Jung K H, Spudich J L
Department of Microbiology and Molecular Genetics, University of Texas Medical School Health Science Center, Houston 77030, USA.
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6557-61. doi: 10.1073/pnas.93.13.6557.
Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.
通过定点诱变，对盐生盐杆菌信号转导蛋白HtrI（用于感光视紫红质I的嗜盐杆菌转导蛋白，即SRI）N端一半区域内的21个酸性和碱性残基进行了中性残基替换。这些替换均发生在HtrI的一个区域内，我们之前通过缺失分析得出该区域包含与趋光性受体SRI的相互作用位点。免疫印迹显示，含有这些突变的htrI - sopI操纵子在质粒中的表达能使细胞以接近野生型的水平产生SRI和突变型HtrI。HtrI的六个突变扰乱了SRI的光化学动力学，其中一个突变逆转了趋光性反应。用中性氨基酸替换Asp - 86、Glu - 87和Glu - 108会加速SRI的光循环，而替换Arg - 70、Arg - 84和Arg - 99则会使其光循环变慢。在含有一个被替换的酸性残基和一个被替换的碱性残基的双突变体中，对光循环速率的相反影响相互抵消。激光闪光光谱表明，动力学扰动是由于视黄醛席夫碱再质子化速率的改变所致。所有这些突变都能使正常的吸引和排斥信号传递得以进行。另一方面，用等电子的谷氨酰胺替换Glu - 56，会使橙色光在中性pH条件下（信号反转）在体内的正常吸引作用转变为排斥信号。低pH值可纠正由于Glu - 56突变为Gln导致的信号反转，当在第56位替换为精氨酸时，信号反转的表观pK值会升高。结果表明，跨膜螺旋2的胞质端和胞质结构域的起始部分包含与SRI的相互作用位点。为了解释这些结果以及之前的结果，我们提出了一个模型，其中：（i）此处鉴定出的HtrI区域形成了一个静电结合网络的一部分，该网络贯穿SRI蛋白并包括其光活性位点；（ii）光异构化诱导的席夫碱去质子化和再质子化导致该网络的改变，使HtrI在吸引和排斥构象之间转换；（iii）HtrI突变和细胞外pH值改变了这些构象的平衡比例。
