Substituted Cysteine Accessibility Method (SCAM)
Substituted Cysteine Accessibility Method (SCAM)
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取代半胱氨酸可及性方法 (SCAM)
DOI：
10.1002/9780470749210.ch12
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发表时间：
2010
影响因子：
4.8
通讯作者：
J. Javitch
中科院分区：
文献类型：
--
作者：
G. Liapakis;J. Javitch
取代的半胱氨酸可及性方法（SCAM）[1]提供了一种系统地绘制蛋白质水可及表面残基的方法。这些残基通过用半胱氨酸（Cys）取代它们并评估带电的亲水性巯基试剂与取代的Cys（工程化Cys）的反应来鉴定。通过应用SCAM，已经绘制了各种离子通道和转运蛋白中的通道衬里残基，包括烟碱乙酰胆碱受体[1-3]、GABAA受体[4，5]、囊性纤维化跨膜电导调节剂[6]、UhpT转运蛋白[7]和钾通道[8]。SCAM还被用于研究蛋白质不同功能状态的结构改变，如β2-肾上腺素能受体（见第12.2节）。7)使用这种方法，我们系统地绘制了多巴胺D2受体（G蛋白偶联受体（GPCR）超家族成员）结合位点裂缝表面上的残基[9-15]。D2和视紫红质样亚家族中其他GPCR的结合位点裂缝是在它们的七个大部分疏水的跨膜片段（TMx，x= 1− 7）之间形成的水可接近的裂缝，并且从受体的细胞外表面延伸到跨膜结构域[16]。该裂缝的表面由可接触特异性激动剂和/或拮抗剂的残基（结合位点残基）和可发挥结构作用并间接影响结合的其他残基形成。
The substituted cysteine accessibility method (SCAM)[1] provides an approach to map systematically the residues on the water-accessible surface of a protein. These residues are identified by substituting them with cysteine (Cys), and assessing for the reaction of charged, hydrophilic, sulfhydryl reagents with the substituted Cys (engineered Cys). By applying SCAM, channel-lining residues in a variety of ion channels and transporters have been mapped, including the nicotinic acetylcholine receptor [1–3], the GABAA receptor [4, 5], the cystic fibrosis membrane-spanning conductance regulator [6], the UhpT transporter [7] and potassium channels [8]. SCAM has also been used to investigate structural alterations in different functional states of proteins, such as the β2-adrenergic receptor (see Section 12.2. 7) Using this approach we mapped systematically residues on the surface of the binding-site crevice in the dopamine D2 receptor, a member of the G protein-coupled receptor (GPCR) superfamily [9–15]. The binding-site crevice of D2 and the other GPCRs in the rhodopsin-like subfamily is a water-accessible crevice formed among their seven, mostly hydrophobic, membrane-spanning segments (TMx, x= 1− 7) and extending from the extracellular surface of the receptor into the membrane-spanning domain [16]. The surface of this crevice is formed by residues that can contact specific agonists and/or antagonists (binding-site residues) and by other residues that may play a structural role and affect binding indirectly.