Protein DNA Interactions Probed by UV Resonance Raman Spectroscopy

9507241 Mukerji这项研究使用光谱和生物化学方法相结合的方法，研究由蛋白质结合介导的遗传调控过程中的分子识别和特异性。通过使用紫外共振拉曼光谱(UVRR)直接研究定义蛋白质-核苷酸界面的分子接触，可以了解这些过程。这项光谱技术通过监测核苷酸、外环、氨基和羰基的氢键配对来研究蛋白质-DNA相互作用的序列特异性。此外，通过调谐到吸收带来增强拉曼效应；因此，可以通过改变激发波长来探测二元络合物的不同区域。因此，除了识别赋予结合位点特异性的接触，UVRR光谱还监测蛋白质和DNA的构象变化。对HU和整合宿主因子(IHF)这两种蛋白质进行了研究，这两种蛋白质都是一类原核组蛋白样蛋白。这些蛋白质通过结合到小沟和弯曲DNA来促进调节蛋白的结合和作用。以前描述的大多数蛋白质-核酸相互作用通常涉及蛋白质-螺旋和主沟之间的直接接触。在HU和IHF中，结合和识别发生在-链和小槽之间，代表了不同类别的相互作用。到目前为止，这种类型的蛋白质-DNA相互作用的晶体结构很少，这项研究提供了一种机制来识别赋予核小体复合体特异性和稳定性的因素。理解细胞的生长和发育的核心是对调节生长的因素的理解。在最基本的层面上，这包括了解基因是如何工作的，以及导致它们“开启或关闭”的因素。在这项研究中，通过检查基因调控蛋白和基因或DNA之间的界面来研究调控的机制。这些蛋白质与基因结合，促进或抑制基因的功能。要了解这些蛋白质是如何发挥作用的，重要的是详细了解它们是如何与DNA相互作用的。激光光谱技术，紫外共振拉曼光谱，被用来研究蛋白质和DNA之间的界面。这种方法监测分子的内部振动，这是对分子结构和成键的敏感测量。使用这项技术，可以选择性地检查蛋白质或DNA的不同部分，从而可以直接调查重要区域和接触点。这些实验探索调节蛋白和基因之间形成的复合体，并有助于理解遗传功能是如何在细胞中调节的。***

9507241 Mukerji This research addresses molecular recognition and specificity in genetic regulatory processes which are mediated by protein binding, using a combination of spectroscopic and biochemical approaches. An understanding of these processes is gained by directly studying the molecular contacts defining the protein-nucleotide interface, using UV resonance Raman (UVRR) spectroscopy. This spectroscopic technique investigates the sequence specificity of the protein-DNA interaction by monitoring the H-bond pairings of nucleotide exocyclic amino and carbonyl groups. Additionally, the Raman effect is enhanced by tuning into an absorption band; therefore, different regions of the binary complex can be probed by varying the excitation wavelength. Thus, in addition to identifying the contacts which confer specificity to the binding site, UVRR spectroscopy also monitors conformational changes in both the protein and DNA. Investigations are conducted on two proteins, HU and Integration Host Factor(IHF), both members of a class of prokaryotic histone- like proteins. These proteins facilitate the binding and action of regulatory proteins by binding to the minor groove and bending the DNA. The majority of previously characterized protein-nucleic acid interactions have typically involved direct contact between the protein -helix and the major groove. In HU and IHF, binding and recognition occurs between -strands and the minor groove, representative of a different class of interaction. To date very few crystal structures exist for this type of protein-DNA interaction and this research provides a mechanism for identifying the factors which confer specificity and stability to the nucleosome complex. %%% Understanding the growth and development of cells centers on an understanding of the factors that regulate growth. At the most basic level this involves knowing how genes work and the factors that cause them to "turn on or turn off." In this research the mec hanism of regulation is studied by examining the interface between genetic regulatory proteins and the gene or the DNA. These proteins bind to the gene and either promote or repress its function. To understand how these proteins function, it is important to know in detail how they interact with DNA. A laser spectroscopic technique, UV resonance Raman spectroscopy, is used to investigate the interface between the proteins and the DNA. This method monitors the internal vibrations of molecules, which are a sensitive measure of the structure and bonding of a molecule. Using this technique, different parts of the protein or the DNA can be selectively examined, thereby allowing direct investigation of the important regions and contact points. These experiments probe the complex formed between regulatory proteins and genes and contribute towards understanding how genetic function is regulated in the cell. ***