TIME RESOLVED FLUORESCENCE SPECTROSCOPY

时间分辨荧光光谱是生物化学研究的有力工具。荧光测定法可以提供对复杂大分子的结构、组装和灵活性的独特见解。我们继续为这类研究开发新的基于激光的技术1)今年，我们继续合作研究HIV整合酶的寡聚和DNA结合，HIV整合酶是艾滋病病毒用来将自己整合到人类DNA中的酶。我们使用含有单一色氨酸的突变蛋白来对Trp和病毒LTR(长末端重复序列)35bp寡核苷酸末端的荧光团之间的距离进行定点观察。我们意外地发现，无论是位于“DNA结合”结构域还是位于“催化核心”结构域中心附近的TRP，都不会被滨蓝标记的DNA显著猝灭，但含有N结构域的锌指结构域(被认为是介导齐聚)的Trp会被显著猝灭。因此，低聚结构域，而不是核心，在病毒掺入的第一步--“3Prime加工”后，距离DNA末端约26A。这些共振能量转移(距离测量)实验旨在帮助我们解析整个DNA-蛋白质复合体的结构。有了其他方法，这项任务是不可能完成的。我们目前的距离限制很快将被使用标记的单半胱氨酸蛋白质版本的类似测量所取代(突变体已经准备好并最近被提纯)。人们可以想象我们的任务就像我们正在建立一个距离的“脚手架”，这个复合体适合我们。希望这种结构洞察力能帮助我们设计合适的药物。2)我们完成并发表了对脱脂蛋白和G蛋白“熔化球状”状态的合作研究，使用DAS(衰变相关光谱)和时间分辨的各向异性来寻找色氨酸环境和运动的变化。我们发现，脱脂肌红蛋白A螺旋侧链的柔性不仅在天然状态下受到限制，而且在“熔融球状”状态下也受到限制。我们发现，详细的时间分辨数据可以解析和鉴定GB1(蛋白G的B1结构域)的几个折叠中间体的性质，这是其他方法所没有的。3)我们完成并发表了与R67 DHFR的pH相关的多聚体的类似研究，R67 DHFR是一种导致抗生素(甲氧嘧啶)耐药性的酶。我们还准备对这种蛋白质进行双动力学研究(在折叠反应中测量荧光寿命和时间分辨各向异性)。4)我们建立并校准了两个新的飞秒激光驱动的上转换荧光仪(一种用于Trp荧光的激光频闪光)，其时间分辨率优于100飞秒。我们开始测量色氨酸荧光中非常早期的事件(溶剂松弛、内部转换、皮秒旋转)，这些事件反映了色氨酸周围的极性，以帮助我们更好地解释我们在蛋白质中观察到的色氨酸行为。我们发现由于激发色氨酸的电偶极子强度的突然增加，出现了皮秒的溶剂弛豫(色移)。我们还发现，Trp测量只在几十飞秒后就能有效地反映环境-所以当它被用于交叉检验分子动力学模拟时，荧光将是一个有效的基准。

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.