这种结构洞察力可以帮助我们设计合适的药物。2）我们完成并发表了对脱辅基肌球蛋白和蛋白G的“熔融球”状态的合作研究，使用DAS（衰变相关光谱）和时间分辨各向异性来寻找色氨酸环境和运动的变化。我们发现，不仅在天然的，但也在“熔融球”的状态下，限制的灵活性的脱辅基肌红蛋白A螺旋侧链。我们发现详细的时间分辨数据可以解析和识别GB 1（蛋白G的B1结构域）的几个折叠中间体的性质，这是其他方法所看不到的。3）我们完成并发表了关于R67 DHFR（一种负责抗生素（甲氧苄啶）抗性的酶）的pH依赖性多聚化的类似研究。 我们还准备对这种蛋白质进行“双动力学”研究（折叠反应期间测量的荧光寿命和时间分辨各向异性）。4）我们构建并校准了两个新的新型飞秒激光驱动的“上转换”荧光计（一种激光频闪灯用于Trp荧光）具有优于100飞秒的时间分辨率。我们开始测量色氨酸荧光中反映其周围极性的非常早期的事件（溶剂弛豫，内部转换，皮秒旋转），以帮助我们更好地解释我们在蛋白质中观察到的Trp行为。 我们发现皮秒溶剂松弛（色移）由于激发色氨酸的电偶极子强度的突然增加。 我们还发现，色氨酸测量是一个有效的反映环境后，只有几十飞秒-所以荧光将是一个有效的基准时，它被用来交叉检查分子动力学模拟。