Siebert H C, Adar R, Arango R, Burchert M, Kaltner H, Kayser G, Tajkhorshid E, von der Lieth C W, Kaptein R, Sharon N, Vliegenthart J F, Gabius H J
Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität, München, Germany.
Eur J Biochem. 1997 Oct 1;249(1):27-38. doi: 10.1111/j.1432-1033.1997.00027.x.
For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-1 and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-1/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of bovine galectin-1 is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82/Tyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When single-site mutants of EcorL ([Ala106]EcorL, [Ala108]EcorL, [Ala229]EcorL) were subjected to molecular-dynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.
对于溶液中的蛋白质，某些晶体学参数的有效性可以通过分子动力学（MD）模拟和核磁共振光谱相结合来确定。使用激光光化学诱导动态核极化（photo-CIDNP）技术来测量组氨酸、酪氨酸和色氨酸的表面可及性，牛半乳糖凝集素-1和刺桐凝集素（EcorL）的光谱很容易与这两种蛋白质的晶体学数据相吻合。结果强调了牛半乳糖凝集素-1/鸡半乳糖凝集素中Trp68/Trp69以及小鼠半乳糖凝集素-3中Trp194在碳水化合物结合中的作用。源自牛半乳糖凝集素-1晶体结构的这一特征在溶液中对于原型人类同源物、两种禽类半乳糖凝集素和嵌合型小鼠半乳糖凝集素-3得以保留，因为光谱证实了结合位点结构的四个计算模型中CIDNP可推断的空间参数。在EcorL中，Tyr106/Tyr108是扩展结合口袋的组成部分，在溶液中其可能会因配体的存在而被屏蔽。建模结果与CIDNP测量结果之间的差异主要涉及人类和禽类原型半乳糖凝集素中组氨酸残基以及植物凝集素中Tyr82/Tyr229缺乏反应性。假定对EcorL进行定点诱变可以提供有关特定残基在功能方面作用的信息。当EcorL的单点突变体（[Ala106]EcorL、[Ala108]EcorL、[Ala229]EcorL）进行分子动力学（MD）模拟时，即使是空间上分离的氨基酸侧链的表观表面可及性也可能受到非均匀影响。这一结论得到了突变体蛋白质光谱评估的支持。基于这些CIDNP结果，对凝集素结合位点结构的建模表明Tyr106/Tyr108苯环的取向发生了显著变化。单点突变对蛋白质构象特征的潜在影响对于比较野生型和突变型蛋白质的研究解释值得关注。
