Cianci M, Rizkallah P J, Olczak A, Raftery J, Chayen N E, Zagalsky P F, Helliwell J R
Department of Chemistry, University of Manchester, Manchester M13 9PL, England.
Acta Crystallogr D Biol Crystallogr. 2001 Sep;57(Pt 9):1219-29. doi: 10.1107/s0907444901009350. Epub 2001 Aug 23.
The molecular basis of the camouflage colouration of marine crustacea is often provided by carotenoproteins. The blue colour of the lobster carapace, for example, is intricately associated with a multimacromolecular 16-mer complex of protein subunits each with a bound astaxanthin molecule. The protein subunits of crustacyanin fall into two distinct subfamilies, CRTC and CRTA. Here, the crystal structure solution of the A(1) protein of the CRTC subfamily is reported. The problematic nature of the structure solution of the CRTC proteins (both C(1) and A(1)) warranted consideration and the development of new approaches. Three putative disulfides per protein subunit were likely to exist based on molecular-homology modelling against known lipocalin protein structures. With two such subunits per crystallographic asymmetric unit, this direct approach was still difficult as it involved detecting a weak signal from these sulfurs and suggested the use of softer X-rays, combined with high data multiplicity, as reported previously [Chayen et al. (2000), Acta Cryst. D56, 1064-1066]. This paper now describes the structure solution of CRTC in the form of the A(1) dimer based on use of softer X-rays (2 A wavelength). The structure solution involved a xenon derivative with an optimized xenon L(I) edge f" signal and a native data set. The hand of the xenon SIROAS phases was determined by using the sulfur anomalous signal from a high-multiplicity native data set also recorded at 2 A wavelength. For refinement, a high-resolution data set was measured at short wavelength. All four data sets were collected at 100 K. The refined structure to 1.4 A resolution based on 60 276 reflections has an R factor of 17.7% and an R(free) of 22.9% (3137 reflections). The structure is that of a typical lipocalin, being closely related to insecticyanin, to bilin-binding protein and to retinol-binding protein. This A(1) monomer or dimer can now be used as a search motif in the structural studies of the oligomeric forms alpha- and beta-crustacyanins, which contain bound astaxanthin molecules.
海洋甲壳类动物伪装色的分子基础通常由类胡萝卜素蛋白提供。例如，龙虾外壳的蓝色与一种多聚大分子的16聚体复合物紧密相关，该复合物由每个都结合有虾青素分子的蛋白质亚基组成。虾青素蛋白的蛋白质亚基分为两个不同的亚家族，CRTC和CRTA。在此，报道了CRTC亚家族A(1)蛋白的晶体结构解析。CRTC蛋白（C(1)和A(1)）结构解析的困难性质值得考虑并开发新方法。基于与已知脂质运载蛋白结构的分子同源性建模，每个蛋白质亚基可能存在三个推定的二硫键。由于每个晶体学不对称单元有两个这样的亚基，这种直接方法仍然很困难，因为它涉及检测来自这些硫的微弱信号，并建议使用更软的X射线，结合高数据多重性，如先前报道的那样[Chayen等人（2000年），《晶体学报》D56，1064 - 1066]。本文现在描述了基于使用更软的X射线（波长2 Å）以A(1)二聚体形式对CRTC的结构解析。结构解析涉及一种具有优化的氙L(I)边缘f"信号的氙衍生物和一个天然数据集。氙SIROAS相的手性通过使用也在2 Å波长记录的高多重性天然数据集的硫反常信号来确定。为了精修，在短波长下测量了一个高分辨率数据集。所有四个数据集均在100 K下收集。基于60276个反射，精修至1.4 Å分辨率的结构的R因子为17.7%，R(free)为22.9%（3137个反射）。该结构是典型的脂质运载蛋白结构，与昆虫青素、胆红素结合蛋白和视黄醇结合蛋白密切相关。现在，这种A(1)单体或二聚体可作为搜索基序用于对含有结合虾青素分子的α - 和β - 虾青素寡聚体形式的结构研究。
