INFRARED STUDIES OF GUANIDINE HC1 PROTEIN DENATURATION

蛋白质结构敏感的酰胺I带的红外光谱 将应用于盐酸胍诱导蛋白质变性的研究 种类繁多的蛋白质。我们将特别强调 变性状态下的结构分析。考虑到增加的兴趣 蛋白质的变性状态在其折叠过程中的可能作用 这类数据将引起人们的广泛兴趣。胍-盐酸变性 是研究蛋白质热力学最常用的方法之一。 因此，关于盐酸胍-变性状态的结构信息是 非常重要。制药业目前也非常 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 蛋白质可能对这个行业至关重要。红外光谱学有一个 研究蛋白质变性的一些优点。它很快， 价格低廉，不受构象平均效应的影响 核磁共振波谱。 蛋白质将使用红外线技术进行分析以进行监测 使用我们实验室以前开发的盐酸胍变性方法 ISO-1-细胞色素c.所选择的蛋白质，14溶菌酶，核糖核酸酶A，核糖核酸酶 T1、去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，并涵盖一系列天然状态结构类型。 在水中用傅里叶变换红外光谱方法获得光谱 解决方案使用6微米路径长度的单元。缓冲区和水蒸气将是 减去，然后仔细减去盐酸胍的红外光谱 乐队。在Savistsky-Golay平滑后，谱将被去卷积 使用二阶导数方法。结构类型的分配将是 基于现有的酰胺I带的经验指认。 为了提高该方法的序列特异性分辨率，制备 还提出了用半合成方法标记~(13)C的蛋白质。这 这项工作将包括诱变iso-1-细胞色素c以产生氰化物。 C-末端α-螺旋前面的溴化物裂解位点。使用 研究较多的溴化氰在蛋氨酸残基上的裂解方法 随后是自催化片段降级，13C标记的羰基将 在C-末端α-螺旋的特定位置引入。37岁的 由该标记产生的酰胺I带中的cm(-1)移动将允许 由C-末端α-产生的墨带的直接指定 螺旋。这项任务将对澄清现有的 酰胺I带二级结构指认中的歧义。这个 标记的蛋白质也将允许盐酸胍的作用 待观察的iso-1-细胞色素c特定片段的变性 直接去吧。

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.