Hiller Sebastian, Joss Rosmarie, Wider Gerhard
Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
J Am Chem Soc. 2008 Sep 10;130(36):12073-9. doi: 10.1021/ja803161d. Epub 2008 Aug 19.
This paper describes an automated method for sequence-specific NMR assignment of the aliphatic resonances of protein side chains in small- and medium-sized globular proteins in aqueous solution. The method requires the recording of a five-dimensional (5D) automated projection spectroscopy (APSY-) NMR experiment and the subsequent analysis of the APSY peak list with the algorithm ALASCA (Algorithm for local and linear assignment of side chains from APSY data). The 5D APSY-HC(CC-TOCSY)CONH experiment yields 5D chemical shift correlations of aliphatic side chain C-H moieties with the backbone atoms H(N), N, and C'. A simultaneous variation of the TOCSY mixing times and the projection angles in this APSY-type TOCSY experiment gives access to all aliphatic C-H moieties in the 20 proteinogenic amino acids. The correlation peak list resulting from the 5D APSY-HC(CC-TOCSY)CONH experiment together with the backbone assignment of the protein under study is the sole input for the algorithm ALASCA that assigns carbon and proton resonances of protein side chains. The algorithm is described, and it is shown that the aliphatic parts of 17 of the 20 common amino acid side chains are assigned unambiguously, whereas the remaining three amino acids are assigned with a certainty of above 95%. The overall feasibility of the approach is demonstrated with the globular 116-residue protein TM1290, for which reference assignments are known. For this protein, 97% of the expected side chain carbon atoms and 87% of the expected side chain protons were detected with the 5D APSY-HC(CC-TOCSY)CONH experiment in 24 h of spectrometer time, and all these resonances were correctly assigned by ALASCA. Based on the experience with TM1290, we expect that the approach presented in this work is routinely applicable to globular proteins with sizes up to at least 120 amino acids.
本文描述了一种用于在水溶液中对中小型球状蛋白质的蛋白质侧链脂肪族共振进行序列特异性核磁共振（NMR）归属的自动化方法。该方法需要记录一个五维（5D）自动化投影光谱（APSY-）NMR实验，并随后使用算法ALASCA（从APSY数据进行侧链局部和线性归属的算法）对APSY峰列表进行分析。5D APSY-HC(CC-TOCSY)CONH实验产生脂肪族侧链C-H基团与主链原子H(N)、N和C'的5D化学位移相关性。在这个APSY型TOCSY实验中同时改变TOCSY混合时间和投影角度，可以获取20种蛋白质原氨基酸中的所有脂肪族C-H基团。5D APSY-HC(CC-TOCSY)CONH实验产生的相关峰列表以及所研究蛋白质的主链归属是用于指定蛋白质侧链碳和质子共振的算法ALASCA的唯一输入。文中描述了该算法，并表明20种常见氨基酸侧链中的17种的脂肪族部分被明确归属，而其余三种氨基酸的归属确定性高于95%。通过已知参考归属的116个残基的球状蛋白质TM1290证明了该方法的整体可行性。对于该蛋白质，在24小时的光谱仪时间内，通过5D APSY-HC(CC-TOCSY)CONH实验检测到了97%的预期侧链碳原子和87%的预期侧链质子，并且所有这些共振都被ALASCA正确归属。基于对TM1290的经验，我们预计本文提出的方法通常适用于大小至少为120个氨基酸的球状蛋白质。
