Structures and Dynamics of DNA-binding Protein Motifs

9419049识别特定DNA序列的帕尔默蛋白质基序是调节基因表达的蛋白质中普遍存在的成分；因此，解释特定识别的分子基础对于发展细胞转化的分子理论是必要的。这项研究的长期目标是定量阐明蛋白质结构域识别DNA的动力学和熵方面。本研究的初始阶段将使用核磁共振波谱来表征Gcn4 bZip同源二聚体在DNA存在和不存在的情况下的构象和动力学性质。在bZip类转录激活剂中，基本结构域识别螺旋只有在DNA存在的情况下才是稳定的。DNA识别的动力学特征被假设包括：(I)表面暴露的，推测是可移动的，游离蛋白质中的侧链在络合时被埋在DNA的主槽中，(Ii)蛋白质结合时DNA的低能量变形意味着变形部位的游离DNA的构象灵活性增加，(Iii)蛋白质主干的构象变化可能是必要的，以将-螺旋容纳到DNA的主槽中，以及(Iv)与小槽或磷酸骨架相互作用的辅助氨基酸残基在没有DNA的情况下可能无序和有序。在没有DNA的情况下，Gcn4 bZip结构域的(新生)结构和构象动力学将通过J耦合测量和自旋驰豫技术来表征，包括同核NOESY和ROESY实验以及异核R1、R2、R1p和NOE实验。在DNA存在的情况下，自旋弛豫实验将被用来获得关于络合物动力学性质的信息，这是对现有晶体结构结果的补充。从单个氨基酸残基的构型熵比较DNA结合自由能的动力学性质。与特定DNA序列结合的蛋白质对基因表达的调控至关重要。解释序列特异性识别的分子基础对于从根本上理解细胞转化至关重要。在过去，人们已经研究了DNA结合蛋白在DNA存在和不存在的情况下的三维结构，以确定蛋白质与DNA之间的相互作用，从而赋予序列特异性。本研究的主要假设是，蛋白质和DNA的内部运动(即分子结构的随时间变化)在调节序列特异性识别中也是重要的。这一假说将针对蛋白质Gcn4的DNA结合域进行检验，Gcn4是一种名为bZip转录激活因子的蛋白质。GCN4是检验上述假说的理想候选者，因为识别DNA分子的蛋白质部分在没有DNA的情况下被认为是无序的(大幅度运动)，而在DNA存在的情况下是刚性的(小幅度运动)。核磁共振光谱分析原子在极强磁场中的性质，将被用来表征GCN4的动力学性质。***

9419049 Palmer Protein motifs recognizing specific DNA sequences are ubiquitous components of proteins that regulate gene expression; consequently, explication of the molecular basis for specific recognition is necessary for development of a molecular theory of cellular transformation. The long term goal of the research is to elucidate quantitatively the dynamical and entropic aspects of DNA recognition by protein domains. The initial phase of this research will use NMR spectroscopy to characterize the conformational and dynamical properties of the GCN4 bZip homodimer, in the presence and absence of DNA. In the bZip classes of transcriptional activators, the basic domain recognition helices are stable only in the presence of DNA. Dynamical features of DNA-recognition are hypothesized to include: (i) surface exposed, presumably mobile, side chains in the free protein are buried in the major groove of the DNA upon complexation, (ii) low energy deformation of the DNA upon protein binding implies increased conformational flexibility of the free DNA at the site of the deformation, (iii) conformational changes in the backbone of the protein may be necessary to accommodate the -helix into the major groove of the DNA, and (iv) ancillary amino acid residues that interact with the minor groove or phosphate backbone may be disordered in the absence of DNA and ordered in the complex. The (nascent) structure and conformational dynamics of the GCN4 bZip domain in the absence of DNA will be characterized using J-coupling measurements and spin relaxation techniques, including homonuclear NOESY and ROESY experiments, and heteronuclear R1, R2, R1p, and NOE experiments. In the presence of DNA, spin relaxation experiments will be used to obtain information on the dynamical properties of the complex that is complementary to the existing crystallographic structural results. Comparison of the dynamical properties of the free energy of DNA binding from configurational entropy of individual amino acid residues. %%% Proteins that bind to specific DNA sequences are critical for regulation of gene expression. Explication of the molecular basis for sequence-specific recognition is crucial to a fundamental understanding of cellular transformation. In the past, the three- dimensional structures of DNA-binding proteins in the absence and presence of DNA have been studied to determine the interactions between the protein and DNA that confer sequence specificity. The main hypothesis of the present research is that internal motions of the proteins and DNA (i.e. time-dependent changes in the structures of the molecules) also are important in modulating sequence- specific recognition. The hypothesis will be tested for the DNA- binding domain of the protein GCN4, a member of class of proteins called bZip transcriptional activators. GCN4 is an ideal candidate for testing the above hypothesis, because the portions of the protein that recognize the DNA molecule are presumed to be disordered (with large amplitude motions) in the absence of DNA and rigid (with small amplitude motions) in the presence of DNA. Nuclear magnetic resonance spectroscopy (NMR), which analyzes the properties of atomic in very strong magnetic fields, will be used characterize the dynamical properties of GCN4. ***