本发明涉及一种哺乳类细胞呼吸链超级复合物蛋白,尤其是具有高分辨原子分辨率冷冻电镜的结构,以及该蛋白的制备方法。
背景技术:
呼吸作用发生在几乎所有的生命体中,并且随着生物进化在越高等的生物体中进行呼吸作用的蛋白复合物越复杂。在哺乳动物中,进行呼吸作用的场所是线粒体。线粒体基质中,经过三羧酸循环(tca循环)稳定的化学能被转换成不稳定的化学能储存在nadh或fadh2中,这些分子则通过氧化呼吸链在线粒体内膜两侧形成质子梯度,而最终线粒体内膜上的atp合成酶则利用质子梯度形成的电化学势能合成高能分子atp。人们对形成质子梯度的氧化呼吸链的认识经历了一个漫长的过程,目前认为,氧化呼吸链由4个功能相对独立的蛋白复合物组成,即呼吸链复合物i(ci,即nadh脱氢酶复合物i),呼吸链复合物ii(cii,即琥珀酸脱氢酶复合物ii),呼吸链复合物iii(ciii,即细胞色素bc1复合物iii),呼吸链复合物iv(civ,即细胞色素c氧化酶复合物iv)。最新的研究显示,这四个蛋白复合物之间也具有相互作用,可以形成更高级的结构:呼吸链超级复合物(respiratorysupercomplex),而具有完整呼吸活性(消耗电子供体和氧气并且产生水)的呼吸链超级复合物又被称为呼吸体(respirasome)。
从20世纪90年代开始,随着x射线晶体学和结构生物学的发展,这些蛋白复合物的原子分辨率结构被相继解析,呼吸链中电子传递和能量转换的分子机理也逐渐变得清晰。在此基础上,关于线粒体内膜上呼吸链复合物组织形式的流动态模型也被大多数人所接受。
最早被解析结构的呼吸链复合物是civ。civ催化电子传递的最后一步,从细胞色素c中接受电子并将电子传递给氧气分子生成水,并且利用电子传递所释放的能量将质子从线粒体基质泵入线粒体膜间隙。1995年,iwatas等提取出了原核生物中的细胞色素c氧化酶并解析了它的晶体结构,最早鉴定出两种质子通道:k(赖氨酸)通道和d(天冬氨酸)通道(nature1995,376:660–669)。1996年,tsukiharat等首次从牛心线粒体中提取出了civ,并解析了首个完整的civ的晶体结构。
不久之后,呼吸链复合物ciii的晶体结构也被解析。ciii从还原态的泛醌qh2中得到电子,并将电子传递给细胞色素c,同时利用释放的能量将质子泵入膜间隙。1997年,xiad等获得了牛心中ciii的部分晶体结构(science1997,277,60-66)。
呼吸链复合物ci是线粒体呼吸链的起点,也是结构最为复杂的蛋白。从2006年到2013年,sazanovla等逐步解析了原核生物中ci的完整晶体结构,揭示了ci传递电子和转运质子的核心功能。但是与哺乳动物线粒体中具有45个亚基的ci相比,原核生物中ci只有14个核心蛋白亚基。考虑到哺乳动物ci庞大的分子量和复杂的亚基组成,传统的x射线晶体学方法不适合解析其晶体结构。2014年,hirstj等通过冷冻电镜的方法解析了牛心中ci的中等分辨率结构。
2000年,
呼吸链超级复合物的功能障碍会引起诸如疾病:阿尔茨海默病又名老年痴呆、帕金森病、多发性硬化症、共济失调症、肌萎缩性脊髓侧索硬化症、嗜酸细胞型腺瘤、遗传性视神经病变、婴儿致死性线粒体病、leigh综合征、复合物i功能减退以及糖尿病等疾病(biochimicaetbiophysicaacta,2014,1368–1379)。
知母皂甙是来源于知母(也叫毛知母,百合科,多年生草本植物)的化合物,已知有知母皂甙a-ⅰ、a-ⅱ、a-ⅲ、a-ⅳ,b-ⅰ和b-ⅱ(科技导报,2010,28(12)110-115)。已有研究表明知母皂甙具有抗病原微生物、降低血糖、治疗阿尔茨海默病以及用于肿瘤治疗的用途(科技导报,2010,28(12)110-115)。但是,对于知母皂甙的作用机理还不清楚。
技术实现要素:
本发明人采用冷冻电镜技术,解析了哺乳类呼吸链超级复合物蛋白i1iii2iv1的冷冻电镜三维结构,其分辨率高于4埃达到了原子分辨率水平。
本发明人提供了一种提取高纯度哺乳类呼吸链超级复合物蛋白的方法。
本发明提供了一种哺乳类呼吸链超级复合物蛋白的冷冻电镜三维结构,由一个nadh脱氢酶复合物i、两个细胞色素bc1复合物iii和一个细胞色素c氧化酶复合物iv组成超级复合物i1iii2iv1,超级复合物i1iii2iv1长300埃,高190埃;超级复合物整体relion(j.struct.biol.2012,180,519–530)软件报告分辨率高于4埃;
其中nadh脱氢酶复合物i包含如下45个蛋白亚基:
蛋白1具有seqidno:1的蛋白序列;或者与seqidno:1具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:1具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白2具有seqidno:2的蛋白序列;或者与seqidno:2具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:2具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白3具有seqidno:3的蛋白序列;或者与seqidno:3具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:3具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白4具有seqidno:4的蛋白序列;或者与seqidno:4具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:4具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白5具有seqidno:5的蛋白序列;或者与seqidno:5具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:5具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白6具有seqidno:6的蛋白序列;或者与seqidno:6具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:6具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白7具有seqidno:7的蛋白序列;或者与seqidno:7具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:7具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白8具有seqidno:8的蛋白序列;或者与seqidno:8具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:8具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白9具有seqidno:9的蛋白序列;或者与seqidno:9具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:9具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白10具有seqidno:10的蛋白序列;或者与seqidno:10具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:10具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白11具有seqidno:11的蛋白序列;或者与seqidno:11具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:11具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白12具有seqidno:12的蛋白序列;或者与seqidno:12具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:12具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白13具有seqidno:13的蛋白序列;或者与seqidno:13具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:13具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白14具有seqidno:14的蛋白序列;或者与seqidno:14具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:14具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白15具有seqidno:15的蛋白序列;或者与seqidno:15具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:15具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白16具有seqidno:16的蛋白序列;或者与seqidno:16具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:16具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白17具有seqidno:17的蛋白序列;或者与seqidno:17具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:17具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白18具有seqidno:18的蛋白序列;或者与seqidno:18具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:18具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白19具有seqidno:19的蛋白序列;或者与seqidno:19具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:19具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白20具有seqidno:20的蛋白序列;或者与seqidno:20具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:20具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白21具有seqidno:21的蛋白序列;或者与seqidno:21具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:21具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白22具有seqidno:22的蛋白序列;或者与seqidno:22具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:22具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白23具有seqidno:23的蛋白序列;或者与seqidno:23具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:23具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白24具有seqidno:24的蛋白序列;或者与seqidno:24具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:24具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白25具有seqidno:25的蛋白序列;或者与seqidno:25具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:25具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白26具有seqidno:26的蛋白序列;或者与seqidno:26具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:26具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白27具有seqidno:27的蛋白序列;或者与seqidno:27具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:27具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白28具有seqidno:28的蛋白序列;或者与seqidno:28具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:28具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白29具有seqidno:29的蛋白序列;或者与seqidno:29具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:29具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白30具有seqidno:30的蛋白序列;或者与seqidno:30具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:30具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白31具有seqidno:31的蛋白序列;或者与seqidno:31具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:31具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白32具有seqidno:32的蛋白序列;或者与seqidno:32具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:32具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白33具有seqidno:33的蛋白序列;或者与seqidno:33具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:33具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白34具有seqidno:34的蛋白序列;或者与seqidno:34具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:34具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白35具有seqidno:35的蛋白序列;或者与seqidno:35具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:35具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白36具有seqidno:36的蛋白序列;或者与seqidno:36具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:36具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白37具有seqidno:37的蛋白序列;或者与seqidno:37具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:37具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白38具有seqidno:38的蛋白序列;或者与seqidno:38具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:38具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白39具有seqidno:39的蛋白序列;或者与seqidno:39具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:39具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白40具有seqidno:40的蛋白序列;或者与seqidno:40具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:40具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白41具有seqidno:41的蛋白序列;或者与seqidno:41具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:41具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白42具有seqidno:42的蛋白序列;或者与seqidno:42具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:42具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白43具有seqidno:43的蛋白序列;或者与seqidno:43具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:43具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白44具有seqidno:44的蛋白序列;或者与seqidno:44具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:44具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;和
蛋白45具有seqidno:45的蛋白序列;或者与seqidno:45具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:45具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
其中细胞色素bc1复合物iii包含如下11个蛋白序列(本发明的超级复合物包含两个相同的细胞色素bc1复合物iii,每一个细胞色素bc1复合物iii都包含如下11个蛋白序列):
蛋白46具有seqidno:46的蛋白序列;或者与seqidno:46具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:46具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白47具有seqidno:47的蛋白序列;或者与seqidno:47具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:47具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白48具有seqidno:48的蛋白序列;或者与seqidno:48具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:48具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白49具有seqidno:49的蛋白序列;或者与seqidno:49具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:49具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白50具有seqidno:50的蛋白序列;或者与seqidno:50具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:50具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白51具有seqidno:51的蛋白序列;或者与seqidno:51具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:51具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白52具有seqidno:52的蛋白序列;或者与seqidno:52具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:52具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白53具有seqidno:53的蛋白序列;或者与seqidno:53具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:53具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白54具有seqidno:54的蛋白序列;或者与seqidno:54具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:54具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白55具有seqidno:55的蛋白序列;或者与seqidno:55具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:55具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白56具有seqidno:56的蛋白序列;或者与seqidno:56具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:56具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
其中细胞色素c氧化酶复合物iv包含如下13个蛋白序列:
蛋白57具有seqidno:57的蛋白序列;或者与seqidno:57具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:57具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白58具有seqidno:58的蛋白序列;或者与seqidno:58具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:58具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白59具有seqidno:59的蛋白序列;或者与seqidno:59具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:59具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白60具有seqidno:60的蛋白序列;或者与seqidno:60具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:60具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白61具有seqidno:61的蛋白序列;或者与seqidno:61具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:61具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白62具有seqidno:62的蛋白序列;或者与seqidno:62具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:62具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白63具有seqidno:63的蛋白序列;或者与seqidno:63具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:63具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白64具有seqidno:64的蛋白序列;或者与seqidno:64具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:64具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白65具有seqidno:65的蛋白序列;或者与seqidno:65具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:65具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白66具有seqidno:66的蛋白序列;或者与seqidno:66具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:66具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白67具有seqidno:67的蛋白序列;或者与seqidno:67具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:67具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;
蛋白68具有seqidno:68的蛋白序列;或者与seqidno:68具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:68具有一个或几个氨基酸的取代、缺失或替换的蛋白序列;和
蛋白69具有seqidno:69的蛋白序列;或者与seqidno:69具有至少95%氨基酸序列同一性的蛋白;或者与seqidno:69具有一个或几个氨基酸的取代、缺失或替换的蛋白序列。
在优选的实施方式中,本发明的蛋白冷冻电镜三维结构中还包含1个fmn分子(位于ndufv1亚基)、10个铁硫簇、一个nadph分子(位于ndufa9亚基)、一个锌离子(位于ndufa6亚基)、3个磷脂酰胆碱(散布于复合物i的跨膜区)、4个磷脂酰乙醇胺(散布于复合物i的跨膜区)、4个心磷脂(散布于复合物i的跨膜区)、2个细胞色素bl(位于复合物iii的cytb亚基)、2个细胞色素bh(位于复合物iii的cytb亚基)、两个细胞色素c1(位于位于复合物iii的cyt1亚基)、cua中心(位于复合物ivcox2亚基)、cub中心位于(位于复合物ivcox1亚基)、1个血红素a(位于复合物ivcox1亚基)和1个血红素a3(位于复合物ivcox1亚基)。
在优选的实施方式中,本发明蛋白,其中nadh脱氢酶复合物i包含如下45个蛋白质序列:seqidno:1(即ndufs2亚基),seqidno:2(即ndufs3亚基),seqidno:3(即ndufv2亚基),seqidno:4(即mt-nd1亚基),seqidno:5(即ndufs7亚基),seqidno:6(即ndufb8亚基),seqidno:7(即ndufa6亚基),seqidno:8(即ndufab1a亚基),seqidno:9(即ndufab1b亚基),seqidno:10(即ndufs4亚基),seqidno:11(即ndufa2亚基),seqidno:12(即ndufa1亚基),seqidno:13(即ndufc1亚基),seqidno:14(即nd4l亚基),seqidno:15(即ndufs8亚基),seqidno:16(即nd2亚基),seqidno:17(即nd3亚基),seqidno:18(即nd4亚基),seqidno:19(即nd5亚基),seqidno:20(即nd6亚基),seqidno:21(即ndufs1亚基),seqidno:22(即ndufs5亚基),seqidno:23(即ndufs6亚基),seqidno:24(即ndufv1亚基),seqidno:25(即ndufa3亚基),seqidno:26(即ndufa5亚基),seqidno:27(即ndufa7亚基),seqidno:28(即ndufa8亚基),seqidno:29(即ndufa9亚基),seqidno:30(即ndufa10亚基),seqidno:31(即ndufa11亚基),seqidno:32(即ndufa12亚基),seqidno:33(即ndufa13亚基),seqidno:34(即ndufb1亚基),seqidno:35(即ndufb2亚基),seqidno:36(即ndufb3亚基),seqidno:37(即ndufb4亚基),seqidno:38(即ndufb5亚基),seqidno:39(即ndufb6亚基),seqidno:40(即ndufb7亚基),seqidno:41(即ndufb9亚基),seqidno:42(即ndufb10亚基),seqidno:43(即ndufb11亚基)和seqidno:44(即ndufc2亚基),和seqidno:45(即ndufv3亚基);
细胞色素bc1复合物iii包含如下11个蛋白质序列:seqidno:46(即uqcrfs1亚基),seqidno:47(即mt-cyb亚基),seqidno:48(即cyc1亚基),seqidno:49(即uqcrc1亚基),seqidno:50(即uqcrc2亚基),seqidno:51(即uqcrh亚基),seqidno:52(即uqcrb亚基),seqidno:53(即uqcrq亚基),seqidno:54(即uqcr10亚基),seqidno:55(即uqcr11亚基),和seqidno:56(即uqcrfs1信号肽亚基);
细胞色素c氧化酶复合物iv包含如下13个蛋白质序列:seqidno:57(即mt-co3亚基),seqidno:58(即mt-co1亚基),seqidno:59(即mt-co2亚基),seqidno:60(即cox4亚基),seqidno:61(即cox5a亚基),seqidno:62(即cox5b亚基),seqidno:63(即cox6a亚基),seqidno:64(即cox6b亚基),seqidno:65(即cox6c亚基)和seqidno:66(即cox7a亚基)seqidno:67(即cox7b亚基),seqidno:68(即cox7c亚基),seqidno:69(即cox8h亚基)。
在一个优选的实施方案中,本发明的呼吸链超级复合物蛋白由80个蛋白序列组成,由于细胞色素bc1复合物iii有2个,因此该80个蛋白序列为上述seqidnos:1-69以及另一个细胞色素bc1复合物iii的序列seqidnos:46-56。优选地,本发明所述的nadh脱氢酶复合物i为哺乳动物来源,尤其是来源于猪(susscrofa)。
在进一步优选的实施方案中,本发明的蛋白冷冻电镜三维结构采用原子坐标来限定。
本发明从猪心线粒体中提取超级复合物,其结合形式与牛心中基本一致,最主要的形式为i1iii2iv1,也就是呼吸体。通过冷冻电镜单颗粒三维重构的方法,我们研究组在全世界范围内首次拿到了呼吸体的高分辨率蛋白结构。在我们的结构中,不仅清晰地解析了单独的ci和ciii的原子分辨率结构,而且精确地确定了ci、ciii和civ之间的相对位置以及它们之间发生相互作用的蛋白亚基。我们的结构为建立全新的电子传递和底物通道理论奠定了坚实的基础,让我们可以更为深入地理解呼吸体中能量转换的机制。
ci是呼吸体中最大的单体,亲水臂和跨膜臂连接呈l形。与ci单独存在时其跨膜臂并呈一条直线不同,在呼吸体中ci的跨膜臂在膜平面内略微弯曲呈弧形,其亲水臂也更加靠近中部。ciii以二聚体的形式在呼吸体中存在,结合在ci跨膜臂的凹面,二聚体中的一个单体面向ci,而另一个单体指向呼吸体之外。这一结构上的差异暗示了二者在功能上是有区别的。civ结合在ci跨膜臂的远端,也就是说ci的跨膜臂一端连接亲水臂,而另一端与civ结合。由于ci跨膜臂呈弧形,civ与ciii之间的相对位置非常接近,使它们之间的相互作用成为可能。ci、ciii和civ的跨膜区位于同一个平面,相互靠近构成一个盘状结构。ci与ciii之间的相互作用非常紧密。在跨膜区内,ci中的蛋白亚基ndufb4、ndufb8和ndufa11可以与ciii中的蛋白亚基uqcrb和uqcrq发生相互作用,同时,由于ci非常庞大,处于跨膜区中段的ndufa11在ci自身的组装过程中也发挥着非常巨大的作用,ndufa11主要与ci的nd5、nd2、nd4和ndufb5亚基相互作用,使得ndufa11与ci紧密相连成为其重要的组成部分。另外,ci中的ndufb4、ndufb9还与ciii中的uqcrc1发生相互作用,进一步加强ci与ciii之间的联系。并且,磷脂酰胆碱分子也会介导复合物i的ndufa11,ndufb4,nd5同复合物iiiuqcrq相互作用。civ与ci和ciii之间的相互作用则相对较弱。civ中的cox7c与ci中的nd5有相互作用,ndufb3,ndufb7和ndufb8同复合物iv的cox7c和cox8在空间位置上相互靠近。同时,civ中的cox7a则与ciii中的uqcrc1、uqcr11和uqcrb都有相互作用。在ci与ciii的跨膜区之间有一个缝隙,我们认为会被线粒体内膜中的脂质填充,同时,也是辅酶q储存和流动的场所。辅酶q被限制在这一区域会极大地提高ci与ciii之间电子传递的效率。ciii和civ结合细胞色素c的位点都位于线粒体膜间隙,在呼吸体中这两个位点处于同一平面且非常接近,这将促进ciii与civ之间细胞色素c的传递。
本发明还涉及一种制备所述呼吸链超级复合物蛋白的方法,该方法包括以下步骤:a.线粒体的提取;b.从线粒体中抽提呼吸链超级复合物蛋白;c.纯化步骤b获得的超级复合物蛋白;d.对步骤c的纯化产物进行bn-page检测;和e.进行凝胶排阻层析。
在优选的实施方案中,步骤b采用洋地黄皂苷来抽提呼吸链超级复合物蛋白。
在优选的实施方案中,在步骤e中,以三羟基氨基甲烷、氯化钾和洋地黄皂苷为溶质配制层析缓冲液,将含有超级复合物的样品载入到凝胶排阻层析柱上,进行层析,并收取峰尖。优选层析缓冲液具有7-8的ph值,进一步优选为7.3-7.5的ph值。优选地,其中所述的峰尖是采用的aktapurifier层析仪器,层析柱为superose610/300gl,收集在280nm波长下检测到的蛋白峰的最高值。
在优选的实施方案中,呼吸链超级复合物蛋白是来源于人或猪。
附图说明
图1为哺乳类来源的超级复合物i1iii2iv1的三维冷冻电镜结构
a为冷冻电镜得到的呼吸链超级复合物的密度图,各个复合物均已表明;
b为密度图和卡通结构图放在一起的得到的整体结构图;
c为超级复合物整体结构的模型。
图2为哺乳类来源超级复合物i1iii2iv1中复合体i与复合物iii相互作用关系。
a为位于复合物i和iii之间介导两者相互作用的ndufa11,ndufb4和ndufb8的位置示意图。
b为ndufa11一侧与复合物i的nd5亚基相互作用,具体作用示意图。
c为ndufa11c端同复合物i的nd2,nd4和ndufb5的作用关系示意图。
d为介导复合物i的ndufa11,ndufb4,nd5同复合物iiiuqcrq相互作用的磷脂酰胆碱示意图。
e为复合物indufa11同复合物iii相互作用示意图。
f为复合物iii的uqcrc1同复合物i的ndufb4、ndufb9作用示意图。
图3为哺乳类来源超级复合物i1iii2iv1中复合体iv与复合物iii或复合物i相互作用关系。
a为复合物iv同复合物i的相互作用关系,复合物i的nd5亚基503位的谷氨酸同复合物iv的cox7c亚基20位的精氨酸形成氢键。此外复合物i的ndufb3,ndufb7和ndufb8同复合物iv的cox7c和cox8在空间位置上相互靠近。
b为复合物iv同复合物iii的相互作用关系。复合物ivcox7a同复合物iii的uqcrc1,uqcr11的n端和uqcrb的c端相互作用。
图4为电镜原始数据。
图5为电镜数据处理流程。
发明详述
蛋白
“多肽”、“蛋白”和“亚基”在本文中含义相同,都是指氨基酸残基的聚合物。氨基酸残基可以是天然产物或者是化学类似物。
呼吸链复合物蛋白
呼吸链复合物蛋白是指锚定在线粒体内膜上,行使将电子从nadh或fadh2传递给氧气,并使得线粒体内膜两侧产生质子梯度的多亚基蛋白复合物。总共包含四种复合物,分别是:呼吸链复合物i(ci,nadh脱氢酶),呼吸链复合物ii(cii,琥珀酸脱氢酶),呼吸链复合物iii(ciii,细胞色素bc1复合体),呼吸链复合物iv(civ,细胞色素c氧化酶)。
同一性
由参数“同一性”描述两个氨基酸序列之间的相关性。
就本发明而言,两个氨基酸序列之间的同一性程度使用如emboss软件包(emboss:theeuropeanmolecularbiologyopensoftwaresuite,rice等,2000,trendsingenetics16:276-277)的needle程序,优选3.0.0版或更高版本中执行的needleman-wunsch算法(needleman和wunsch,1970,j.mol.biol.48:443-453)来确定。使用的任选参数为缺口罚分(gappenalty)10,缺口延伸罚分(gapextensionpenalty)0.5和eblosum62取代矩阵(blosum62的emboss版)。使用needle标记为“最高同一性(longestidentity)”(使用-nobrief选项获得)的输出结果作为百分比同一性,并计算如下:
(相同的残基×100)/(比对长度-比对中缺口的总数)。
原子坐标
“原子坐标”或“结构坐标”指描述原子在proteindatabank(pdb)格式(format)中位置的数学坐标,包括每个原子的x、y、z和b坐标。本领域技术人员应理解,由x射线结晶学确定的一组结构坐标并不是没有标准偏差的。在本发明中,任何来源的线粒体呼吸链超级复合物i1iii2iv1结构坐标,当叠加在原子坐标的非氢原子位置上时,其非氢原子位置的均方根偏差小于
实施例
实施例1.呼吸链超级复合物蛋白i1iii2iv1的纯化
线粒体的提取
100g新鲜猪心组织,切成2cm×2cm×2cm小块,用100ml缓冲液a(100mmtrisph7.4,225mm山梨醇,60mmkcl,1mmegta和0.1%牛血清蛋白)悬起。用大容量组织匀浆机破碎300秒。将匀浆液转移至50ml离心管中,3000g离心10分钟(min),弃去细胞碎片等沉淀,留取上清。将上清转移至高速离心管中,20,000离心30分钟,得到沉淀,此沉淀即为粗提的线粒体。配制缓冲液b(100mmtrisph7.4,250mm蔗糖,60mmkcl,40%percoll(经过聚乙烯吡咯烷酮处理的硅胶颗粒混悬液)和0.1mmegta),用100ml缓冲液b重悬粗提的线粒体,60,000离心50分钟,而后会形成明晰的线粒体层,用吸管吸取悬浮的线粒体,加入到缓冲液c(100mmtrisph7.4,100mm山梨醇,60mmkcl,0.05mmegta)中。将重悬液20,000g离心30分钟,得到的沉淀即为超纯线粒体。
超级复合物蛋白从线粒体中抽提
将提纯的线粒体用缓冲液c重悬,然后加入终浓度为1%的洋地黄皂苷(digitonin),放置在旋转摇床上4℃抽提过夜。收取抽提液,150,000g离心30分钟。收取上清,将上清加入到100kda超滤浓缩管中浓缩至1ml。
超级复合物蛋白的纯化
配制缓冲液d(25mmtris,ph7.4,60mmkcl,0.1%洋地黄皂苷,0.3m蔗糖)和缓冲液e(25mmtris,ph7.4,60mmkcl,0.1%洋地黄皂苷,1.8m蔗糖),用两种缓冲液配制成11ml连续的0.3m-1.8m的浓度梯度,装载到sw41转子专用的离心管中,而后将1ml超级复合物粗提蛋白轻轻加到11ml连续梯度之上,采用sw41转子,150,000g,4℃,离心20小时。
bn-page检测
将离心完毕的离心管轻轻取出,1ml一管收取样品,分别制样,进行3%-10%梯度bn-page电泳,检测哪些样品中含有超级复合物。
凝胶排阻层析
以10mmtrisph7.4,60mmkcl,0.1%洋地黄皂苷为层析缓冲液,将含有超级复合物的样品载入到凝胶排阻层析柱superose610/300gl上,进行层析,收取峰尖,此为超纯的超级复合物。
小子药物添加与浸泡
在提纯的超级复合物中添加终浓度1μm的知母皂甙,4℃孵育3小时。
实施例2.呼吸链超级复合物蛋白i1iii2iv1冷冻电镜数据的收集及蛋白结构解析
电镜样品制备
取4μl浸泡过小分子药物的超级复合物蛋白滴加到事先铺好碳膜的400目quantifoilr1.2/1.3铜网(购自quantifoil,microtoolsgmbh,germany)上,静置1.5秒。然后用feimarkivvitrobot制样机器制取冷冻样品。
电镜数据采集
采用300kvtitankrios进行高分辨率图像数据的收集,检测器为falconiidirectelectrondetector(fei公司)。数据采集的控制软件为epu(fei公司)。电镜的放大倍数是75,000×,像素大小为1.08埃,欠焦值为-1.3μm到-2.3μm,电子剂量为35个/像素/秒,曝光时间为1.5秒,每个显微照片栈包含28幅照片。
结果见图4.电镜原始数据,图5为电镜数据处理流程
电镜图像处理
图像漂移校正采用dosefgpu_driftcorrprogram(2013,naturemethods10,584–590.)。使用eman2.1(2007,j.struct.biol.157,38–46)和relion1.4(2012,j.struct.biol.180,519–530)这两个程序进行图像筛选、单颗粒挑选、归一化。使用ctffind3(2003,j.struct.biol.142,334–347)进行衬度传递函数(ctf)校正。单颗粒的二维、三维分类以及优化是用relion程序完成的。从4660张图片中挖出的424,002个单颗粒分子,进行一系列二维和三维分类,而后161,912个相对比较均一的单颗粒分子被拿来用作最终的优化。通过局部优化以及加入各种柔性表面限定模型(softmask),最终的得到3.6埃分辨率的复合体i的电镜数据,3.97埃超级复合物i1iii2iv1的电镜数据,3.9埃超级复合物i1iii2的电镜数据,3.6埃复合体iii的电镜数据,7.5埃复合体iv的电镜数据。最终报告分辨率基于fsc=0.143的标准。最终的电子云密度图采用
三维结构模型的搭建及优化
采用5.0埃低分辨率的牛心来源的复合体i(pdb号4uq8)为初始模型,通过coot软件(2004,actacrystallogr.d60,2126–2132),以3.6埃的电子云密度图为基础,将各氨基酸的侧链搭进密度图并进行位置的优化。复合物i的14个核心亚基以及30个附属亚基搭建进了3.6埃的电子云密度图。对于超级复合物中原子分辨率的复合体iii结构的解析,采用pdb号为1bgyx-ray结构作为初始模型,采用coot软件进行模型搭建及修正。磷脂三维结构通过phenix(2010,actacryst.d66,213-221)的elbow组件以simile文件生成。最后,所有的小分子以及蛋白的整体结构通过phenix进行整体优化。最终得到完整的超级复合物i1iii2iv1以及各组分的结构模型。
sequencelisting
<110>清华大学
<120>一种呼吸链超级复合物蛋白
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metileleuvalthralaasnasnleupheglnleupheileglytrp
130135140
gluglyvalglyilemetserpheleuleuileglytrptrphisgly
145150155160
argthraspalaasnthralaalaleuglnalaileleutyrasnarg
165170175
ileglyaspileglyphevalleusermetalatrppheleuthrhis
180185190
serasnalatrpaspleuglnglnilephemetleuasnasnglucys
195200205
proasnmetproleuileglyleuleuleualaalaalaglylysser
210215220
alaglnpheglyleuhisprotrpleuproseralametgluglypro
225230235240
thrprovalseralaleuleuhisserserthrmetvalvalalagly
245250255
valpheleuleuileargphetyrproleumetgluthrasnlysleu
260265270
valglnthrmetthrleucysleuglyalailethrthrleuphethr
275280285
alaleucysalailethrglnasnaspilelyslysilevalalaphe
290295300
serthrserserglnleuglyleumetmetvalthrileglyileasn
305310315320
glnprohisleualapheleuhisilecysmethisalaphephelys
325330335
alametleuphemetcysserglyserileilehisserleuasnasp
340345350
gluglnaspilearglysmetglyglyleutyrlysalametprophe
355360365
thrthrthralaleuileileglyserleualaleuthrglymetpro
370375380
tyrleuthrglyphetyrserlysaspleuileileglualavalasn
385390395400
metsertyrthrasnalatrpalaleuleumetthrleuilealathr
405410415
serleuthralaalatyrserthrargileilephephealapheleu
420425430
glylysproargpheproproleuvalleuileasngluasnasnpro
435440445
leuleuileasnserilelysargleuleuileglyserilepheala
450455460
glypheileileserasnasnileproprometthrvalproasnthr
465470475480
thrmetproleutyrmetlysmetthralaleuilevalthrilemet
485490495
glyphemetleualaleugluleuasnasnthrthrtyrtyrleulys
500505510
phelystyrproserglnthrtyrlyspheserasnmetleuglytyr
515520525
tyrproserilemethisargleuprothrtyrhisasnleusermet
530535540
serglnlysseralaserserleuleuaspleuiletrpleugluthr
545550555560
ileleuprolysthrthrserpheileglnmetlysmetserilemet
565570575
valserasnglnlysglyleuilelysleutyrpheleuserpheleu
580585590
ilethrilemetilesermetthrleupheasntyrhisglu
595600605
<210>20
<211>175
<212>prt
<213>susscrofa
<400>20
metthrmettyrilealapheileleuserthrilephevalilegly
151015
phevalglypheserserlysproserproiletyrglyglyleugly
202530
leuilevalserglyglyvalglycysglyilevalleuasnphegly
354045
glyserpheleuglyleumetvalpheleuiletyrleuglyglymet
505560
leuvalvalpheglytyrthrthralametalathrglumettyrpro
65707580
gluvaltrpvalserasnlysthrvalpheglyalaphevalsergly
859095
leumetmetgluphecysmetvaltyrtyralaleulysglugluglu
100105110
valgluileilephelyspheasnglyleuglyasptrpvaliletyr
115120125
aspthrglyaspserglyphephesergluglualametglyileala
130135140
alaleutyrsertyrglythrtrpleuvalilevalthrglytrpser
145150155160
leuleuileglyvalvalvalilemetgluilethrargglyasn
165170175
<210>21
<211>704
<212>prt
<213>susscrofa
<400>21
thralathralaalaserasnleuilegluvalphevalaspglygln
151015
servalmetvalgluproglythrthrvalleuglnalacysglulys
202530
valglymetglnileproargphecystyrhisgluargleuserval
354045
alaglyasncysargmetcysleuvalgluileglulysalaprolys
505560
valvalalaalacysalametprovalmetlysglytrpasnileleu
65707580
thrasnserglulysserlyslysalaarggluglyvalmetgluphe
859095
leuleualaasnhisproleuaspcysproilecysaspglnglygly
100105110
glucysaspleuglnaspglnsermetmetpheglyseraspargser
115120125
argpheleugluglylysargalavalgluasplysasnileglypro
130135140
leuvallysthrilemetthrargcysileglncysthrargcysile
145150155160
argphealasergluilealaglyvalaspaspleuglythrthrgly
165170175
argglyasnaspmetglnvalglythrtyrileglulysmetphemet
180185190
sergluleuserglyasnileileaspilecysprovalglyalaleu
195200205
thrserlysprotyralaphethralaargprotrpgluthrarglys
210215220
thrgluserileaspvalmetaspalavalglyserasnilevalval
225230235240
serthrargthrglygluvalmetargileleuproargmethisglu
245250255
aspileasngluglutrpileserasplysthrargphealatyrasp
260265270
glyleulysargglnargleuthrglnprometileargasnglulys
275280285
glyleuleuthrtyrthrthrtrpgluaspalaleuserargvalala
290295300
glymetleuglnserpheglnglyasnaspvalalaalailealagly
305310315320
glyleuvalaspalaglualaleuvalalaleulysaspleuleuasn
325330335
argvalaspseraspserleucysthrglugluvalpheprothrala
340345350
glyalaglythraspleuargserasntyrleuleuasnthrthrile
355360365
alaglyvalgluglualaaspvalileleuleuvalglythrasnpro
370375380
argpheglualaproleupheasnalaargilearglyssertrpleu
385390395400
hisasnaspleulysvalalaleuileglyserprovalaspleuthr
405410415
tyrargtyrasphisleuglyaspserprolysileleuglnaspile
420425430
alaserglyasnhispropheserglnileleulysglualalyslys
435440445
prometvalvalleuglyserseralaleuglnargseraspglythr
450455460
alaileleualaalavalserasnilealaglnasnileargleuser
465470475480
serglyvalthrglyasptrplysvalmetasnileleuhisargile
485490495
alaserglnvalalaalaleuaspleuglytyrlysproglyvalglu
500505510
alailearglysasnproprolysvalleupheleuleuglyalaasp
515520525
glyglycysilethrargglnaspleuprolysaspcyspheileile
530535540
tyrglnglyhishisglyaspvalglyalaprometalaaspvalile
545550555560
leuproglyalaalatyrthrglulysseralathrtyrvalasnthr
565570575
gluglyargalaglnglnthrlysvalalavalthrproproglyleu
580585590
alaarggluasptrplysileileargalaleusergluilealagly
595600605
metthrleuprotyraspthrleuaspglnvalargserargleuglu
610615620
gluvalserproasnleuvalargtyraspaspvalgluglyalaasn
625630635640
tyrpheglnglnalaasngluleuserlysleuvalasnglnglnleu
645650655
leualaaspproleuvalproproglnleuthrilelysaspphetyr
660665670
metthraspserileserargalaserglnthrmetalalyscysval
675680685
lysalavalthrgluglyileglnalavalglugluproserilecys
690695700
<210>22
<211>105
<212>prt
<213>susscrofa
<400>22
prophepheaspvalglnlysargleuglyleuaspleuaspargtrp
151015
metthrileglnseralagluglnprohislysileproglyargcys
202530
hisalapheglulysglutrpileglucysalahisglyileglygly
354045
ileargalaglulysglucyslysileglupheaspaspphevalglu
505560
cysleuleuargglnlysthrmetlysargleuseralailelysarg
65707580
glnargasplysleuilelysgluglylystyrthrproproprohis
859095
hisleuglylysgluaspproargpro
100105
<210>23
<211>96
<212>prt
<213>susscrofa
<400>23
glyvalargthrserprothrglyglulysvalthrhisthrglygln
151015
alatyraspaspglyasptyrargargvalargpheserasparggln
202530
lysgluvalasngluasnphealaileaspleuilealagluglnpro
354045
valsergluvalglyserargvalilesercysaspglyglyglygly
505560
alaleuglyhisproargvaltyrileasnleuasplysgluthrlys
65707580
thrglythrcysglytyrcysglyleuglnpheargglnprohishis
859095
<210>24
<211>444
<212>prt
<213>susscrofa
<400>24
serglyaspthrthralaprolyslysthrserpheglyserleulys
151015
aspgluaspargilephethrasnleutyrglyarghisasptrparg
202530
leulysglyalaglnserargglyasptrptyrlysthrlysgluile
354045
leuleulysglyproasptrpileleuglygluvallysthrsergly
505560
leuargglyargglyglyalaglypheprothrglyleulystrpser
65707580
phemetasnlysproseraspglyargprolystyrleuvalvalasn
859095
alaaspgluglygluproglythrcyslysasparggluileilearg
100105110
hisaspprohislysleuvalgluglycysleuvalglyglyargala
115120125
metglyalaargalaalatyriletyrileargglygluphetyrasn
130135140
glualaserasnleuglnvalalaileargglualatyrglualagly
145150155160
leuileglylysasnalacysglyserglytyrasppheaspvalphe
165170175
valvalargglyalaglyalatyrilecysglyglugluthralaleu
180185190
ilegluserilegluglylysglnglylysproargleulyspropro
195200205
pheproalaaspvalglyvalpheglycysprothrthrvalalaasn
210215220
valgluthrvalalavalserprothrilecysargargglyglyala
225230235240
trpphealaserpheglyarggluargasnserglythrlysleuphe
245250255
asnileserglyhisvalasnhisprocysthrvalglugluglumet
260265270
servalproleulysgluleuileglulyshisalaglyglyvalile
275280285
glyglytrpaspasnleuleualavalileproglyglyserserthr
290295300
proleuileprolysservalcysgluthrvalleumetasppheasp
305310315320
alaleuvalglnalaglnthrglyleuglythralaalavalileval
325330335
metaspargserthraspilevallysalailealaargleuileglu
340345350
phetyrlyshisglusercysglyglncysthrprocysargglugly
355360365
valasptrpmetasnlysvalmetalaargphevallysglyaspala
370375380
argproalagluileaspserleutrpgluileserlysglnileglu
385390395400
glyhisthrilecysalaleuglyaspglyalaalatrpprovalgln
405410415
glyleuilearghispheargprogluleuglugluargmetglngln
420425430
phealaleuglnhisglnalaargglnalaalaser
435440
<210>25
<211>83
<212>prt
<213>susscrofa
<400>25
alaglyargilealaserpheleulysasnalatrpalalysglupro
151015
valleuvalalaserphealaileglyglyleualaileileleupro
202530
serleuserprotyrthrasntyralaileargileasnargalathr
354045
protyrasntyrprovalproleuargaspaspglyasnmetproasp
505560
valproserhisproglnaspproglnglyproserleuglutrpleu
65707580
lysasnleu
<210>26
<211>115
<212>prt
<213>susscrofa
<400>26
alaglyleuleulyslysthrthrglyleuvalglyleualavalcys
151015
gluthrprohisgluargleulysileleutyrthrlysileleuasp
202530
valleuglyglnileprolysasnalaalatyrarglystyrthrglu
354045
glnilethrasnglulysleuglymetvallysalagluproaspval
505560
lyslysleuglugluglnleuglnglyglyglnileglugluvalile
65707580
leuglnalagluasngluleuserleualaarglysmetleuargtrp
859095
lysprotrpgluproleuvalglugluproproalaasnglntrplys
100105110
trpproile
115
<210>27
<211>112
<212>prt
<213>susscrofa
<400>27
alaseralathrargvalileglnleuleuargasntrpalasergly
151015
argaspleuglnalalysleuglnleuargtyrglngluileserlys
202530
argthrglnproproprolysleuprovalglyproserhislysleu
354045
serasnasntyrtyrcysthrargaspglyargargglualametpro
505560
proserilevalmetserserglnlysvalleualaserglylyspro
65707580
alagluserseralavalalagluthrglulyslysalavalthrpro
859095
alaproproilelysargtrpgluleuserlysaspglnprotyrleu
100105110
<210>28
<211>171
<212>prt
<213>susscrofa
<400>28
proglyilevalgluleuprothrleugluaspleulysvalglnglu
151015
vallysvalserserservalleulysalaalaalahishistyrgly
202530
alaglncysasplysproasnlysgluphemetleucysargtrpglu
354045
glulysaspproargargcysleuglugluglylysleuvalasngln
505560
cysalaleuaspphepheargglnilelysarghiscysalaglupro
65707580
phethrglutyrtrpthrcysileasptyrserglyleuglnleuphe
859095
arghiscysarglysglnglnalalyspheaspglucysvalleuasp
100105110
lysleuglytrpvalargproaspleuglygluleuserlysvalthr
115120125
lysvallysthraspargproleuprogluasnprotyrhisserarg
130135140
alaargprogluproasnproglualagluglyaspleulysproala
145150155160
lyshisglyserargleuphephetrpthrmet
165170
<210>29
<211>342
<212>prt
<213>susscrofa
<400>29
leuhishisalaleuileprohisglylysglyglyargserserval
151015
serglyilevalalathrvalpheglyalathrglypheleuglyarg
202530
tyrvalvalasnhisleuglyargmetglyserglnvalilevalpro
354045
tyrargcysgluprotyraspthrmethisleuargprometglyasp
505560
leuglyglnileilephemetglutrpasnglylysasplysaspser
65707580
ilearglysvalvalgluhisserasnvalvalileasnleuvalgly
859095
argglutrpgluthrlysasnpheaspphegluaspvalphevallys
100105110
ileprohisalailealaglnvalserlysglualaglyvalglulys
115120125
leuilehisileserhisleuasnalaaspilelysserproserarg
130135140
tyrleuargserlysalavalglyglulysgluvalargalaalaphe
145150155160
proglualathrileilelysproseraspilepheglyarggluasp
165170175
argpheleuasntyrphealasermetargtrppheglyglyvalpro
180185190
leuileserleuglylysgluthrvallysglnprovaltyrileval
195200205
aspvalserlysglyileileasnalailelysaspproaspalalys
210215220
glylysthrphealaphevalglyproasnargtyrleuleupheasp
225230235240
leuvalglntyrilephealavalalatyrargpropheleuprotyr
245250255
proleuprohisphealatyrargtrpvalglyargleuphegluval
260265270
serprophegluprotrpthrthrargasplysvalgluargvalhis
275280285
metseraspmetthrleuprohisleuproglyleugluaspleugly
290295300
ileglnalathrproleugluleulysalailegluvalleuargarg
305310315320
hisargthrtyrargtrpleuthrserglumetgluaspvallyspro
325330335
alalysthrvalasnile
340
<210>30
<211>320
<212>prt
<213>susscrofa
<400>30
leuglntyrglyproleualaphevalleuglygluargthrthrarg
151015
lysleuthrgluthrserlysvalilethrvalaspglyasnilecys
202530
serglylysglyargleualaarggluilealaglulysleuglyleu
354045
arghispheproglualaglyilehistyralaaspserthrthrgly
505560
aspglylysproleuaspvalglnleuserglyasncysserleuglu
65707580
lysphetyraspaspprolysserasnaspglyasnsertyrargleu
859095
glnsertrpleutyralaserargleuleuglntyralaaspalaleu
100105110
gluhisleuleuserthrglyglnglyvalvalleugluargserile
115120125
tyrseraspphevalpheleuglualamettyrargglnglypheile
130135140
arglysglncysvalgluhistyrasngluvallyslysvalthrala
145150155160
cysglutyrleuproprohisvalvalvaltyrvalaspvalproval
165170175
progluileglnserargileglnlyslysglyasnprohisglumet
180185190
lysilethralaalatyrleuglnaspilegluasnalatyrlyslys
195200205
thrpheleuproglumetserglulyscysgluvalleuglntyrser
210215220
alaargglualagluaspalaglulysvalvalgluaspileglutyr
225230235240
leulyscysasplysglyprotrpproaspglnaspaspargthrphe
245250255
hisargleuargmetleuvalglnasnlysleugluvalleuasntyr
260265270
thrthrileprovaltyrleuprogluilethrileglyalahisgln
275280285
seraspargvalpheglnlysphethrgluleuproglyarglystyr
290295300
serproglytyrasngluaspvalglyasplystrpiletrpleulys
305310315320
<210>31
<211>140
<212>prt
<213>susscrofa
<400>31
alalysthrleuleuhislystyrseraspileprogluglythrglu
151015
cyshisarglysalatyralaserthrserileglyglyalathrgly
202530
leuilevalseralatyrserilealaleulysproproalaserphe
354045
leugluglyvalalaargthrglyargtyrthrphethrseralaala
505560
ileglyalailepheglyleuthrsercysileseralaglnvalarg
65707580
glulysproaspaspproleuasntyrpheileglyglycysalagly
859095
glyleuthrleuglyalaargthrargsertyrglyileglyalaala
100105110
alacysalatyrmetglyleuthralaalaleuvallysmetglygln
115120125
leugluglytrpglnvalphealagluprolysval
130135140
<210>32
<211>145
<212>prt
<213>susscrofa
<400>32
metgluleuvalglnvalleuargargglyleuglnglnvalsergly
151015
hisglyglyleuargglytyrleuargvalleupheargalaasnasp
202530
valargvalglythrleuvalglygluasplystyrglyasnlystyr
354045
tyrgluaspasnlysglnphepheglyarghisargtrpvaliletyr
505560
thrthrglumetasnglyargaspthrphetrpaspvalaspglyser
65707580
metvalproproglutrphisargtrpleuhiscysmetthraspasp
859095
proprothrthrlysproprothralaarglystyriletrpthrasn
100105110
hislyspheasnvalserglythrproglnglntyrvalprotyrser
115120125
thrthrarglyslysileglnglutrpvalproproserthrprotyr
130135140
lys
145
<210>33
<211>143
<212>prt
<213>susscrofa
<400>33
alaalaserlysvallysglnaspmetproproproglyglytyrgly
151015
proileasptyrlysargasnleuproargargglyleuserglytyr
202530
sermetphealavalglyileglythrleuleupheglytyrtrpser
354045
metmetlystrpasnarggluargargargleuglnilegluaspphe
505560
glualaargilealaleumetproleupheglnalaglulysasparg
65707580
argvalleuglnmetleuarggluasnleuglugluglualaileile
859095
metlysaspvalproasptrplysvalglygluservalphehisthr
100105110
thrargtrpvalthrprometmetglygluleutyrglyleuargthr
115120125
asnglugluileleuseralathrtyrglypheiletrptyrthr
130135140
<210>34
<211>57
<212>prt
<213>susscrofa
<400>34
metasnvalleuglnilevalargasphistrpvalhisileleuval
151015
provalglyphevalpheglycystyrleuaspargargseraspglu
202530
lysleuthralapheargasnlysserleuleuphelysarggluleu
354045
argproasnglugluvalthrtrplys
5055
<210>35
<211>72
<212>prt
<213>susscrofa
<400>35
alaglyglyglyvalhisilegluproargtyrargglnpheprogln
151015
leuthrargserglnleuileglnalagluphepheseralathrmet
202530
trpphetrpileleutrpargphetrphisaspseraspalavalleu
354045
glyhispheprotyrproaspproserglntrpthraspglugluleu
505560
glyileleuproaspaspgluasp
6570
<210>36
<211>97
<212>prt
<213>susscrofa
<400>36
alahisglyhisglyhisgluhisglyhisserlysmetgluleupro
151015
asptyrlysglntrplysilegluglythrproleugluthrvalgln
202530
glulysleualaalaargglyleuargaspprotrpglyargasnglu
354045
alatrpargtyrserglyglyphealaasnasnvalserphevalgly
505560
alaleuleulysglyphelystrpglyphealaalaphevalvalala
65707580
valglyalaglutyrtyrleugluserglnasnlysasplyslyshis
859095
his
<210>37
<211>128
<212>prt
<213>susscrofa
<400>37
serpheprolystyrlysproserargleualathrleuproalathr
151015
leuaspproalaglutyraspileserprogluthrarglysalagln
202530
alagluargleualaileargserargleulysargglutyrleuleu
354045
glntyrasnaspproasnargleuglyvalilegluaspproalaleu
505560
ileargtrpthrtyralaargseralaasniletyrproasnphearg
65707580
prothrprolysthrserleuleuglyalaleupheglyileglypro
859095
leuphephetrptyrtyrvalphelysthraspargasplyslysglu
100105110
lysleuileglngluglylysleuaspglnthrpheasnilesertyr
115120125
<210>38
<211>143
<212>prt
<213>susscrofa
<400>38
serglyasphisglylysargleupheileilelysproserglyphe
151015
tyraspargargpheleulysleumetargphetyrileleuleuthr
202530
glyileprovalalaileglyilethrleuvalasnvalpheilegly
354045
glualagluleualaaspileprogluglytyrvalprogluhistrp
505560
glutyrphelyshisproileserargtrpilealaargthrphetyr
65707580
aspglyproglulysasntyrglulysthrmetalaileleuglnile
859095
glualaglulysalagluleuargleulysgluleugluvalargarg
100105110
leumetargalaargglyaspglyprotrptyrglntyrprothrile
115120125
asplysalaleuileasphisserprolysthrthrproaspasn
130135140
<210>39
<211>127
<212>prt
<213>susscrofa
<400>39
serglytyrthrproaspglulysleuargleuglnglnleuargglu
151015
leuargargargtrpleulysaspglngluleuserproargglupro
202530
valleuproproargargvaltrpprometgluglnphetrpasnlys
354045
pheleuglnaspglyalaprotrplysasnvaliletyrlysthrtyr
505560
arghisserilephealavalthrhisvalleuileprovaltrpile
65707580
ilehistyrtyrleulystyrhisvalthralalysprotyrthrval
859095
valgluarglysproargilepheproglyaspthrileleugluthr
100105110
glygluvalileproleumetlysglupheproaspglnhishis
115120125
<210>40
<211>136
<212>prt
<213>susscrofa
<400>40
glyalahisleualaargargtyrleuglyaspalaserlysglupro
151015
aspproleuargmetprothrpheproproasptyrglypheproglu
202530
arglysgluargglumetvalalathrglnglnglumetasnaspala
354045
glnleumetleuglnglnargasptyrcysalahistyrleuilegln
505560
leuleulyscyslysargaspserpheproasnpheleualacyslys
65707580
hisgluglnhisasptrpasptyrcysgluhisleuasptyrvallys
859095
argmetlysgluphegluarggluargargleuleuglnarglyslys
100105110
argarggluglnargglualaglumetalaargglyglnglyprogly
115120125
gluvalalaproglumetalaleu
130135
<210>41
<211>178
<212>prt
<213>susscrofa
<400>41
alapheseralaproalaalatyrleuthrhisglnglnlysvalleu
151015
argleutyrlysargalaleuarghisleuglusertrpcysvalhis
202530
argasplystyrargtyrphealacysleumetargalaargpheasp
354045
gluhislysasnglulysaspmetvallysalathrglnleuleuarg
505560
glnalagluglugluphetrptyrglyglnhisproglnprotyrile
65707580
pheprogluserproglyglythrsertyrgluargtyrglucystyr
859095
lysvalproglutrpcysleuaspasptrphisproserglulysala
100105110
mettyrproasptyrphealalysarggluglntrplysargleuarg
115120125
argglusertrpgluarggluvallysglnleuglnglugluthrpro
130135140
proglyglyproargthrglualaleuproproalaarglysglugly
145150155160
aspleuproproleutrptrphisilevalthrargproarggluarg
165170175
promet
<210>42
<211>175
<212>prt
<213>susscrofa
<400>42
proaspsertrpasplysaspvaltyrprogluproproargargthr
151015
proalaproalaproglnthrserleuproasnprovalthrtyrleu
202530
thrlysilepheaspleuleuvalaspargprovalthrleualaarg
354045
glupheilegluglnglnhisalalysasnargtyrtyrtyrtyrhis
505560
argglupheargargvalproaspilethrglucysgluglulysasp
65707580
ileleucysmetpheglualaglumetglntrpargargasptyrlys
859095
valaspglngluilevalasnileileglngluargleulysalacys
100105110
glnglnarggluglyglusertyrargglnasncysalalysgluleu
115120125
gluglnphethrglnvalserlysalapheglnaspargtyrserasp
130135140
leuglyalahistyrseralaarglyscysleualalysglnlysgln
145150155160
argmetleualagluarglysalaalalysglualaalaalaala
165170175
<210>43
<211>125
<212>prt
<213>susscrofa
<400>43
gluserglyserserargalavalilealaproseralaleuvalgly
151015
lysargproprogluprothrileargleuglngluaspproasppro
202530
gluaspgluasnleutyrglulysasnproaspserhisglytyrasp
354045
lysaspproilevalaspleutrpasnmetargvalvalphephephe
505560
glypheserilevalleuvalleuglyserthrphevalalatyrleu
65707580
proasptyrargmetglnglutrpalaargargglualagluargleu
859095
vallystyrargglualaasnglyleuproleumetgluserasncys
100105110
pheaspproasnlysileglnleuprogluaspgluasp
115120125
<210>44
<211>122
<212>prt
<213>susscrofa
<400>44
metthrmetmetserglyargproglyargvalproleuglnpheleu
151015
proasnglualaargserleuproproprolysleuthraspproarg
202530
leuvaltyrmetglypheleuglytyrcysserglyleuileaspasn
354045
alaileargargargprovalvalseralaglyleuhisargglnleu
505560
leutyrvalthrserphevalphepheglytyrtyrleuleulysarg
65707580
glnasptyrmettyralaleuargasphisaspmetphealatyrval
859095
lysserhisprogluasppheproglulysasplyslysthrtyrgly
100105110
gluileleuglugluphehisprovalarg
115120
<210>45
<211>471
<212>prt
<213>susscrofa
<400>45
metalaalaserleuleuleuargglnglyargalaargalaleulys
151015
thrvalleuleugluglyglyvalpheargglyleualaproalaval
202530
serleuseralagluserglyargasnglulysgluargproproasn
354045
prolyslysglnserproprolysasnvalvalalaalaglnglugly
505560
glythrproleualathrproalaalaprogluleuserglnglnleu
65707580
serserprothrserproproalaalaalaargproglyglythrleu
859095
alaproproasnproglyaspserthrleuphethraspargglyarg
100105110
proargpheproserarglysthrleuvalglupheproarglysval
115120125
leuserpropheserglnglnglyseraspseralaalathrglnala
130135140
serargglyargalaglyaspalathrserserserserserserser
145150155160
serseraspsergluseraspaspgluglugluglyaspglyserglu
165170175
alavalargprovallysserthrserproglyglyserprolysala
180185190
glualaproargserleualaglyglyalaproglnthrglyvalpro
195200205
alaglualaargproglyproglnglnprohisproaspleualaala
210215220
alagluargproargglnalalysglylysglyglyserglyalapro
225230235240
gluaspargargalaprolysalaalaalaproglyserargglygly
245250255
proglyalaalaargglnsergluthrproserglnlysileproarg
260265270
sergluglualaaspalagluserglnlysproprogluvallysthr
275280285
serleuproaspprothrlysserglyleuserthralaproarggly
290295300
glyproalaseralagluproalaalathrproalaglyalaargleu
305310315320
proalathrproleugluthrglygluargproleuglygluargval
325330335
progluproglyglyglnvalalaserproleupheserargalapro
340345350
leuglyglyglnalaalagluglyvalalaglualalysglygluleu
355360365
leugluglyargproleuvalglnglyprolysalavalproaspgly
370375380
glnaspgluglyalaglulyslysalaleuargprogluglugluala
385390395400
glyilealaglyaspalaalaproglythralaglyargaspalathr
405410415
glngluprothrproalaalaalaalaalaglupropheaspasnser
420425430
thrtyrargasnleuglnhishisglutyrserthrtyrthrpheleu
435440445
aspleuasnvalgluleuserlyspheargmetproglnproserser
450455460
glyargglnserproarghis
465470
<210>46
<211>196
<212>prt
<213>susscrofa
<400>46
serhisthraspileargvalproasppheserasptyrargargala
151015
gluvalleuaspserthrlysserserlysgluserseraspalaarg
202530
lysglyphesertyrleuilethralathrthrthrvalglyvalala
354045
tyralaalalysasnalavalserglnphevalsersermetserala
505560
seralaaspvalleualametserlysilegluilelysleuserasp
65707580
ileprogluglylysasnmetalaphelystrpargglylysproleu
859095
phevalarghisargthrlyslysgluileaspglnglualaalaval
100105110
gluvalserglnleuargaspproglnhisaspleugluargvallys
115120125
lysproglutrpvalileleuileglyvalcysthrhisleuglycys
130135140
valproilealaasnalaglyasppheglyglytyrtyrcysprocys
145150155160
hisglyserhistyraspalaserglyargilearglysglyproala
165170175
proleuasnleugluvalprothrtyrgluphethrseraspaspleu
180185190
valilevalgly
195
<210>47
<211>379
<212>prt
<213>susscrofa
<400>47
metthrasnilearglysserhisproleumetlysileileasnasn
151015
alapheileaspleuproalaproserasnilesersertrptrpasn
202530
pheglyserleuleuglyilecysleuileleuglnileleuthrgly
354045
leupheleualamethistyrthrseraspthrthrthralapheser
505560
servalthrhisilecysargaspvalasntyrglytrpvalilearg
65707580
tyrleuhisalaasnglyalasermetphepheilecysleupheile
859095
hisvalglyargglyleutyrtyrglysertyrmetpheleugluthr
100105110
trpasnileglyvalvalleuleuphethrvalmetalathralaphe
115120125
metglytyrvalleuprotrpglyglnmetserphetrpglyalathr
130135140
valilethrasnleuleuseralaileprotyrileglythraspleu
145150155160
valglutrpiletrpglyglypheservalasplysalathrleuthr
165170175
argphephealaphehispheileleupropheileilethralaleu
180185190
alaalavalhisleuleupheleuhisgluthrglyserasnasnpro
195200205
thrglyileserseraspmetasplysileprophehisprotyrtyr
210215220
thrilelysaspileleuglyalaleuphemetmetleuileleuleu
225230235240
ileleuvalleupheserproaspleuleuglyaspproaspasntyr
245250255
thrproalaasnproleuasnthrproprohisilelysproglutrp
260265270
tyrpheleuphealatyralaileleuargserileproasnlysleu
275280285
glyglyvalleualaleuvalalaserileleuileleuileleumet
290295300
prometleuhisthrserlysglnargsermetmetpheargproleu
305310315320
serglncysleuphetrpmetleuvalalaaspleuilethrleuthr
325330335
trpileglyglyglnprovalgluhispropheileileileglygln
340345350
leualaserileleutyrpheleuileileleuvalleumetproile
355360365
thrserileilegluasnasnleuleulystrp
370375
<210>48
<211>326
<212>prt
<213>susscrofa
<400>48
metseralaalaalaalaalaserleuargglyalavalleuglypro
151015
argglyalaglyleuproglyalaargalaargglyleuleucysgly
202530
proargproglyglnleuproleuargthrproglnalaleuserleu
354045
serserlysserglyleuserargglyarglysvalileleuserala
505560
leuglymetleualaalaglyglyalaglyleualavalalaleuhis
65707580
seralavalseralathraspleugluleuhisalaprosertyrpro
859095
trpserhisargglyleuleuserserleuasphisthrserilearg
100105110
argglypheglnvaltyrlysglnvalcyssersercyshissermet
115120125
asptyrvalalatyrarghisleuvalglyvalcystyrthrgluglu
130135140
glualalysalaleualaglugluvalgluvalglnaspglyproasn
145150155160
gluaspglyglumetphemetargproglylysleuserasptyrphe
165170175
prolysprotyrproasnproglualaalaargalaalaasnasngly
180185190
alaleuproproaspleusertyrilevalargalaarghisglygly
195200205
gluasptyrvalpheserleuleuthrglytyrcysgluproprothr
210215220
glyvalserleuarggluglyleutyrpheasnprotyrpheprogly
225230235240
glnalailealametalaproproiletyrasngluvalleugluphe
245250255
aspaspglythrproalathrmetserglnvalalalysaspvalcys
260265270
thrpheleuargtrpalasergluprogluhisasphisarglysarg
275280285
metglyleulysmetleumetmetmetglyleuleuleuproleuval
290295300
tyralametlysarghislystrpservalleulysserarglysleu
305310315320
alatyrargproprolys
325
<210>49
<211>480
<212>prt
<213>susscrofa
<400>49
metalaalaseralavalcysargvalalaglythrglyserargval
151015
leuleuargthrcysargserproalaleuleuargserproalaleu
202530
argglythralathrtyralaglnalaleuglnservalprogluthr
354045
glnvalserglnleuaspasnglyleuargvalalasergluglnser
505560
serglnprothrcysthrvalglyvaltrpileaspalaglyserarg
65707580
tyrgluasnglulysasnasnglyalaglytyrphevalgluhisleu
859095
alaphelysglythrlysasnargproglyseralaleuglulysglu
100105110
valglusermetglyalahisleuasnalatyrserthrarggluhis
115120125
thralatyrtyrilelysalaleuserlysaspleuprolysalaval
130135140
gluleuleualaaspilevalglnasncysserleugluaspsergln
145150155160
ileglulysgluargaspvalileleuglngluleuglngluasnasp
165170175
sersermetargaspvalvalpheasptyrleuhisalathralaphe
180185190
glnglythrproleualaglnservalgluglyprosergluasnval
195200205
arglysleuserargalaaspleuthrglutyrvalserglnhistyr
210215220
lysalaproargmetvalleualaalaalaglyglyvalgluhisarg
225230235240
glnleuleuaspleualaglnlyshispheserserleuserglythr
245250255
tyrvalgluaspalavalproalaphethrprocysargphethrgly
260265270
sergluilearghisargaspaspalaleuproleualahisvalala
275280285
ilealavalgluglyproglytrpalaasnproaspasnvalproleu
290295300
glnvalalaasnalaileileglyhistyraspserthrtyrglygly
305310315320
glythrhismetserserthrleualaservalalaalathrarglys
325330335
leucysglnserpheglnthrpheasnilecystyralagluthrgly
340345350
leuleuglyalahisphevalcysaspasnmetserileaspaspmet
355360365
metphepheleuglnglyglntrpmetargleucysthrseralathr
370375380
glusergluvalvalargglylysasnileleuargasnalaleuval
385390395400
serhisleuaspglythrthrprovalcysgluaspileglyargser
405410415
leuleuthrtyrglyargargileproleualaglutrpgluserarg
420425430
ilealagluvalaspalaservalvalarggluvalcysserlystyr
435440445
phetyraspglncysproalavalalaglyleuglyproileglugln
450455460
leuproasptyrasnargileargserglymetphetrpleuargphe
465470475480
<210>50
<211>453
<212>prt
<213>susscrofa
<400>50
metlysleuilethrargalaglyserpheserargphetyrserleu
151015
lysvalalaprolysalaleualaseralaalaproalaglyvalpro
202530
leuglnproglnaspleugluphethrargleuproasnglyleuval
354045
ilealaserleugluasntyralaproalaserargileglyleuphe
505560
ilelysalaglyserargtyrgluaspserasnasnleuglythrser
65707580
hisleuleuargleualaserserleuthrthrlysglyalaserser
859095
phelysilethrargglyileglualavalglyglylysleuserval
100105110
thrserthrargglusermetalatyrthrvalglucysleuargasp
115120125
aspilegluileleumetglupheleuleuasnvalthralaalapro
130135140
glupheargargtrpgluvalalaalaleuglnserglnleuargile
145150155160
asplysalavalalapheglnasnproglnalaglnvalleugluasn
165170175
leuhisalaalaalatyrargasnalaleualaasnserleutyrcys
180185190
proasptyrargileglylysvalthrproaspglnleuhistyrtyr
195200205
valglnasnhisphethrseralaargmetalaleuileglyleugly
210215220
valserhisprovalleulysglnvalalagluargpheleuasnmet
225230235240
argglyglyleuglyleuserglyalalysalalystyrargglygly
245250255
gluileargaspglnasnglyaspserleuvalhisalaalaleuval
260265270
alagluseralaalathrglyseralaglualaasnalapheserval
275280285
leuglnhisvalleuglyalaglyprohisvallysargglyserasn
290295300
alathrserserleutyrglnalavalalalysglyvalhisglnpro
305310315320
pheaspvalseralapheasnalasertyrseraspserglyleuphe
325330335
glyiletyrthrileserglnalaalaseralaglyaspvalilelys
340345350
seralatyraspglnvallysalailealaglnglyasnleuserasn
355360365
thraspvalglnalaalalysasnlysleulysalaglytyrleumet
370375380
servalglusersergluglypheleuaspgluvalglyserglnala
385390395400
leuvalalaglysertyrmetglnproserthrvalleuglnglnile
405410415
aspservalalaaspalaaspvalileasnalaalalyslyspheval
420425430
serglyarglyssermetalaalaserglyasnleuglyhisthrpro
435440445
phevalaspgluleu
450
<210>51
<211>91
<212>prt
<213>susscrofa
<400>51
metglyleugluaspgluglnargmetleuthrglyserglyasppro
151015
lysglugluglugluglugluglugluleuvalaspproleuthrthr
202530
valarggluglncysgluglnileglulyscysilelysalaargglu
354045
argleugluleucysaspglnargvalserserargserglnthrglu
505560
gluaspcysthrglugluleupheasppheleuhisalaargasphis
65707580
cysvalalahislysleupheasnserleulys
8590
<210>52
<211>111
<212>prt
<213>susscrofa
<400>52
metalaserargproalavalalaalaserserlystrpleuglugly
151015
ilearglystrptyrtyrasnalaalaglypheasnlysleuglyleu
202530
metargaspaspthriletyrgluaspaspaspvallysglualaile
354045
argargleuprogluasnleutyrasnaspargvalpheargilelys
505560
argalaleuaspleuthrmetargglnglnileleuprolysglugln
65707580
trpthrlystyrglugluasplysphetyrleugluprotyrleulys
859095
gluvalilearggluarglysgluarggluglutrpalalyslys
100105110
<210>53
<211>82
<212>prt
<213>susscrofa
<400>53
metglyargglupheglyhisleuthrargmetarghisvalilethr
151015
tyrserleuserprophegluglnargalapheprohistyrphethr
202530
lysglyileproasnvalleuargargthrargalacysileleuarg
354045
valalaproprophevalvalphetyrleuvaltyrthrtrpglythr
505560
glnglupheglulysserlysarglysasnproalaalatyrgluasn
65707580
asplys
<210>54
<211>64
<212>prt
<213>susscrofa
<400>54
metalaalaprothrleuthralaargleutyrserleuleuphearg
151015
argthrserthrphealaleuthrilealavalglyalaleuphephe
202530
gluargalapheaspglnglyalaaspalailetyrgluhisileasn
354045
glnglylysleutrplyshisilelyshislystyrgluasnlysglu
505560
<210>55
<211>56
<212>prt
<213>susscrofa
<400>55
metleuserargpheleuglyproargtyrarggluleualaargasn
151015
trpileprothralasermettrpglyalavalglyalavalglyleu
202530
valtrpalathrasptrpargleuileleuasptrpvalprotyrile
354045
asnglylysphelyslysgluasp
5055
<210>56
<211>78
<212>prt
<213>susscrofa
<400>56
metleuservalalaserargserglyprophealaprovalleuser
151015
alathrserargglyvalalaglyalaleuargproleuvalglnala
202530
alaleuproalathrsergluserprovalleuaspalalysargser
354045
pheleucysarggluserleuserglyglnalaalaglyargproleu
505560
valalaservalglyleuasnvalproalaservalargtyr
657075
<210>57
<211>261
<212>prt
<213>susscrofa
<400>57
metthrhisglnthrhisalatyrhismetvalasnproserprotrp
151015
proleuthrglyalaleuseralaleuleumetthrserglyleuthr
202530
mettrpphehispheasnsermetleuleuleuserleuglyleuleu
354045
thrasnthrleuthrmettyrglntrptrpargaspileileargglu
505560
serthrpheglnglyhishisthrservalvalglnlysglyleuarg
65707580
tyrglymetileleupheileilesergluvalleuphephethrgly
859095
phephetrpalaphetyrhisserserleualaprothrprogluleu
100105110
glyglycystrpproprothrglyilehisproleuasnproleuglu
115120125
valproleuleuasnthrserileleuleualaserglyvalserile
130135140
thrtrpalahishisserleumetgluglyasparglyshismetile
145150155160
glnalaleuserilethrilealaleuglyvaltyrphethrleuleu
165170175
glnalaserglutyrtyrglualaprophethrileseraspglyval
180185190
tyrglyserthrphephevalalathrglyphehisglyleuhisval
195200205
ileileglyserthrpheleualavalcysleuleuargglnleulys
210215220
phehisphethrserasnhishispheglypheglualaalaalatrp
225230235240
tyrtrphisphevalaspvalvaltrpleupheleutyrvalserile
245250255
tyrtrptrpglyser
260
<210>58
<211>514
<212>prt
<213>susscrofa
<400>58
metphevalasnargtrpleutyrserthrasnhislysaspilegly
151015
thrleutyrleuleupheglyalatrpalaglymetvalglythrala
202530
leuserleuleuileargalagluleuglyglnproglythrleuleu
354045
glyaspaspglniletyrasnvalilevalthralahisalapheval
505560
metilephephemetvalmetproilemetileglyglypheglyasn
65707580
trpleuvalproleumetileglyalaproaspmetalapheproarg
859095
metasnasnmetserphetrpleuleuproproserpheleuleuleu
100105110
leualasersermetvalglualaglyalaglythrglytrpthrval
115120125
tyrproproleualaglyasnleualahisalaglyalaservalasp
130135140
leuthrilepheserleuhisleualaglyvalserserileleugly
145150155160
alaileasnpheilethrthrileileasnmetlysproproalamet
165170175
serglntyrglnthrproleuphevaltrpservalleuilethrala
180185190
valleuleuleuleuserleuprovalleualaalaglyilethrmet
195200205
leuleuthraspargasnleuasnthrthrphepheaspproalagly
210215220
glyglyaspproileleutyrglnhisleuphetrpphepheglyhis
225230235240
progluvaltyrileleuileleuproglypheglymetileserhis
245250255
ilevalthrtyrtyrserglylyslysglupropheglytyrmetgly
260265270
metvaltrpalametmetserileglypheleuglypheilevaltrp
275280285
alahishismetphethrvalglymetaspvalaspthrargalatyr
290295300
phethrseralathrmetileilealaileprothrglyvallysval
305310315320
phesertrpleualathrleuhisglyglyasnilelystrpserpro
325330335
alametleutrpalaleuglypheilepheleuphethrvalglygly
340345350
leuthrglyilevalleualaasnserserleuaspilevalleuhis
355360365
aspthrtyrtyrvalvalalahisphehistyrvalleusermetgly
370375380
alavalphealailemetglyglyphevalhistrppheproleuphe
385390395400
serglytyrthrleuasnglnalatrpalalysilehisphevalile
405410415
metphevalglyvalasnmetthrphepheproglnhispheleugly
420425430
leuserglymetproargargtyrserasptyrproaspalatyrthr
435440445
alatrpasnthrilesersermetglyserpheileserleuthrala
450455460
valmetleumetilepheileiletrpglualaphealaserlysarg
465470475480
gluvalseralavalgluleuthrserthrasnleuglutrpleuhis
485490495
glycysproproprotyrhisthrpheglugluprothrtyrileasn
500505510
leulys
<210>59
<211>229
<212>prt
<213>susscrofa
<400>59
metalatyrpropheglnleuglypheglnaspalathrserproile
151015
metglugluleuleuhisphehisasphisthrleumetilevalphe
202530
leuileserserleuvalleutyrileileserleumetleuthrthr
354045
lysleuthrhisthrserthrmetaspalaglngluvalgluthrile
505560
trpthrileleuproalaileileleuileleuilealaleuproser
65707580
leuargileleutyrmetmetaspgluileasnasnproalaleuthr
859095
vallysthrmetglyhisglntrptyrtrpsertyrglutyrthrasp
100105110
tyrgluaspleuthrpheaspsertyrmetileprothrseraspleu
115120125
lysproglyglumetargleuleugluvalaspasnargvalvalleu
130135140
prometglumetthrileargmetleuvalsersergluaspvalleu
145150155160
hissertrpalavalproserleuglyleulysthraspalailepro
165170175
glyargleuasnglnthrthrleumetserthrargproglyleutyr
180185190
tyrglyglncyssergluilecysglyserasnhisserphemetpro
195200205
ilevalleugluleuvalproleulystyrpheglulystrpserthr
210215220
sermetleuthrgly
225
<210>60
<211>169
<212>prt
<213>susscrofa
<400>60
metleualathrargvalpheasnleuileglyargargalaileser
151015
thrservalcysvalargalahisglyservalvallyssergluasp
202530
tyralaleuprovaltyrvalaspargargasptyrproleuproasp
354045
valalahisvallysasnleuseralaserglnlysalaleulysglu
505560
lysglulysalasertrpserserleusermetaspglulysvalglu
65707580
leutyrargleulyspheasngluserphealaglumetasnargser
859095
thrasnglutrplysthrilevalglythralaleuphepheilegly
100105110
phethralaleuileleuiletrpglulyshistyrvaltyrglypro
115120125
ileprohisthrpheglugluglutrpvalalalysglnthrlysarg
130135140
metleuaspmetlysvalalaproileglnglypheseralalystrp
145150155160
asptyrasplysasnglutrplyslys
165
<210>61
<211>152
<212>prt
<213>susscrofa
<400>61
metleuglyalaalavalargargcysservalalaalaalaalaval
151015
alaargalaserproargglyleuleuhisprothrprothrprogly
202530
proalaalaalaileglnserileargcystyrserhisglyserhis
354045
gluthraspgluglupheaspalaargtrpvalthrtyrpheasnlys
505560
proaspileaspalatrpgluleuarglysglymetasnthrleuval
65707580
glytyraspleuvalprogluprolysileileaspalaalaleuarg
859095
alacysargargleuasnaspphealaseralavalargileleuglu
100105110
valvallysasplysalaglyprohislysgluiletyrprotyrval
115120125
ileglngluleuargprothrleuasngluleuglyileserthrpro
130135140
glugluleuglyleuasplysval
145150
<210>62
<211>129
<212>prt
<213>susscrofa
<400>62
metalaserargleuleuargglyalaglyalaleualaalaglnthr
151015
leuargalaargglyproasnglyvalalavalvalargsermetala
202530
serglyglyglyvalprothraspglugluglnalathrglyleuglu
354045
arggluvalmetmetalaalaarglysglyleuaspprotyrasnile
505560
leualaprolysalaalaserglythrlysgluaspproasnleuval
65707580
proserilethrasnlysargilevalglycysilecysglugluasp
859095
asnserthrvaliletrpphetrpvalhislysglygluthrglnarg
100105110
cysprosercysglythrhistyrlysleuvalserhisglnleuala
115120125
his
<210>63
<211>109
<212>prt
<213>susscrofa
<400>63
metalaalaalaalaglyserargvalserglyleuleuargargser
151015
glnleuglnleuargargprometserserseralahisglygluglu
202530
glyseralaargmettrplysthrleuthrtyrphevalalaleupro
354045
glyvalglyvalsermetleuasnvalpheleulysserhishisala
505560
gluglugluargprogluphevalprotyrprohisleuargilearg
65707580
serlyspropheprotrpglyaspglyasnhisthrleuphehisasn
859095
prohisvalasnproleuprothrglytyrgluaspglu
100105
<210>64
<211>86
<212>prt
<213>susscrofa
<400>64
metalagluaspileglnthrlysilelysasntyrglnthralapro
151015
pheaspserargpheproasnglnasnglnthrargasncystrpgln
202530
asntyrleuaspphehisargcysglulysalametthralalysgly
354045
glyaspvalservalcysglutrptyrargargvaltyrlysserphe
505560
cysproilesertrpvalseralatrpaspaspargargalaglugly
65707580
thrpheproglylysile
85
<210>65
<211>76
<212>prt
<213>susscrofa
<400>65
metalathrserserleuthrlysproglnmetargtrpprosergly
151015
proargargleuargphehisilevalglyalapheilevalserleu
202530
glyvalalathrphetyrlysphealavalalagluproarglyslys
354045
alatyralaaspphetyrargasntyraspsermetlysasppheglu
505560
glumetarglysalaglyilepheglnseralalys
657075
<210>66
<211>83
<212>prt
<213>susscrofa
<400>66
metleuargasnleuleualaleuargglnilealaglnargthrile
151015
serseralaserargargglnleugluasnlysvalalaglulysgln
202530
lysleupheglngluaspasnglyileprovalhisleulysglygly
354045
ileglyaspalapheleutyrargalathrmetileleuthrvalgly
505560
glythralatyrthriletyrglnleualavalalaserleuprolys
65707580
lysglnasp
<210>67
<211>80
<212>prt
<213>susscrofa
<400>67
metpheproleualalysasnalaleuserargleuargvalargasn
151015
ileglnglnthrmetalaargglnasnhisglnlysargalaproasp
202530
phehisasplystyrglyasnalaileleualaserglyalathrphe
354045
cysvalalavaltrpalatyrthralathrglnileglyileglutrp
505560
asnleuserprovalglyargvalthrprolysglutrparggluglu
65707580
<210>68
<211>63
<212>prt
<213>susscrofa
<400>68
metleuglyglnserileargargphethrthrservalvalargarg
151015
serhistyrglugluglyproglylysasnleupropheservalglu
202530
asnlystrpargleuleualametmetthrleutyrpheglysergly
354045
phealaalaprophepheilevalarghisglnleuleulyslys
505560
<210>69
<211>70
<212>prt
<213>susscrofa
<400>69
metleuargleualaprothrvalargleuleuglnalaproleugly
151015
glytrpvalvalprolysalahisiletyralalysproalaargthr
202530
prothrserprothrgluglnalaileglyleuservalthrpheleu
354045
serpheleuileproalaglytrpvalleuserhisleuasphistyr
505560
lysargserseralaala
6570