Perálvarez-Marín Alex, Bourdelande José-Luis, Querol Enric, Padrós Esteve
Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Barcelona, Spain.
Mol Membr Biol. 2006 Mar-Apr;23(2):127-35. doi: 10.1080/09687860500435019.
Proline residues in transmembrane helices have been found to have important roles in the functioning of membrane proteins. Moreover, Pro residues occur with high frequency in transmembrane alpha-helices, as compared to alpha-helices for soluble proteins. Here, we report several properties of the bacteriorhodopsin mutants P50A (helix B), P91A (helix C) and P186A (helix F). Compared to wild type, strongly perturbed behaviour has been found for these mutants. In the resting state, increased hydroxylamine accessibility and altered Asp-85 pKa and light-dark adaptation were observed. On light activation, hydroxylamine accessibility was increased and proton transport activity, M formation kinetics and FTIR difference spectra of M and N intermediates showed clear distortions. On the basis of these alterations and the near identity of the crystalline structures of mutants with that of wild type, we conclude that the transmembrane proline residues of bacteriorhodopsin fulfil a dynamic role in both the resting and the light-activated states. Our results are consistent with the notion that mutation of Pro to Ala allows the helix to increase its flexibility towards the direction originally hindered by the steric clash between the ring Cgamma and the carbonyl O of the i-4 residue, at the same time decreasing the mobility towards the opposite direction. Due to their properties, transmembrane Pro residues may serve as transmission elements of conformational changes during the transport process. We propose that these concepts can be extended to other transmembrane proteins.
人们发现跨膜螺旋中的脯氨酸残基在膜蛋白的功能发挥中具有重要作用。此外，与可溶性蛋白的α螺旋相比，脯氨酸残基在跨膜α螺旋中出现的频率较高。在此，我们报告了细菌视紫红质突变体P50A（螺旋B）、P91A（螺旋C）和P186A（螺旋F）的几个特性。与野生型相比，已发现这些突变体表现出强烈的行为扰动。在静止状态下，观察到羟胺可及性增加、Asp-85的pKa改变以及明暗适应变化。在光激活时，羟胺可及性增加，质子转运活性、M形成动力学以及M和N中间体的傅里叶变换红外差光谱显示出明显的畸变。基于这些变化以及突变体与野生型晶体结构的近乎相同，我们得出结论，细菌视紫红质的跨膜脯氨酸残基在静止和光激活状态下均发挥动态作用。我们的结果与以下观点一致：脯氨酸突变为丙氨酸使螺旋能够朝着最初因环Cγ与i-4残基的羰基O之间的空间冲突而受阻的方向增加其柔韧性，同时降低朝着相反方向的流动性。由于其特性，跨膜脯氨酸残基可能在转运过程中作为构象变化的传递元件。我们提出这些概念可以扩展到其他跨膜蛋白。
