Ratakonda Sireesha, Anand Arvind, Dikshit Kanak, Stark Benjamin C, Howard Andrew J
Biology Division, Department of Biological and Chemical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Mar 1;69(Pt 3):215-22. doi: 10.1107/S1744309112044818. Epub 2013 Feb 22.
Site-directed mutants of the gene encoding wild-type Vitreoscilla hemoglobin were made that changed Tyr29 (B10) of the wild-type Vitreoscilla hemoglobin (VHb) to either Phe or Ala. The wild-type and the two mutant hemoglobins were expressed in Escherichia coli and purified to homogeneity. The binding of the two mutants to CO was essentially identical to that of wild-type VHb as determined by CO-difference spectra. Circular-dichroism spectra also showed the two mutants to be essentially the same as wild-type VHb regarding overall helicity. All three VHbs were crystallized and their structures were determined at resolutions of 1.7-1.9 Å, which are similar to that of the original wild-type structure determination. The Tyr29Phe mutant has a structure that is essentially indistinguishable from that of the wild type. However, the structure of the Tyr29Ala mutant has significant differences from that of the wild type. In addition, for the Tyr29Ala mutant it was possible to determine the positions of most of the residues in the D region, which was disordered in the originally reported structure of wild-type VHb as well as in the wild-type VHb structure reported here. In the Tyr29Ala mutant, the five-membered ring of proline E8 (Pro54) occupies the space occupied by the aromatic ring of Tyr29 in the wild-type structure. These results are discussed in the context of the proposed role of Tyr29 in the structure of the oxygen-binding pocket.
构建了编码野生型透明颤菌血红蛋白基因的定点突变体，将野生型透明颤菌血红蛋白（VHb）的Tyr29（B10）分别替换为Phe或Ala。野生型和两种突变型血红蛋白在大肠杆菌中表达并纯化至均一。通过CO差光谱测定，两种突变体与CO的结合与野生型VHb基本相同。圆二色光谱也表明，在整体螺旋度方面，两种突变体与野生型VHb基本相同。所有三种VHb均结晶，并以1.7 - 1.9 Å的分辨率测定其结构，这与原始野生型结构测定的分辨率相似。Tyr29Phe突变体的结构与野生型基本无法区分。然而，Tyr29Ala突变体的结构与野生型有显著差异。此外，对于Tyr29Ala突变体，可以确定D区域中大多数残基的位置，在最初报道的野生型VHb结构以及本文报道的野生型VHb结构中，该区域是无序的。在Tyr29Ala突变体中，脯氨酸E8（Pro54）的五元环占据了野生型结构中Tyr29芳香环所占据的空间。本文结合Tyr29在氧结合口袋结构中所提出的作用对这些结果进行了讨论。
