Vancraenenbroeck Renée, Kunzelmann Simone, Webb Martin R
The Francis Crick Institute, London, United Kingdom.
Structural Biology Science Technology Platform, The Francis Crick Institute, London, United Kingdom.
PLoS One. 2017 Jun 21;12(6):e0179547. doi: 10.1371/journal.pone.0179547. eCollection 2017.
The range of ATP concentrations that can be measured with a fluorescent reagentless biosensor for ATP has been increased by modulating its affinity for this analyte. The ATP biosensor is an adduct of two tetramethylrhodamines with MatB from Rhodopseudomonas palustris. Mutations were introduced into the binding site to modify ATP binding affinity, while aiming to maintain the concomitant fluorescence signal. Using this signal, the effect of mutations in different parts of the binding site was measured. This mutational analysis revealed three variants in particular, each with a single mutation in the phosphate-binding loop, which had potentially beneficial changes in ATP binding properties but preserving a fluorescence change of ~3-fold on ATP binding. Two variants (T167A and T303A) weakened the binding, changing the dissociation constant from the parent's 6 μM to 123 μM and 42 μM, respectively. Kinetic measurements showed that the effect of these mutations on affinity was by an increase in dissociation rate constants. These variants widen the range of ATP concentration that can be measured readily by this biosensor to >100 μM. In contrast, a third variant, S170A, decreased the dissociation constant of ATP to 3.8 μM and has a fluorescence change of 4.2 on binding ATP. This variant has increased selectivity for ATP over ADP of >200-fold. This had advantages over the parent by increasing sensitivity as well as increasing selectivity during ATP measurements in which ADP is present.
通过调节荧光无试剂ATP生物传感器对该分析物的亲和力，可测量的ATP浓度范围得以扩大。该ATP生物传感器是由两个四甲基罗丹明与来自沼泽红假单胞菌的MatB形成的加合物。在结合位点引入突变以改变ATP结合亲和力，同时旨在保持伴随的荧光信号。利用该信号，测量了结合位点不同部位突变的影响。这种突变分析特别揭示了三个变体，每个变体在磷酸结合环中都有一个单一突变，它们在ATP结合特性方面有潜在的有益变化，但在结合ATP时保持约3倍的荧光变化。两个变体（T167A和T303A）减弱了结合，将解离常数从亲本的6 μM分别变为123 μM和42 μM。动力学测量表明，这些突变对亲和力的影响是通过解离速率常数的增加。这些变体将该生物传感器可轻松测量的ATP浓度范围扩大到>100 μM。相比之下，第三个变体S170A将ATP的解离常数降低到3.8 μM，结合ATP时荧光变化为4.2。该变体对ATP的选择性比对ADP增加了>200倍。在存在ADP的ATP测量中，这通过提高灵敏度和选择性比亲本具有优势。
