Effective concentrations of amino acid side chains in an unfolded protein.
Effective concentrations of amino acid side chains in an unfolded protein.
复制标题
未折叠蛋白质中氨基酸侧链的有效浓度。
DOI：
10.1021/bi00233a010
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
B. Nall
中科院分区：
文献类型：
--
作者：
K. Muthukrishnan;B. Nall
研究了未折叠细胞色素c中链段之间的优先相互作用。该方法利用了血红素连接在未折叠的蛋白质，一个独特的功能与共价连接血红素的蛋白质。该方法允许估计一个多肽链段相对于另一个多肽链段的有效浓度，并且成功地检测由线性序列中不同数目的残基分开的肽链段的差异。该方法使用质子NMR光谱监测咪唑对组氨酸血红素配体的置换，因为盐酸胍未折叠细胞色素c用氘代咪唑滴定。当咪唑浓度超过组氨酸配体的有效（局部）浓度时，蛋白质配体被氘代咪唑取代。在位移时，组氨酸环质子共振从光谱的顺磁性区域移动到反磁性区域。已进行了滴定的线粒体细胞色素c家族的成员，含有不同数量的组氨酸残基。这些包括来自金枪鱼的细胞色素c（2）、酵母iso-2（3）和酵母iso-1-MS（4）。在高咪唑浓度下，出现在NMR谱的组氨酸环C2 H区域中的质子共振的数目比蛋白质中组氨酸残基的数目少一个。因此，一个组氨酸，可能是His-18，仍然是血红素配体。组氨酸-26、-33和-39相对于血红素（位置14-17）的有效局部浓度估计为（3-16）× 10（-3）M。（250字处删节）
Preferential interactions between chain segments are studied in unfolded cytochrome c. The method takes advantage of heme ligation in the unfolded protein, a feature unique to proteins with covalently attached heme. The approach allows estimation of the effective concentration of one polypeptide chain segment relative to another, and is successful in detecting differences for peptide chain segments separated by different numbers of residues in the linear sequence. The method uses proton NMR spectroscopy to monitor displacement of the histidine heme ligands by imidazole as guanidine hydrochloride unfolded cytochrome c is titrated with deuterated imidazole. When the imidazole concentration exceeds the effective (local) concentration of histidine ligands, the protein ligands are displaced by deuterated imidazole. On displacement, the histidine ring proton resonances move from the paramagnetic region of the spectrum to the diamagnetic region. Titrations have been carried out for members of the mitochondrial cytochrome c family that contain different numbers of histidine residues. These include cytochromes c from tuna (2), yeast iso-2 (3), and yeast iso-1-MS (4). At high imidazole concentration, the number of proton resonances that appear in the histidine ring C2H region of the NMR spectrum is one less than the number of histidine residues in the protein. So one histidine, probably His-18, remains as a heme ligand. The effective local concentrations of histidines-26, -33, and -39 relative to the heme (position 14-17) are estimated to be (3-16) X 10(-3) M.(ABSTRACT TRUNCATED AT 250 WORDS)