Kossiakoff A A, Randal M, Guenot J, Eigenbrot C
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080.
Proteins. 1992 Sep;14(1):65-74. doi: 10.1002/prot.340140108.
The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.
比较了五个基本胰蛋白酶抑制剂（BPTI）分子的结构，以确定由晶体堆积效应导致的构象变异性的程度和性质。BPTI是评估此类因素的理想体系，因为有五个不同BPTI结构的高分辨率X射线模型，每个模型处于不同的晶体堆积环境中。尽管显示出最大变异性的区域与环结构（残基14 - 17和24 - 29）相关，但在这些结构之间观察到的差异分布在整个分子中。与五个堆积基序的平均值相比，显示出最大均方根偏差的结构区域与晶格中的分子间接触的存在密切相关。对于大多数分子，分子间接触数量较多与系统地更高的B因子之间也存在对应关系，尽管尚不清楚这是由晶体接触引起的，还是由于接触主要通过表面环形成这一事实导致的。在X射线模型中看到的构象差异不仅仅是晶格接触表面的局部位移，实际上在某些情况下还涉及分子内氢键的形成和断裂。堆积模型之间的差异幅度明显大于通常与诱变引起的变化相关的幅度；例如，从分子中去除一个内部二硫键时在突变位点观察到的结构差异明显小于与晶格接触效应相关的差异。将晶体堆积构象与在96皮秒分子动力学（MD）模拟过程中生成的BPTI代表性结构进行了比较。这种比较表明，X射线和MD分析所表明的蛋白质灵活性之间，特别是在那些最可变的区域之间，存在高度的对应关系。这表明在晶体堆积模型中显示出最大变异性的区域不是结晶的假象，而是代表了由结晶因素优先选择的真正的低能构象体。
