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INFRARED STUDIES OF GUANIDINE HC1 PROTEIN DENATURATION

盐酸胍蛋白质变性的红外研究

基本信息

批准号：
2193664
负责人：
BRUCE E BOWLER
金额：
$ 10.15万
依托单位：
UNIVERSITY OF DENVER (COLORADO SEMINARY)
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-06-01 至 1998-08-31
项目状态：
已结题

项目摘要

Infrared spectroscopy of the structure-sensitive amide I band of proteins will be applied to the study of guanidine-HCl-induced protein denaturation of a broad range of proteins. Particular emphasis will be placed on analysis of structure in the denatured state. Given the increased interest in the possible role of the denatured states of proteins in their folding properties such data will be of wide interest. Guanidine-HCl denaturation is one of the most used methods to study protein thermodynamics. Structural information on the guanidine-HCl-denatured state is therefore very important. The pharmaceutical industry is also currently very interested in the effects of protein denaturation in drug formulations. Improved spectroscopic methods and understanding of the denatured state of proteins could be crucial to this industry. Infrared spectroscopy has a number of advantages for studying protein denaturation. It is fast, inexpensive and not subject to conformational averaging effects as with NMR spectroscopy. Proteins will be analyzed using the infrared technique for monitoring guanidine-HCl denaturation previously developed in our laboratory using iso-1-cytochrome c. The proteins of choice, 14 lysozyme, RNase A, RNase T1, apomyoglobin and staphylococcal nuclease are all widely studied models for protein folding and cover a range of native state structural types. Spectra will be acquired by Fourier transform infrared methods in H2O solution using 6 mu m pathlength cells. Buffer and water vapor will be subtracted, followed by careful subtraction of the guanidine-HCl infrared band. After Savistsky-Golay smoothing the spectra will be deconvoluted using second derivative methods. Assignment of structural types will be based on existing empirical assignments of the amide I band. To enhance the sequence-specific resolution of this method, preparation of 13C-labelled proteins using semisynthesis methods is also proposed. This work will involve mutagenesis of iso-1-cytochrome c to produce cyanogen bromide cleavage sites in front of the C-terminal alpha-helix. Using the well-studied methods of cyanogen bromide cleavage at methionine residues followed by autocatalytic fragment relegation, 13C-labelled carbonyls will be introduced at specific positions in the C-terminal alpha-helix. The 37 cm(-1) shift in the amide I band produced by this labelling will allow direct assignment of the inked band resulting from the C-terminal alpha- helix. This assignment will be important in clarifying existing ambiguities in secondary structure assignment of the amide I band. The labelled proteins will also allow the effects of guanidine-HCl denaturation on a specific segment of iso-1-cytochrome c to be observed directly.

蛋白质酰胺I结构敏感带的红外光谱研究将应用于盐酸胍诱导蛋白质变性的研究一系列蛋白质。将特别强调在变性状态下的结构分析。鉴于人们对蛋白质的变性状态在其折叠过程中的可能作用这些数据将引起广泛的兴趣。盐酸胍变性是研究蛋白质热力学最常用的方法之一。因此，关于胍-HCl变性状态的结构信息是非常重要的.制药行业目前也非常对药物制剂中蛋白质变性的影响感兴趣。改进的光谱方法和对变性状态的理解蛋白质对这个行业至关重要。红外光谱具有研究蛋白质变性的许多优点。它很快，便宜，不受构象平均效应的影响，核磁共振波谱法。蛋白质将使用红外技术进行分析，盐酸胍变性以前在我们的实验室开发，使用异-1-细胞色素c。选择的蛋白质，14溶菌酶，核糖核酸酶A，核糖核酸酶 T1、脱辅基肌红蛋白和葡萄球菌核酸酶都是被广泛研究的模型用于蛋白质折叠，并覆盖一系列天然状态的结构类型。在水溶液中用傅里叶变换红外方法获得光谱解决方案使用6 μ m光程的细胞。缓冲液和水蒸气将减去，然后仔细减去盐酸胍红外乐队Savistsky-Golay平滑后，将对光谱进行去卷积使用二阶导数方法。结构类型的分配将基于酰胺I谱带的现有经验归属。为了提高该方法的序列特异性分辨率，制备了还提出了使用半合成方法的13 C-标记蛋白质。这工作将涉及异-1-细胞色素c的诱变以产生氰溴裂解位点在C-末端α-螺旋的前面。使用在甲硫氨酸残基上进行溴化氰裂解的研究方法随后是自催化片段降级，13 C标记的羰基将在C-末端α-螺旋的特定位置引入。的37 通过这种标记产生的酰胺I带的cm（-1）位移将允许直接归属C-末端α- 螺旋。这项任务将是重要的澄清现有的酰胺I带二级结构归属的不明确性。的标记的蛋白质也将允许盐酸胍的作用，观察异-1-细胞色素c特定片段的变性直接.