Luisi D L, Wu W J, Raleigh D P
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY, 11794-3400, USA.
J Mol Biol. 1999 Mar 26;287(2):395-407. doi: 10.1006/jmbi.1999.2595.
There is considerable interest in the structure of the denatured state and in the role local interactions play in protein stability and protein folding. Studies of peptide fragments provide one method to assess local conformational preferences which may be present in the denatured state under native-like conditions. A set of peptides corresponding to the individual elements of secondary structure derived from the N-terminal domain of the ribosomal protein L9 have been synthesized. This small 56 residue protein adopts a mixed alpha-beta topology and has been shown to fold rapidly in an apparent two-state fashion. The conformational preferences of each peptide have been analyzed by proton nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. Peptides corresponding to each of the three beta-stands and to the first alpha-helix are unstructured as judged by CD and NMR. In contrast, a peptide corresponding to the C-terminal helix is remarkably structured. This 17 residue peptide is 53 % helical at pH 5.4, 4 degrees C. Two-dimensional NMR studies demonstrate that the helical structure is distributed approximately uniformly throughout the peptide, although there is some evidence for fraying at the C terminus. Detailed analysis of the NMR spectra indicate that the helix is stabilized, in part, by a native N-capping interaction involving Thr40. A mutant peptide which lacks Thr40 is only 32 % helical. pH and ionic strength-dependent studies suggested that charge charge interactions make only a modest net contribution to the stability of the peptide. The protein contains a trans proline peptide bond located at the first position of the C-terminal helix. NMR analysis of the helical peptide and of a smaller peptide containing the proline residue indicates that only a small amount of cis proline isomer (8 %) is likely to be populated in the unfolded state.
人们对变性状态的结构以及局部相互作用在蛋白质稳定性和蛋白质折叠中所起的作用非常感兴趣。对肽片段的研究提供了一种方法来评估在类似天然条件下变性状态中可能存在的局部构象偏好。已经合成了一组对应于核糖体蛋白L9 N端结构域二级结构各个元件的肽。这种由56个残基组成的小蛋白采用混合α-β拓扑结构，并且已显示以明显的两态方式快速折叠。通过质子核磁共振光谱和圆二色光谱分析了每种肽的构象偏好。根据圆二色光谱（CD）和核磁共振（NMR）判断，对应于三个β链和第一个α螺旋的肽是无结构的。相比之下，对应于C端螺旋的肽具有明显的结构。这个由17个残基组成的肽在pH 5.4、4℃时螺旋含量为53%。二维核磁共振研究表明，螺旋结构在整个肽中大致均匀分布，尽管有一些证据表明C端存在解旋现象。对核磁共振光谱的详细分析表明，该螺旋部分是通过涉及苏氨酸40的天然N-封端相互作用而稳定的。缺乏苏氨酸40的突变肽螺旋含量仅为32%。pH和离子强度依赖性研究表明，电荷相互作用对肽的稳定性仅产生适度的净贡献。该蛋白在C端螺旋的第一个位置含有一个反式脯氨酸肽键。对螺旋肽和含有脯氨酸残基的较小肽的核磁共振分析表明，在未折叠状态下可能只有少量的顺式脯氨酸异构体（8%）存在。
