This work was supported by National Institutes of Health Grant R21MH104471 and R01MH106511 (D.M.C.), Brain Research Foundation SG 2012-01 (D.M.C.), Northwestern University Clinical and Translational Sciences Institute 8UL1TR000150 (Y.H.), Chicago Biomedical Consortium HTS-004 (Y.H. and D.M.C.), and National Institutes of Health Grant 2T32MH067564 (K.L.). A part of this work was performed by the Northwestern University Medicinal and Synthetic Chemistry Core (ChemCore) at the Center for Molecular Innovation and Drug Discovery (CMIDD), which is funded by the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust and Cancer Center Support Grant P30 CA060553 from the National Cancer Institute awarded to the Robert H. Lurie Comprehensive Cancer Center. The high throughput screen work was performed in the High Throughput Analysis Laboratory which is also a core facility of the Robert H. Lurie Comprehensive Cancer Center.

1. TRIP8b（241-602）蛋白的纯化<ol><li>变换含TRIP8b（241-602）中的细菌蛋白表达载体pGS21 3到感受态质粒大肠杆菌用于根据制造商的说明蛋白的表达。板300微升上卢里亚肉汤（LB）文化 - 琼脂用5微克/毫升氯霉素和氨苄西林。孵育在37℃的板16小时。 </li><li>第二天，挑取单个菌落接种50毫升LB用50微克/毫升氯霉素和氨苄西林。孵育在37℃下培养16小时（振荡）。 </li><li> 50ml培养添加到1升的LB用50微克/毫升氨苄青霉素的。在37℃孵育振荡。 </li><li>一旦培养达到的OD 600读数的0.8-1.2，培养箱的温度改变到18℃，并添加IPTG（异丙基-β-D-硫代半乳糖苷）至1mM的终浓度。允许蛋白质表达进行16小时。 </li><li>降速E.大肠杆菌在4℃6000×g离心15分钟以沉淀细菌。从离心机取出管后，保持细菌在冰上的过程的剩余。重悬于36毫升缓冲液A的细菌与0.25毫米的氟苯甲（PMSF）。 </li><li>超声处理用扁平螺栓在冰上再悬浮细菌5-10分钟，"上"在高功率和30秒"关"30秒之间交替。 </li><li>离心机以12,000 xg离心在4℃下15分钟。离心额外15分钟，如果上清液尚不清楚。 </li><li>适用上清液至2ml的Ni-NTA珠的柱。孵育在4℃下轻轻摇动60分钟。 </li><li>使未结合的材料通过柱流动并用500ml缓冲液A洗</li><li>用250毫升缓冲液B洗柱</li><li>洗柱用125毫升缓冲液A中添加5 mM咪唑。 </li><li>洗脱蛋白质来回回米，20毫升洗脱缓冲液的柱。 </li><li>洗脱的蛋白添加至透析盒（分子量截留值 - 10 kDa）的使用注射器。透析的蛋白质在4℃下在4升冷磷酸盐缓冲盐水（PBS）中的60分钟。 </li><li>移动透析盒冷PBS的一个新的4升一桶。在4℃透析蛋白16小时</li><li>第二天早上，检查用Coomassie蛋白质检测试剂盒中的蛋白质浓度，按照制造商的说明进行操作。使用蛋白浓缩，按照制造商的说明，如果观察到低于40μM的浓度浓缩蛋白质。等分试样并冷冻在-80℃下贮存。 </li></ol> 2.小​​型荧光偏振检测，以表征两个蛋白片段相互作用<ol><li>解冻将200μl40μM的TRIP8b（241-602），并把它添加到1.5ml管中。加入100微升的PBS至11追加15毫升管。 </li><li>通过从原始管中的一系列转印100μl的TRIP8b（241-602）的下管，上下吹打，并重复该过程执行连续稀释。这将产生从0.01-40微米，12点系列TRIP8b（241-602）的2倍稀释的。 </li><li>制备0.1μMHCN1 FITC（6.5微升10μM的等分试样的）和2mM二硫苏糖醇（DTT）在PBS中的650微升主混合物。 注:主结构为2倍。 </li><li>设立了12个新的1.5 mL管。加入50μl含HCN1 FITC每个管的主混合物，然后从每个12系列稀释的加入50微升。 注意:这将产生12管，各含0.05微米HCN1 FITC和TRIP8b（241-602）的浓度的一半从原来的连续稀释。 </li><li>稀释系列加入测定板，30微升每孔，一式三份。使用低结合实心黑色384孔板。 </li><li>旋转的托盘f或在RT 900 xg离心2分钟。 </li><li>用酶标仪，按照制造商的指示读板。使用以下测量参数测量荧光偏振:485nm激发，530nm处发射，在505nm分色镜，100毫秒的积分时间。 </li><li>绘制极化（MP）的值相对于TRIP8b对数标度（241-602）的浓度。与希尔方程来确定亲和力（K D）TRIP8b（241-602）的用于标记的HCN1肽拟合数据。 注意:为了制备系列稀释液，多孔板也可以用来代替管。 </li></ol> 3.检查蛋白质相互作用使用阳性对照<ol><li>加入200μl未标记HCN1肽（200μM）的到1.5ml管中。加入100微升的PBS至11追加15毫升管。通过从含有200μl的肽以在第一管的管转移100μl的执行连续稀释液的100微升PBS中。重复这个过程，产生12管与未标记HCN1肽的2倍稀释。 </li><li>准备0.2μMHCN1 FITC和4μMTRIP8b（241-602）的650μl反应混合液。 注:这是一个2倍的解决方案。 </li><li>加入50μl反应混合液到12新的1.5 mL管。 </li><li>加入50μl的每个系列稀释的到1.5ml管中。 </li><li>装载在一式三份的黑色384孔微量滴定板，每孔加入30微升。 </li><li>离心板在室温900 XG 2分钟。 </li><li>阅读使用能够FP的酶标仪板。如步骤2.7中所述使用相同的设置。 </li><li>计算使用Cheng-Prusoff方程为TRIP8b未标记的肽（241-602）的亲和力:K d 1 的 = IC 50 /（1+ [L] / K D 2），其中K d 1 的代表未标记的亲和肽为TRIP8b（241-602），IC 50是在前面的步骤确定的并代表未标记的配位体的置换标记的配体的能力，[L]为标记的配体的在步骤3.2的浓度，和K D 2是TRIPb（241-602）的该标记配体的亲和力。 </li></ol> 4.评估分析性能（计算Z参数） <ol><li>通过使FP缓冲的12 ml溶液与2微米TRIP8b（241-602），50nM的HCN1 FITC，和1mM DTT中的FP缓冲制备主混合物。 注:这将需要60微升40μMTRIP8b（241-602）的60微升10μMHCN1 FITC，而1080微升FP缓冲。 </li><li>添加30微升主混合物到一个黑色384孔微量滴定板的每个孔中。 </li><li>加入1微升1毫米未标记HCN1肽对192口井作为阳性对照。 </li><li>加入1μlFP缓冲液至192孔充当阴性对照。 </li><li>离心板在室温900 XG 2分钟。 </li><li>阅读使用MI板croplate读卡器。 </li><li>使用下列公式计算测定在Z因子Z = 1 - 3 *（σPOS-σ 负 ）/（μ 负 -μ 正 ）其中σPOS和σ 负是阳性和阴性对照孔的标准偏差，μPOS和μ 负表示在阳性和阴性对照孔的平均信号。 </li></ol> 5.高通量筛选<ol><li>解冻TRIP8b（241-602）和HCN1 FITC的等份，并保持他们在冰上。 </li><li>在FP缓冲准备10毫升TRIP8b（2μM）和HCN1 FITC（50纳米）。 </li><li>分配25微升混合物的进入低结合使用移液管384孔黑色微量滴定板的每个孔中。 </li><li>用于文库筛选中，添加化合物成使用声液体处理器列的各孔3〜22（40μM终浓度为每）。 注:由于relativel与此法观察ý低命中率，两种不同的化合物被汇集成一个单一的孔。从每个活动以及这两种化合物随后单独进行测试，以确定哪些所赋予的活性。 </li><li>添加100 NL二甲亚砜（DMSO）加入到1列和各板23作为阴性对照的每个孔中。 </li><li>添加未标记HCN1肽成2列和各板24作为阳性对照的各孔中。 </li><li>孵育在室温将板2小时。读板或阅读之前，在4℃孵育16小时。 </li><li>测量酶标仪荧光偏振。如步骤2.7中所述使用相同的设置。 </li><li>通过使用从每个平板为100％的平均阳性和阴性对照归一化信号计算抑制百分比。使用方程XN = 100％*（（X 负 - X）/（X 负 - X 正 ）），其中X，N是对应于信号X的归一化抑制百分率，X POS </sub&gt;是从阳性对照孔的平均信号，和X 负距离阴性对照孔的信号。 </li></ol> 6.点击确认使用TAMRA标记肽HCN <ol><li>验证命中使用TAMRA标记HCN1肽（HCN1 TAMRA）超过50％的抑制。 <ol><li>准备12×2微米TRIP8b（241-602）和50纳米HCN1 TAMRA在FP缓冲的主结构。 </li><li>分配25微升主混合物的成黑色384孔微量滴定板的每个孔中。 </li><li>转移的每个活性化合物的系列稀释液到相应的孔中。使两倍稀释，使得每个的浓度击中范围从0.2至200μm。 </li><li>为阴性对照的反应，转移100 NL DMSO中到每个孔中。为阳性对照添加100 NL系列稀释未标记的HCN1肽的终浓度范围从200μM至0.1μM。 </li><li>孵育在室温在板2小时。读板或在4孵育16小时 阅读前°以下。 </li><li>使用以下测量参数测量荧光偏振（MP）:535毫微米激发; 580 nm发射; 535纳米分色镜，20毫秒的积分时间。 </li><li>通过使用四参数非线性回归模型23拟合各化合物的浓度依赖的FP数据计算的IC 50值。 </li></ol></li></ol> 7.基于微珠的亲近试验的剂量 - 反应验证<ol><li>解冻1等分试样His-标记TRIP8b（241-602）（他-TRIP8b）和GST标签HCN1（GST-HCN1 C40）融合蛋白的，并保持他们在冰上。 </li><li>在400nm的His-TRIP8b和40纳米的GST HCN1 C40的浓度结合的His-TRIP8b与GST-HCN1 C40在测定缓冲液。制备300微升混合物的每种化合物进行测试。 注:化合物一式三份在11个不同浓度的加了DM运行SO控制（无化合物）。 </li><li>对于对照孔，准备30微升他-TRIP8b（200纳米），而GST-HCN1C40，然后分别在分析缓冲液无他，TRIP8b准备30微升GST-HCN1C40（20纳米）。 </li><li>使用16通道移液器的GST-HCN1 C40混合物（在上面的步骤7.2的方法制备）至36孔的板:为每个要测试的化合物中，添加7微升的His-TRIP8b的。为了测试单一化合物，安排行AL的12个不​​同浓度，并在列1-3三次重复。新增7微升了他的-TRIP8b溶液（7.3编写），以列排M的1-3和7微升GST-C40 HCN1溶液（7.3编写），以排N. 1-3列短暂离心板确保液体是在每个孔的底部，以除去任何气泡。 </li><li>分配的命中化合物待测试入盘，使得每个的浓度击中范围为200微米（在A行）向下至0.1微米（在排K）。分配的体积DMSO中以行LN匹配化合物的行的AK分配的量。 </li><li>摇板2分钟，在室温下孵育2.5小时。 </li><li>稀释抗GST受体珠1:50用测定缓冲液。 </li><li>添加3.5微升的稀释受体珠粒以16通道移液器板的各孔中，轻轻吹打，以避免产生气泡混合。 </li><li>将培养板在室温在黑暗中1小时。 </li><li>稀释镍螯合供体珠1:50与分析缓冲液。不要混合物置于阴暗处。 </li><li>添加3.5微升的稀释供体珠以16通道移液器板的各孔中，轻轻吹打，以避免产生气泡混合。 </li><li>将培养板在室温在黑暗中1.5小时。 </li><li>阅读酶标仪。使用具有以下测量参数酶标仪:40毫秒激发时间，100毫秒发射时间为0.1mm检测高度。 </li><li>由选管会数据拟合计算IC 50值使用四参数非线性回归模型23小时化合物。 </li></ol>

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Y., Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+dorsal+hippocampal+activity+by+knockdown+of+HCN1+channels+leads+to+anxiolytic-+and+antidepressant-like+behaviors.">Enhancement of dorsal hippocampal activity by knockdown of HCN1 channels leads to anxiolytic- and antidepressant-like behaviors.</a> Neuron. 75 (3), 503-516 (2012).</li><li>Wahl-Schott, C., Biel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN+channels:+structure,+cellular+regulation+and+physiological+function.">HCN channels: structure, cellular regulation and physiological function.</a> Cell Mol Life Sci. 66 (3), 470-494 (2009).</li><li>DeBerg, H. A., Bankston, J. R., Rosenbaum, J. C., Brzovic, P. S., Zagotta, W. N., Stoll, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Mechanism+for+the+Regulation+of+HCN+Ion+Channels+by+the+Accessory+Protein+TRIP8b.">Structural Mechanism for the Regulation of HCN Ion Channels by the Accessory Protein TRIP8b.</a> Structure. 23 (4), 734-744 (2015).</li><li>Weiss, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hill+equation+revisited:+uses+and+misuses.">The Hill equation revisited: uses and misuses.</a> FASEB J. 11 (11), 835-841 (1997).</li><li>Prinz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hill+coefficients,+dose-response+curves+and+allosteric+mechanisms.">Hill coefficients, dose-response curves and allosteric mechanisms.</a> J Chem Biol. 3 (1), 37-44 (2009).</li><li>Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Guide+to+Simple+and+Informative+Binding+Assays.">A Guide to Simple and Informative Binding Assays.</a> Mol Biol Cell. 21 (23), 4061-4067 (2010).</li><li>Rossi, A. M., Taylor, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein-ligand+interactions+by+fluorescence+polarization.">Analysis of protein-ligand interactions by fluorescence polarization.</a> Nat Protoc. 6 (3), (2011).</li><li>Ryan, A. J., Gray, N. M., Lowe, P. N., Chung, C. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Detergent+on+"Promiscuous"+Inhibitors.">Effect of Detergent on "Promiscuous" Inhibitors.</a> J Med Chem. 46 (16), 3448-3451 (2003).</li><li>McGovern, S. L., Caselli, E., Grigorieff, N., Shoichet, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+mechanism+underlying+promiscuous+inhibitors+from+virtual+and+high-throughput+screening.">A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening.</a> J Med Chem. 45 (8), 1712-1722 (2002).</li><li>Hertzberg, R. P., Pope, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening:+new+technology+for+the+21st+century.">High-throughput screening: new technology for the 21st century.</a> Curr Opin Chem Biol. 4 (4), 445-451 (2000).</li><li>Sundberg, S. 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The authors have nothing to disclose.

因为其在MDD 24的治疗目标电位的，出现了在拮抗HCN通道功能在中枢神经系统4药理学方法相当大的兴趣。然而，这些努力已经被HCN通道在心脏起搏中的重要作用和心律不齐25的风险停滞不前。我们推断，破坏HCN和其大脑特定辅助亚基，TRIP8b 8之间的相互作用可能是足以产生，而不会影响心脏HCN通道3抗抑郁样作用。这一假说是由缺乏的小鼠表现出TRIP8b抗抑郁样...

超极化激活环核苷酸门控（HCN）频道在心脏和中枢神经系统，其中它们起到调节膜兴奋1中起重要作用表示。 HCN通道有牵连抑郁症（MDD）2，这导致几个组提议限制性HCN通道功能的药理学可以有效地作为一种新型治疗MDD 3的发病机制。然而，直接针对HCN通道是因为它们在心脏动作电位4重要的作用并不可行。伊伐布雷定，唯一的FDA批准的HCN通道拮抗剂，用于心脏衰竭，以产生一个心动过缓作用5的治疗。因此，有必要为限制HCN通道在中枢神经系统专门功能的药理学试剂。 Rab8b相互作用蛋白（TRIP8b）三十四肽重复含，是一种大脑特定FIC HCN通道控制HCN通道6,7的表面表达和定位的辅助亚基。 TRIP8b基因敲除导致而不影响心脏HCN 8表达降低大脑HCN通道7。有趣的是，TRIP8b基因敲除小鼠花强迫游泳任务，尾吊任务7，两种常用的筛选试验的抗抑郁疗效9-11时间少动。这些结果表明，而不是直接靶向HCN通道与HCN通道功能的小分子拮抗剂，扰乱TRIP8b和HCN之间的相互作用可以是足以产生抗抑郁样行为。 TRIP8b结合氰化氢在两个不同的结合位点。 HCN的环核苷酸结合结构域（CNBD）与TRIP8b位于N端的到的TRIP8b 12,13的TPR结构域的保守结构域相互作用。虽然所涉及在此CNBD的残相互作用已经映射14，即所涉及的TRIP8b的区域还没有被缩小之外的80个氨基酸的片段13。 TRIP8b的三十四肽重复（TPR）结构域和HCN的C末端三肽（&#39;SNL&#39;在HCN1，HCN2和HCN4，而是"ANM"在HCN3）3,12之间发生的第二交互作用。本C尾相互作用的最近解析晶体结构15揭示实质结构相似性的过氧化物酶体导入受体之间的相互作用，peroxin 5（PEX5），和其相互作用伙伴，含有1型的过氧化物酶体靶向序列（PTS1）16。 虽然两者相互作用位点所必需的HCN通道功能，TRIP8b的TPR结构域和HCN1的C-末端三肽之间的相互作用作为主导结合位点和调节HCN表面表达。因此，这种相互作用被选为本研究中的定位点。为原稿，当提及到TRIP8b和HCN之间的相互作用制成的其余部分，它是这种相互作用正在提及。这种相互作用是由TRIP8b的对应于含有它的保守C端结合的HCN的C-末端尾部（残基的第1a-4同种型TRIP8b的241-602）3所需的TPR结构域的高可溶性片段概括。 为了开发高通量筛选，以确定能够破坏这种互动的小分子，荧光偏振（FP）为基础的测定采用17。荧光偏振是基于与偏振光，并测量发射的荧光18的偏振度的荧光团标记的配体的激励。在结合伴侣的存在，所述荧光配体的旋转运动被约束和偏振光发射19。在没有一个结合伴侣，T他的配位体的旋转运动导致去极化光的发射。 在封闭的协议，提出了N末端标记（的6xHis）TRIP8b使用镍氮基三乙酸（的Ni-NTA）珠粒纯化（241-602）的方法。一个类似的协议是用来净化该协议的步骤7中使用的谷胱甘肽-S-转移酶（GST）-taggedçHCN1端40个氨基酸（HCN1 C40）。出于空间考虑，省略该步骤的详细描述。 在步骤2到协议的7，高通量筛选工作流提出了（ 见图1）。蛋白质-蛋白质相互作用的高通量筛选出了名的困难的目标，并建议读者去寻找关于这个专题的20个额外的资源。 步骤2和步骤3表征纯化TRIP8b（241-602） 的体外构建亲和力FO岭异硫氰酸荧光素（FITC）-tagged对应HCN1（HCN1 FITC）的C-末端尾部11个氨基酸的肽。基于所述TRIP8b-HCN络合物15的晶体结构该11氨基酸片段是足以产生与TRIP8b（241-602）的结合。在步骤2中，相互作用的K个d由滴定TRIP8b（241-602）插入HCN1 FITC的固定浓度测定。在步骤3，在步骤2中使用的氰化氢肽的未标记的版本滴定到两个TRIP8b（241-602）和HCN1 FITC的固定浓度如果FITC标记具有结合干扰检查。这些实验都是在选择高通量筛选使用TRIP8b（241-602）和HCN1 FITC的适当浓度是必不可少的。 高通量筛选的前提是能够扰乱TRIP8b（241-602）和HCN1 FITC之间的相互作用将产生一个分解的小分子rease在偏振光。在步骤4中，测定的Z因子计算21 TRIP8b（241-602）和HCN1 FITC的给定浓度，以确保该测定是适当的高通量筛选（步骤5）。步骤6和7是验证实验，以确认在初级高通量筛选中鉴定的命中通过破坏TRIP8b（241-602）和HCN1 FITC之间，而不是通过非特异性机制的交互作用。在步骤6中，carboxytetramethylrhodamine（TAMRA）标记的HCN1肽（HCN1 TAMRA）是在其它方面相同的荧光偏振测定法用于筛选危及使用FITC标记的FP试验的荧光化合物。步骤7使用较大HCN1 C端片段（HCN1 C40）并采用基于珠子的亲近测定，这是基于从供体珠子单线态氧的"隧道"由相互作用的蛋白质带到彼此接近的受体珠22。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ni-NTA agarose&#160;</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis cassette&#160;</td>
			<td>ThermoFisher</td>
			<td>66456</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl b-D-1-thiogalactopyranoside&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>I5502-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>384 Well Black plate&#160;</td>
			<td>Corning</td>
			<td>3820</td>
			<td></td>
		</tr>
		<tr>
			<td>Proxiplate&#160;</td>
			<td>Perkin-Elmer&#160;</td>
			<td>6008289</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GST Acceptor beads&#160;</td>
			<td>Perkin-Elmer&#160;</td>
			<td>6760603C</td>
			<td></td>
		</tr>
		<tr>
			<td>NiChelate</td>
			<td>Perkin-Elmer&#160;</td>
			<td>AS101D</td>
			<td></td>
		</tr>
		<tr>
			<td>pGS21-a</td>
			<td>Genscript</td>
			<td>SD0121</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>10837091001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Kit</td>
			<td>ThermoFisher</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein concentrator</td>
			<td>ThermoFisher</td>
			<td>88527</td>
			<td></td>
		</tr>
		<tr>
			<td>Perkin Elmer Enspire Multimode Plate reader</td>
			<td>Perkin-Elmer&#160;</td>
			<td>#2300-001M</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent Cells</td>
			<td>Agilent</td>
			<td>200131</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for identifying small molecule inhibitors of the protein-protein interaction between hcn1 and trip8b

超极化激活环核苷酸门控（HCN）频道整个大脑，在那里它们起作用来调节神经元的兴奋遍在表达。海马CA1区锥体细胞这些通道的亚细胞分布由三十四肽含有重复-Rab8b相互作用蛋白（TRIP8b），辅助亚基调节。 HCN的遗传敲除孔形成亚基或TRIP8b，既导致增加抗抑郁样行为，这表明限制HCN通道的功能可以是作为用于抑郁症（MDD）的治疗是有用的。尽管显著治疗意义，HCN通道也都在心脏，在那里它们调节节律性表达。规避与阻断心脏HCN通道相关联的脱靶的问题，我们的实验室最近提出靶向为了专门破坏HCN通道功能在大脑HCN和TRIP8b之间的蛋白质 - 蛋白质相互作用。TRIP8b结合HCN孔形成在两个不同的相互作用位点亚基，尽管这里的重点是TRIP8b的三十四肽重复（TPR）结构域和HCN1的C-末端尾部之间的相互作用。在这个协议中，一种方法的用于纯化TRIP8b并执行高通量筛选，以鉴定HCN和TRIP8b之间的相互作用的小分子抑制剂的扩展的说明中，进行说明。用于高通量筛选的方法，利用荧光偏振（FP）为基础的测定以监测大TRIP8b片段荧光团标记十一结合氨基酸对应于HCN1 C端尾酸肽。基于在发射的光的偏振的变化，以确定该方法允许&#39;命中&#39;的化合物。然后进行验证试验，以确保&#39;打&#39;的化合物不是人为的。

以避免与我们先前的出版物的版权问题，一个TAMRA标记的探针HCN1 TAMRA用于生成图2和3。需要注意的是，这种替代并没有使在结果有明显的差别，并且这些协议是相同的上述与HCN1 FITC。来评估与HCN1 TAMRA，TRIP8b（241-602）的相互作用滴定成使用在步骤2（ 图2）中概述的协议HCN1 TAMRA的固定浓度。接着，进行在...

Watch this Scientific Journal Video about Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b at JoVE.com

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

HCN通道和其辅助亚基之间的相互作用已被确定为在抑郁症的治疗靶点。这里，用于识别这种蛋白质 - 蛋白质相互作用的小分子抑制剂荧光基于偏振的方法，提出。

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

method-for-identifying-small-molecule-inhibitors-protein-protein

Research

JoVE Journal

Biochemistry

8.5K 次观看.  Feinberg School of Medicine, Northwestern University.  HCN通道和其辅助亚基之间的相互作用已被确定为在抑郁症的治疗靶点。这里，用于识别这种蛋白质 - 蛋白质相互作用的小分子抑制剂荧光基于偏振的方法，提出。 

视频: Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

用利甘德分析（DRACALA）的微分径向毛细管作用识别小配体的结合蛋白

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

一种高通量酶偶联活性测定法，用于探测小分子与 dNTP 酶 SAMHD1 的相互作用

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

开发用于快速定量检测小分子化合物的横向流动免疫色谱带

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...