Checover S, Marantz Y, Nachliel E, Gutman M, Pfeiffer M, Tittor J, Oesterhelt D, Dencher N A
Technische Universitat Darmstadt, Institut fur Biochemie, Darmstadt D-64287, Germany.
Biochemistry. 2001 Apr 10;40(14):4281-92. doi: 10.1021/bi002574m.
The cytoplasmic surface of bacteriorhodopsin is characterized by a group of carboxylates that function as a proton attractive domain [Checover, S., Nachliel, E., Dencher, N. A., and Gutman, M. (1997) Biochemistry 36, 13919-13928]. To identify these carboxylates, we selectively mutated them into cysteine residues and monitored the effects of the dynamics of proton transfer between the bulk and the surface of the protein. The measurements were carried out without attachment of a pH-sensor to the cysteine residue, thus avoiding any structural perturbation and change in the surface charge caused by the attachment of a reporter group, and the protein was in its BR state. The purple membranes were suspended in an unbuffered solution of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) and exposed to a train of 1000 laser pulses (2.1 mJ/pulse, lambda = 355 nm, at 10 Hz). The excitation of the dye ejected the hydroxyl's proton, and a few nanoseconds later, a pair of free protons and ground-state pyranine anion was formed. The experimental observation was the dynamics of the relaxation of the system to the prepulse state. The observed signals were reconstructed by a numeric method that replicates the chemical reactions proceeding in the perturbed space. The detailed reconstruction of the measured signal assigned the various proton-binding sites with rate constants for proton binding and proton exchange and the pK values. Comparison of the results obtained by the various mutants indicates that the dominant proton-binding cluster of the wild-type protein consists of D104, E161, and E234. The replacement of D104 or E161 with cysteine lowered the proton binding capacity of the cluster to approximately 60% of that of the native protein. The replacement of E234 with cysteine disrupted the structure of the cluster, causing the two remaining carboxylates to function as isolated residues that do not interact with each other. The possibility of proton transfer between monomers is discussed.
细菌视紫红质的细胞质表面具有一组羧酸盐，它们作为质子吸引域发挥作用[切科弗，S.，纳赫利尔，E.，登彻尔，N. A.，和古特曼，M.（1997年）《生物化学》36卷，13919 - 13928页]。为了鉴定这些羧酸盐，我们将它们选择性地突变为半胱氨酸残基，并监测质子在蛋白质本体与表面之间转移动力学的影响。测量是在未将pH传感器连接到半胱氨酸残基的情况下进行的，从而避免了因连接报告基团而引起的任何结构扰动和表面电荷变化，并且蛋白质处于其BR状态。紫色膜悬浮在吡喃宁（8 - 羟基芘 - 1,3,6 - 三磺酸盐）的无缓冲溶液中，并暴露于1000个激光脉冲序列（2.1 mJ/脉冲，λ = 355 nm，10 Hz）。染料的激发使羟基的质子逸出，几纳秒后，形成一对游离质子和基态吡喃宁阴离子。实验观察的是系统弛豫到脉冲前状态的动力学。观察到的信号通过一种数值方法重建，该方法复制了在受扰空间中进行的化学反应。对测量信号的详细重建为各种质子结合位点分配了质子结合和质子交换的速率常数以及pK值。通过各种突变体获得的结果比较表明，野生型蛋白质的主要质子结合簇由D104、E161和E234组成。用半胱氨酸取代D104或E161会使该簇的质子结合能力降低至天然蛋白质的约60%。用半胱氨酸取代E234会破坏该簇的结构，导致其余两个羧酸盐作为不相互作用的孤立残基发挥作用。讨论了单体之间质子转移的可能性。
