《第7章镰刀贫血病精品课程课件文档资料.ppt》由会员分享，可在线阅读，更多相关《第7章镰刀贫血病精品课程课件文档资料.ppt（42页珍藏版）》请在三一办公上搜索。
1、Chapter 6 蛋白质的结构与功能的关系,蛋白质的分子的生物学功能是与其化学组成和极其复杂的结构密切相关的。同时，蛋白质功能总是跟蛋白质与其他分子相互作用相联系。相互作用中蛋白质构象有时发生微小变化，有时发生剧烈变化。因此蛋白质的构象不是刚性的、静止的，而是柔性的、动态的。本章主要介绍蛋白质的一级结构与功能的关系一个分子病发现的故事。,一、蛋白质的一级结构与功能的关系遗传病镰刀贫血病的发现(Genetic disease-Sickle-cell anemia)事情发生在1904年，美国东部NewYork,NewYork city,当时有一位黑人大学生Smark(18岁），找一位医生（Ing
2、ram)看病，自觉头昏、发烧、呼吸困难。医生对Smark 进行了检查，发现病人眼睛有黄疸，心跳有杂音，没有发现寄生虫，只发现Red blood cell 350万/mm3，Blood platelet 25万/mm3,White blood cell5000/mm3.根据正常人的数值（RBC500万/mm3，WBC6000/mm3，BP15.6万/mm3）诊断病人为贫血症。,后来Ingram医生又对病人取血，镜检，发现病人的红血球的是半月型的（形状象镰刀）。,以后这位医生对这位黑人大学生进行了精心治疗，也就是加强营养和一般治疗。结果，病人有所好转，但是，医生发现病人的红血球还是镰刀状的。于是医
3、生建议该生休学。Ingram对这位黑人大学生进行了6年的跟踪观察。当时医生判断，这个病很可能是一个genetic disease。后来在美国黑人主要集中的New York,Chicago,Los Angeles,Philadelphia,Detroit and Washington发现了很多这样的病例，发病率占4/1000，都是由于得此病以至30岁以前，就由于此病引起血栓、肾功能减退、心肌梗塞，特别是毛细血管阻塞引起死亡。病人的红血球很容易溶血和破裂，且红血球寿命比正常的要短，结果造成严重贫血和庰发许多种严重疾病。后来诊断此病是先天性的或遗传性的，而且在一定条件下甚至是一种绝症。,久而久之，医
4、生又发现血红蛋白有缺陷，就是去氧血红蛋白溶解度降低，比正常低1/25。由于溶解度的降低，造成了血红蛋白运氧机能大大降低。后来，对不正常血红蛋白进行了研究。血红蛋白是由4条肽链以亚基连接起来（2条-链，2条-链），大家知道去氧血红蛋白对于新陈代谢起着重要作用。它既可以载氧，又可以运走CO2。这位医生把病人的血红蛋白提取出来，用正常人的血红蛋白和病人的血红蛋白进行电泳比较。结果发现有病的与正常的斜率曲线是平行的，也就是说，在电泳时镰刀状的比正常的要快。,+,pH,病例,电泳的泳动速度：V=V速度 E电场强度 Z带电颗粒所带电荷f带电颗粒的大小,E Z,f,快的原因有几种可能性：可能是Z，也可能是f
5、。,后来经过研究推断，泳动速度的加快是由于Z（带电荷）的不同。HbA HbS 差异PI O2-Hb 6.87 7.09 0.22 去氧Hb 6.68 6.91 0.23异常的去氧Hb比正常的Hb的PI高0.23个pH单位。,在上述基础上，进一步的研究表明，病例的多了几个电荷。采用滴定法，也就是根据耗碱量知道氨基的数目，耗酸量了解羧基的数目的原理（NH3+可看作可滴定酸，COO-看作可滴定碱）。结果，改变一个PH就有13个电荷数目变化。设异常带电荷为X1:13=0.23:X X=130.23=2.99 3 这样就决定了异常的比正常的Hb多了3个电荷。,当时认为电荷的增多可能是血红蛋白中铁卜啉所引
6、起的，把铁卜啉环提取出来，结晶，用X-射线衍射，结果没有什么差异。,当时认为电荷的增多可能是血红蛋白中铁卜啉所引起的，把铁卜啉环提取出来，结晶，用X-射线衍射，结果没有什么差异。后来把目标转向了肽链分析。在肽链上有三种可能电荷增多：,正常 异常 中+-+-中,当然，要知道那一种可能，就必须进行研究试验。把血红蛋白的一级结构进行分析。首先用胰蛋白酶水解HB，得到28个肽段，采用纸上层析分离与电泳相结合的方法对这些肽与正常的进行比较。得到了Finger-prints.根据该图的比较，27个肽一样，只有一个肽段是不同的。把这个异常的肽段剪下来，进行测序。结果是链上的一个8肽。Hba:H2N-Val-
7、His-Leu-Thr-Pro-Glu-Glu-Lys-COOHHbs:H2N-Val-His-Leu-Thr-Pro-Val-Glu-Lys-COOH,在正常血红蛋白分子的链中，从N-端开始的 第6个氨基酸残基是GLU，而镰刀壮贫血患者的血红蛋白分子的链中的第6个氨基酸残基则被VAL 所代替，这样就明了了产生电荷增多的原因，是第三种可能性，就是说，原来一个带负电荷的8肽变成了一个中性的8肽,H3N+,COO-,COO-,H3N+,COO-,由此可见，由于HB有4条肽链，而这8肽是在-链上，一个-链多了一个正电荷，而两条-链就多了2个正电荷。多了2个电荷的血红蛋白发生了结构改变，异常的HBS与
8、HBS聚集起来，有时HBS与HBA凝集，形成所谓平行排列的非球形（线形）结构镰刀状结构。,Mutation and molecular interactions changes,这个有574个氨基酸残基的HB中，只要两条-链中的2个GLU残基被VAL残基所代替就能引起如此严重的病理现象，可见蛋白质的结构与功能有着如此密切的联系。清楚了病因以后，INGRAM 医生再治疗就不难了，根据HB的聚集，用脲素可以使这种聚集消失，医生给患者吃大量的尿素，结果是有效的。患者感觉好多了，头昏和呼吸困难都有所改善。但是，由于尿素在体内不能保存太久，就被分解了，这位寻求真理，攀登科学高峰的医生，继续进行了研究。根
9、据：,H2N-CO-NH2 NH4+（N=C=O）-37OC，PH7.4时，有5 10-3M的氰酸盐，证明在体内不是脲素起作用，而是脲素平衡后发生的氰酸盐起作用，后来给患者服氰酸盐，比吃脲素好得多。RNH2+H-N=C=O R-NH-CO-NH2 O RNH2+CO2 R-NH-CO-这样打断了HBS的聚合，矫正了它的构象，使分子重新获得输氧能力。由于这种疾病是先天性的“分子病”，还不能完全治愈。到目前为止还在研究之中。在人类中已发现300多种不同的异常血红蛋白，其中中国人30种。这种疾病只有有赖于基因工程的方法才能彻底根治。,一氧化碳:神秘的杀手,Carbon Monoxide:A Stea
10、lthy Killer Lake Powell莱克.鲍威尔，Arizona亚利桑那州,August 2000.A family was vacationing度假 in a rented租用 houseboat游艇.They turned on the electrical generator发电机 to power an air conditioner and a television.About 15 minutes later,two brothers,aged 8 and 11,jumped off the swim deck at the stern船尾.Situated immed
11、iately below the deck was the exhaust port for the generator.Within two minutes,both boys were overcome征服 by the carbon monoxide in the exhaust 排气口,which had become concentrated in the space under the deck.Both drowned淹死了.These deaths,along with a series of deaths in the 1990s linked to houseboats o
12、f similar design,eventually led to the recall召回 and redesign of the generator exhaust assembly.Carbon monoxide(CO),a colorless,odorless gas,is responsible for more than half of yearly deaths due to poisoning中毒 worldwide.CO has an approximately 250-fold greater affinity for hemoglobin血红蛋白 than does o
13、xygen.Consequently,relatively low levels of CO can have substantial and tragic悲剧 effects.When CO combines with hemoglobin,the complex is referred to as carboxyhemoglobin,or COHb.,Some CO is produced by natural processes,but locally high levels generally result only from human activities.Engine and f
14、urnace炉子 exhausts are important sources,as CO is a byproduct of the incomplete combustion不完全燃烧 of fossil fuels石油.In the United States alone,nearly 4,000 people succumb to CO poisoning each year,both accidentally意外地 and intentionally故意地.Many of the accidental deaths involve undetected CO buildup积累 in
15、 enclosed spaces,such as when a household furnace malfunctions故障 or leaks泄漏,venting排 CO into a home.However,CO poisoning can also occur in open spaces露天场所,as unsuspecting people at work or play休闲 inhale吸入 the exhaust from generators,outboard motors舷外马达,tractor拖拉机 engines,recreational vehicles休闲交通工具,
16、or lawn mowers割草机.Carbon monoxide levels in the atmosphere are rarely dangerous,ranging from less than 0.05 parts per million(ppm)in remote偏僻的 and uninhabited无人居住 areas to 3 to 4 ppm in some cities of the northern hemisphere北半球.In the United States,the government-mandated(Occupational职业 Safety and H
17、ealth Administration,OSHA)limit for CO at worksites工地 is 50 ppm for people working an eight-hour shift轮班.The tight binding of CO to hemoglobin means that COHb can accumulate over,time as people are exposed to a constant low-level source of CO.In an average,healthy individual,1%or less of the total h
18、emoglobin is complexed as COHb.Since CO is a product of tobacco smoke,many smokers have COHb levels in the range of 3%to 8%of total hemoglobin,and the levels can rise to 15%for chain-smokers.COHb levels equilibrate at 50%in people who breathe air containing 570 ppm of CO for several hours.Reliable m
19、ethods have been developed that relate CO content in the atmosphere to COHb levels in the blood(Fig.1).In tests of houseboats with a generator exhaust like the one responsible for the Lake Powell deaths,CO levels reached 6,000 to 30,000 ppm under the swim deck,and atmospheric O2 levels under the dec
20、k declined下降 from 21%to 12%.Even above the swim deck,CO levels of up to 7,200 ppm were detected,high enough to cause death within a few minutes.How is a human affected by COHb?At levels of less than 10%of total hemoglobin,symptoms症状 are rarely observed.At 15%,the individual experiences体验 mild轻微 head
21、aches.At 20%to 30%,the headache is severe剧烈的 and,图1.血液中COHb与环境中的CO浓度的关系。图中显示：在4种不同的条件下，短时间中毒的影响和休息状态和轻度运动状态下的比较。,is generally accompanied by nausea呕心,dizziness头昏眼花,confusion混乱,disorientation方向知觉的丧失,and some visual disturbances视觉混乱;these symptoms are generally reversed恢复 rapidly if the individual is
22、treated with oxygen.At COHb levels of 30%to 50%,the neurological symptoms神经学上的症状 become more severe严重,and at levels near 50%,the individual loses consciousness失去知觉 and can sink into coma昏迷.Respiratory failure 呼吸衰竭may follow.With prolonged exposure,some damage becomes permanent永久的.Death normally occu
23、rs when COHb levels rise above 60%.Autopsy尸体解剖 on the boys who died at Lake Powell revealed显示 COHb levels of 59%and 52%.Binding of CO to hemoglobin is affected by many factors,including exercise(Fig.1)and changes in air pressure related to altitude海拔.Because of their higher base levels of COHb,smoke
24、rs exposed to a source of CO often develop symptoms faster than nonsmokers.Individuals with heart and lung conditions or blood diseases that reduce the availability of oxygen to tissues may also experience symptoms at lower levels of CO exposure.Fetuses胎儿 are at particular risk for CO poisoning,beca
25、use fetal hemoglobin胎儿血红蛋白 has a somewhat稍微 higher affinity for CO than adult hemoglobin.Cases案例 of CO exposure have been recorded in which the fetus died but the mother recovered.,It may seem似乎 surprising that the loss of half of ones hemoglobin to COHb can prove fatal致命的we know that people with an
26、y of several anemic贫血 conditions manage to function reasonably well with half the usual complement 补充of active hemoglobin.However,the binding of CO to hemoglobin does more than remove protein from the pool available to bind oxygen.It also affects影响 the affinity of the remaining剩余 hemoglobin subunits
27、 for oxygen.As CO binds to one or two subunits of a hemoglobin tetramer四聚体,the affinity for O2 is increased substantially充分地 in the remaining subunits(Fig.2).Thus,a hemoglobin tetramer with two bound CO molecules can efficiently bind O2 in the lungsbut it releases very little of it in the tissues.Ox
28、ygen deprivation剥夺 in the tissues rapidly becomes severe严重.To add to the problem,the effects of CO are not limited to interference干涉 with hemoglobin function.CO binds to other heme proteins亚铁血红素蛋白 and a variety of metalloproteins金属蛋白.The effects of these interactions互作 are not yet well understood,bu
29、t they may be responsible for some of the longer-term effects of acute剧烈的 but nonfatal非致命 CO poisoning.,图2.几种氧合曲线。正常的血红蛋白；来自于贫血个体的只有50%血红蛋白功能，和来自于与CO结合了50%的血红蛋白。图示在人的肺和组织中的氧分压pO2,When CO poisoning is suspected怀疑,rapid evacuation撤离 of the person away from the CO source is essential,but this does not
30、always result in rapid recovery.When an individual is moved from the CO-polluted site to a normal,outdoor室外 atmosphere,O2 begins to replace the CO in hemoglobin.The COHb levels drop rather slowly,however;the half-time半衰期 is 2 to 6.5 hours,depending on individual and environmental factors.If 100%oxyg
31、en is administered with a mask面具,the rate of exchange can be increased about fourfold四倍;the halftime for O2-CO exchange can be reduced to tens of minutes if 100%oxygen at a pressure of 3 atm(303 kPa)is supplied.Thus,rapid treatment by a properly equipped medical team is critical急需的.Carbon monoxide d
32、etectors探测器 in all homes are highly recommended强烈推荐.This is a simple and inexpensive measure to avoid possible tragedy灾难.After completing the research for this box,we immediately purchased several new CO detectors for our homes.,二、蛋白质空间结构与功能的关系：蛋白质折叠病(proteinfolding)人们对由于基因突变造成蛋白质分子中仅仅一个氨基酸残基的变化就引起疾
33、病的情况已有所了解，即所谓“分子病”，如地中海镰刀状红血球贫血症就是因为血红蛋白分子中第六位的谷氨酸突变成了颉氨酸。现在则发现蛋白质分子的氨基酸序列没有改变，只是其结构或者说构象有所改变也能引起疾病，那就是所谓“构象病”，或称“折叠病”。您知道“蛋白质折叠病”吗？疯牛病、老年性痴呆症、囊性纤维病变、家族性高胆固醇症、家族性淀粉样蛋白症、某些肿瘤、白内障等等都是“折叠病”。就是相关蛋白质的三维空间结构异常。这种三维空间结构异常是由于致病蛋白质分子通过分子间作用感染正常蛋白质而造成的。请注意，致病蛋白质分子与正常蛋白质分子的构成完全相同，只是空间结构不同。由于蛋白质折叠异常而造成分子聚集甚至沉淀或
34、不能正常转运到位所引起的。,您知道蛋白质折叠有多复杂吗？美国“科学美国人”曾经载文称，用当今最快的计算机模拟计算蛋白质折叠，要花一百年！而当今最快的计算机已经达到每秒几万亿甚至十几万亿次浮点运算的高速了。在正常机体中，Prion(原蛋白)是正常神经活动所需要的蛋白质，造成疯牛病的Prion病蛋白可以感染正常蛋白而在蛋白质之间传染,从而认为蛋白结构的变化来自于序列的变化，而序列的变化来自于基因的变化，生命信息从核酸传递到蛋白。而致病Prion的信息已被诺贝尔奖获得者普鲁辛纳证明不是来自基因的变化，致病蛋白Prion导致正常蛋白Prion转变为致病的折叠状态是通过蛋白分子间的作用而感染！这种相互作
35、用的本质和机制是什么？仅仅改变了折叠状态的分子又如何导致严重的疾病？这些问题都不能用传统的概念给予满意的解释，因此在科学界引起激烈的争论，有关研究的强度和竞争性也随之大大增强。由于分子伴侣在蛋白质折叠中至关重要的作用，分子伴侣本身的突变显然会引起蛋白质折叠异常而引起折叠病。随着蛋白质折叠研究的深入，人们会发现更多疾病的真正病因和更针对性的治疗方法，设计更有效的药物。现在发现有,些小分子可以穿越细胞作为配体与突变蛋白结合，从而使原已失去作战能力的突变蛋白逃逸“蛋白质质量控制系统”而“带伤作战”。这种小分子被称为“药物分子伴侣”，有希望成为治疗“折叠病”的新药。新生肽的折叠问题或蛋白质折叠问题不仅
36、具有重大的科学意义，除了上面提到的在医学上的应用价值外，在生物工程上具有极大的应用价值。基因工程和蛋白工程已经逐渐发展成为产值以数十亿美元计的大产业，进入21世纪后，还将会有更大的发展。但是当前经常遇到的困难，是在简单的微生物细胞内引入异体DNA后所合成的多肽链往往不能正确折叠成为有生物活性的蛋白质而形成不溶解的包含体或被降解。这一“瓶颈”问题的彻底解决有待于对新生肽链折叠更多的认识。蛋白质的折叠是否有“第二遗传密码”？,Death by Misfolding:The Prion Diseases(蛋白质折叠之死:疯牛病)A misfolded protein appears to be th
37、e causative agent of a number of rare degenerative brain diseases in mammals.Perhaps the best known of these is mad cow disease疯牛病(bovine spongiform encephalopathy,牛绵状脑病BSE),an outbreak爆发 of which made international headlines in the spring of 1996.Related diseases include kuru 苦鲁病,新几内亚震颤病and Creutzf
38、eldt-Jakobdisease痉挛性假硬化,克雅病 in humans,scrapie绵羊疯痒病 in sheep,and chronic wasting disease慢性消耗性疾病 in deer and elk麋鹿.These diseases are also referred to as spongiform encephalopathies海绵状脑病,because the diseased brain frequently becomes riddled迷惑 with holes(Fig.1).Typical symptoms include dementia 痴呆and l
39、oss of coordination失去协调.The diseases are fatal致命的.In the 1960s,investigators found that preparations of the disease-causing agents appeared to lack nucleic acids.At this time,Tikvah Alper suggested that the agent was a protein.Initially,the idea seemed heretical异端邪说.All disease-causing agents known
40、up to that timeviruses,bacteria,fungi,and so oncontained nucleic acids,and their virulence was related to genetic reproduction复制 and propagation繁殖.However,four decades四十,年 of investigations,pursued most notably by Stanley Prusiner,have provided evidence that spongiform encephalopathies are different
41、.,The infectious agent has been traced to a single protein(Mr 28,000),which Prusiner dubbed prion(from proteinaceous infectious only)protein(PrP).Prion protein is a normal constituent of brain tissue in all mammals.Its role in the mammalian brain is notknown in detail,but it appears to have a molecu
42、lar signaling function.Strains of mice lacking the gene for PrP(and thus the protein itself)suffer no obvious ill effects.Illness occurs only when the normal cellular PrP,or PrPC,occurs in an altered conformation called PrPSc(Sc denotes scrapie).The interaction of PrPSc with PrPC converts the latter
43、 to PrPSc,initiating a domino effect多米诺效应 in which more and more of the brain protein converts to the disease-causing form.The mechanism by which the presence of PrPSc leads to spongiform encephalopathy is not understood.In inherited forms of prion diseases,a mutation in the gene encoding PrP produc
44、es a change in one amino acid residue that is believed to make the conversion of PrPC to PrPSc more likely.A complete understanding of prion diseases awaits new information about how prionprotein affects brain function.Structural information about PrP is beginning to provide insights into the molecu
45、lar process that allows the prion proteins to interact so as to alter their conformation(Fig.2).,Color Blindness:John Daltons Experimentfrom the GraveThe chemist John Dalton(of atomic theory fame闻名)was color-blind.He thought it probable很可能 that the vitreous humor玻璃体液 of his eyes(the fluid that fills
46、 the eyeball behind the lens透镜)was tinted blue,unlike the colorless fluid of normal eyes.He proposed 提议that after his death,his eyes should be dissected 切开and the color of the vitreous humor determined.His wish was honored尊重.The day after Daltons death in July 1844,Joseph Ransome dissected his eyes
47、and found the vitreous humor to be perfectly完全 colorless.Ransome,like many scientists,was reluctant to throw samples away.He placed Daltons eyes in a jar of preservative防腐剂(Fig.1),where they stayed 保存for a century and a half.Then,in the mid-1990s,molecular biologists in England took small samples of
48、 Daltons retinas视网膜 and extracted提取 DNA.Using the known gene sequences for the opsins视蛋白 of the red and green photopigments感光色素,they amplified the relevant sequences,色盲:来自坟墓的约翰道尔顿实验,and determined that Dalton had the opsin视蛋白 gene for the red photopigment but lacked the opsin gene for the green photopigment.Dalton was a green-dichromat.So,150 years after his death,the experiment Dalton startedby hypothesizing猜测 about the cause of his color blindnesswas finally finished.,THANK YOU,
