Alam S L, Volkman B F, Markley J L, Satterlee J D
Department of Chemistry, Washington State University, Pullman 99164-4630, USA.
J Biomol NMR. 1998 Feb;11(2):119-33. doi: 10.1023/a:1008202621725.
Complete 13C, 15N, and 1H resonance assignments have been obtained for the recombinant, ferrous CO-ligated from of component IV monomeric hemoglobin from Glycera dibranchiata. This 15642 Da myoglobin-like protein contains a large number of glycine and alanine residues (47) and a heme prosthetic group. Coupling constant information has allowed the determination of chi(1) and chi(2) torsion angles, backbone phi angles, as well as 43 of 81 possible assignments to H beta 2/beta 3 pairs. The 13C alpha, 13 beta, 13C', and 1H alpha assignments yield a consensus chemical shift index (CSI) that, in combination with NOE information and backbone torsion angles, defines seven distinct helical regions for the protein's global architecture. Discrepancies between the CSI and NOE/3JHNH alpha-based secondary structure definitions have been attributed to heme ring current shifts on the basis of calculations from a model structure [Alam et al. (1994) J. Protein Chem., 13, 151-164]. The agreement can be improved by correcting the 1H alpha chemical shifts for the ring current contributions. Because the holoprotein was assembled from isotopically enriched globin and natural isotope-abundance heme, data from 13C-filtered/13C-edited and 13C-filtered/13C-filtered 2D NOESY experiments could be used to determine complete heme proton assignments and to position the heme within the protein. The results confirm the unusual presence of Phe31 (B10) and Leu58 (E7) side chains near the heme ligand binding site which may alter the polarity and steric environment and thus the functional properties of this protein.
已获得双分支甘油虫（Glyceradibranchiata）组分IV单体血红蛋白的重组亚铁一氧化碳连接体的完整碳-13、氮-15和氢-1共振归属。这种15642道尔顿的肌红蛋白样蛋白质含有大量甘氨酸和丙氨酸残基（47个）以及一个血红素辅基。耦合常数信息有助于确定χ(1)和χ(2)扭转角、主链φ角，以及81个可能的Hβ2/β3对中的43个归属。碳-13α、碳-13β、碳-13′和氢-1α归属产生了一个共识化学位移指数（CSI），该指数与核Overhauser效应（NOE）信息和主链扭转角相结合，为该蛋白质的整体结构定义了七个不同的螺旋区域。基于模型结构的计算[阿拉姆等人（1994年），《蛋白质化学杂志》，13卷，151 - 164页]，CSI与基于NOE/3JHNHα的二级结构定义之间的差异归因于血红素环电流位移。通过校正氢-1α化学位移的环电流贡献，可以改善这种一致性。由于全蛋白是由同位素富集的珠蛋白和天然同位素丰度的血红素组装而成，碳-13过滤/碳-13编辑和碳-13过滤/碳-13过滤二维NOESY实验的数据可用于确定完整的血红素质子归属，并将血红素定位在蛋白质内。结果证实了在血红素配体结合位点附近异常存在苯丙氨酸31（B10）和亮氨酸58（E7）侧链，这可能会改变极性和空间环境，从而改变该蛋白质的功能特性。
