Laux Valerie, Callow Phil, Svergun Dmitri I, Timmins Peter A, Forsyth V Trevor, Haertlein Michael
ILL-EMBL Deuteration Laboratory, Partnership for Structural Biology, Institut Laue Langevin, 38042, Grenoble Cedex 9, France.
Eur Biophys J. 2008 Jul;37(6):815-22. doi: 10.1007/s00249-008-0280-5. Epub 2008 Feb 15.
We describe methods that have been developed within the ILL-EMBL Deuteration Laboratory for the production of maltose binding protein (MBP) that has been selectively labelled either with deuterated tryptophan or deuterated methionine (single labelling), or both (double labelling). MBP is used as an important model system for biophysical studies, and selective labelling can be helpful in the analysis of small-angle neutron scattering (SANS) data, neutron reflection (NR) data, and high-resolution neutron diffraction data. The selective labelling was carried out in E. coli high-cell density cultures using auxotrophic mutants in minimal medium containing the required deuterated precursors. Five types of sample were prepared and studied: (1) unmodified hydrogenated MBP (H-MBP), (2) perdeuterated MBP (D-MBP), (3) singly labelled MBP with the tryptophan residues deuterated (D-trp MBP), (4) singly labelled MBP with methionine residues deuterated (D-met MBP) and (5) doubly labelled MBP with both tryptophan and methionine residues deuterated (D-trp/met MBP). Labelled samples were characterised by size exclusion chromatography, gel electrophoresis, light scattering and mass spectroscopy. Preliminary small-angle neutron scattering (SANS) experiments have also been carried out and show measurable differences between the SANS data recorded for the various labelled analogues. More detailed SANS experiments using these labelled MBP analogues are planned; the degree to which such data could enhance structure determination by SANS is discussed.
我们描述了ILL-EMBL氘代实验室开发的用于生产麦芽糖结合蛋白（MBP）的方法，该蛋白已用氘代色氨酸或氘代甲硫氨酸进行了选择性标记（单标记），或两者都进行了标记（双标记）。MBP用作生物物理研究的重要模型系统，选择性标记有助于分析小角中子散射（SANS）数据、中子反射（NR）数据和高分辨率中子衍射数据。选择性标记是在含有所需氘代前体的基本培养基中使用营养缺陷型突变体在大肠杆菌高细胞密度培养物中进行的。制备并研究了五种类型的样品：（1）未修饰的氢化MBP（H-MBP），（2）全氘代MBP（D-MBP），（3）色氨酸残基被氘代的单标记MBP（D-trp MBP），（4）甲硫氨酸残基被氘代的单标记MBP（D-met MBP）和（5）色氨酸和甲硫氨酸残基都被氘代的双标记MBP（D-trp/met MBP）。通过尺寸排阻色谱、凝胶电泳、光散射和质谱对标记样品进行了表征。还进行了初步的小角中子散射（SANS）实验，结果表明各种标记类似物记录的SANS数据之间存在可测量的差异。计划使用这些标记的MBP类似物进行更详细的SANS实验；讨论了此类数据可增强SANS结构测定的程度。
