第二章 蛋白质的结构与功能

1、Chapter 3Chapter 3Protein Protein Structure & FunctionStructure & FunctionP24,671. 1. amino acidamino acid L L- - - -氨基酸氨基酸 building blocks：amino acidsproteinhydrolysis (acid or base or enzyme)NH3+COO-Hcarboxyl groupamino groupside chainCOMMOM GROUP 氨基酸通式氨基酸通式C R例p16peptide bone peptide bone （CH+ H3

2、NR1COO-+-H2OCHNR2COO -HHH+CH+ H3NR1COO-CHNR2COH Features of peptide bondFeatures of peptide bondNH3-COO-7.23A3.61AComponents of a polypeptide chain. A polypeptide chain consists of a constant backbone (shown in black) and variable side chains (shown in green).肽链：肽链：主链（主链（backbonebackbone）+ + 侧链（侧链（s

3、ide chainside chain）肽平面肽平面肽平面肽平面-1肽平面肽平面-2多肽链可以看成由多肽链可以看成由C串联起来的无数个肽平面组成串联起来的无数个肽平面组成一级结构级结构（初级结构）（初级结构）v 指多肽链中指多肽链中氨基酸的排列顺序氨基酸的排列顺序称为蛋白质的称为蛋白质的一级结构。一级结构。v 维系一级结构的化学键：维系一级结构的化学键：肽键肽键（主键）（主键）Primary structurePrimary structure牛胰核糖核酸牛胰核糖核酸酶一级结构：酶一级结构：一条肽链一条肽链124个氨基个氨基酸残基，缺色酸残基，缺色氨酸氨酸四个二硫键四个二硫键胰岛素的一级结构：

4、胰岛素的一级结构：两条肽链，两条肽链，A A链和链和B B链链5151个氨基酸残基个氨基酸残基3 3 个二硫键个二硫键Protein space structureProtein space structure空间结构的表示方法：空间结构的表示方法：金属丝模型金属丝模型主链金属丝模型主链金属丝模型Backbone wire model 飘带模型飘带模型Ribbon model香肠模型香肠模型Sausage model空间填充模型空间填充模型 Space-filling model Secondary structureSecondary structureSeveral types of se

5、condary structure are particularly stable and occur widely in proteins. The most prominent are the helix and conformations described below. Using fundamental chemical principles and a few experimental observations, Linus Pauling and Robert Corey predicted the existence of these secondary structures

6、in 1951, several years before the first complete protein structure was elucidated.n定义定义：蛋白质分子中局部肽链的肽：蛋白质分子中局部肽链的肽平面通过平面通过碳原子碳原子旋转，使碳链主链旋转，使碳链主链沿中心轴盘曲成稳定的沿中心轴盘曲成稳定的右手螺旋右手螺旋构象构象n螺旋是主要的二级结构形式，存螺旋是主要的二级结构形式，存在于大多数的蛋白质中，如在于大多数的蛋白质中，如 角蛋角蛋白（皮肤、毛发、指甲、和角）全部白（皮肤、毛发、指甲、和角）全部是是螺旋螺旋 - -螺旋（螺旋（ -helix)-helix) -螺旋

7、（螺旋（ -helix)模型图模型图5.4 A 3.6个残基个残基氢键氢键侧链基团侧链基团Four models of the a helix, showing different aspects of its structure. (a) Formation of a right-handed helix. The planes of the rigid peptide bonds are parallel to the long axis of the helix. (b) Ball-and-stick model of a right-handed helix, showing the

8、intrachain hydrogen bonds. The repeat unit is a single turn of the helix, 3.6 residues.(c) The helix as viewed from one end, looking down the longitudinal axis. Note the positions of the R groups, represented by red spheres. (d) A space-filling model of the helix.Hair -keratin is an elongated a-heli

9、x with somewhat thicker domains near the amino and carboxy termini. Pairs of these helices are interwound, probably in a left-handed sense, to form two-chain coiled coils. 例例1Structure of collagen. (胶原质）The collagen helix is a repeating secondary structure unique to this protein. (a) The repeating t

10、ripeptide sequence Gly-X-Pro or Gly-X-Hyp adopts a left-handed helical structure with three residues per turn. The repeating sequence used to generate this model is Gly-Pro-Hyp. (b) Space-filling model of the collagen helix shown in (a). (c) Three of these helices wrap around one another with a righ

11、t-handed twist. The resulting three-stranded molecule is referred to as tropocollagen. (d) The three-stranded collagen superhelix shown from one end, in a ball-and-stick representation. Glycine residues are shown in red. Glycine, because of its small size, is required at the tight junction where the

12、 three chains are in contact.例例2n定义定义：多肽链的局部肽段多肽链的局部肽段，以肽，以肽键平面为单位折叠成锯齿状的一种键平面为单位折叠成锯齿状的一种较伸展结构较伸展结构. . - -折折叠叠 （ -pleated sheet)-pleated sheet)模型模型图图C A twisted sheet. (A) A ball-and-stick model. (B) A schematic model. (C) The schematic view rotated by 90 degrees to illustrate the twist more clearl

13、y.片层并非是平直而是扭曲的片层并非是平直而是扭曲的-折叠的折叠的要点：要点： - -转角转角 （ -turn)-turn)n定义定义：多肽链中的某局部出现多肽链中的某局部出现180度回折度回折处形成处形成的特定构象。的特定构象。n一般由四个氨基酸组成，常见氨基酸：一般由四个氨基酸组成，常见氨基酸：Pro,Gly,Asp,Asn,Trp。 以氢键维持稳定，以氢键维持稳定，第第1个个氨基酸残基的氨基酸残基的C=O与与第第4个个残基的残基的N-H之间形成之间形成氢键。氢键。n 赋予蛋白质较大的构象柔性，常位于三维结构赋予蛋白质较大的构象柔性，常位于三维结构的表面，有些具有特定功能。的表面，有些具有

14、特定功能。-转角转角 (-turn or reverse turn or hairpin bend) Structure of a reverse turn. The CO group of residue i of the polypeptide chain is hydrogen bonded to the NH group of residue i+3 to stabilize the turn.无规则卷曲（无规则卷曲（ random coil ）n定义定义：没有明确规律性的那部分肽段的结构。：没有明确规律性的那部分肽段的结构。但这些但这些“无规则卷曲无规则卷曲”也象其他二级结构那样是也

15、象其他二级结构那样是明确而稳定的结构。即蛋白质在其多肽链的不同明确而稳定的结构。即蛋白质在其多肽链的不同区段可形成不同的二级结构。区段可形成不同的二级结构。n作用作用：赋予蛋白质较大的构象柔性，常构成蛋：赋予蛋白质较大的构象柔性，常构成蛋白质的特定功能部位。白质的特定功能部位。超二级结构超二级结构Motif 的常见组合的常见组合结构域结构域结构域的特征：结构域的特征：v定义定义：指在二级结构的基础上，多肽链进一步卷：指在二级结构的基础上，多肽链进一步卷曲、折叠而形成具有一定规律的三维空间结构。三曲、折叠而形成具有一定规律的三维空间结构。三级结构是一条多肽链的级结构是一条多肽链的完整的构象完整的

16、构象，包括全部的主，包括全部的主链和侧链的专一性的空间排布。链和侧链的专一性的空间排布。v维系三级结构的化学键维系三级结构的化学键：氢键、疏水键、盐键和氢键、疏水键、盐键和范德华力范德华力等次级键以及等次级键以及二硫键。二硫键。tertiary structuretertiary structureMyohemerythrin (2mhr in the Protein Data Bank) is an oxygen-carrying protein from a sipunculan worm. The four-helix, up-and-down bundle motif is commo

17、n to a number of functionally unrelated proteins. This commonality indicates that this is an energetically favorable arrangement.蚯蚓肌红蛋白三级结构蚯蚓肌红蛋白三级结构（ -螺旋螺旋）激酶相关蛋白激酶相关蛋白半乳糖苷变旋酶半乳糖苷变旋酶三级结构三级结构（-折叠折叠）氧化铁还原蛋白氧化铁还原蛋白三级结构三级结构半乳糖苷激酶三级结构半乳糖苷激酶三级结构（ -螺旋螺旋、-折叠）折叠）Quaternary structureQuaternary structure）Quat

18、ernary structure of hemoglobin Hemoglobin is composed of four poly-peptide chains, each of which is bound to a heme group. The two chains and the two chains are identical. Heme prosthetic groups (1 per subunit, so 4 per Hb tetramer) in red (ball & stick rendering) - the 4 hemes are far apart. 八聚体组蛋白

19、核心八聚体组蛋白核心2(H2A,H2B,H3,H4)蛋白质空间结构的总结蛋白质空间结构的总结细胞内高稠密介质，以及狭窄空间使得伸展的和部细胞内高稠密介质，以及狭窄空间使得伸展的和部分折叠的蛋白质在高浓度时倾向于聚集；无法自我分折叠的蛋白质在高浓度时倾向于聚集；无法自我迅速完成正确折叠；迅速完成正确折叠；“辅助性组装学说辅助性组装学说”（assisted assembling）：新生肽：新生肽链折叠并组装成有功能的蛋白质并非都能自发完成，链折叠并组装成有功能的蛋白质并非都能自发完成，在相当多的情况下是需要其他蛋白质帮助的，这类在相当多的情况下是需要其他蛋白质帮助的，这类帮助蛋白包括帮助蛋白包括分

20、子伴侣分子伴侣（molecular chaperones）和和折折叠酶叠酶（folding enzymes, foldases）。1987,Lasky: molecular chaperone分子伴侣主要的分类分子伴侣主要的分类：v伴侣素家族伴侣素家族（chaperonin,Cpn）v应激蛋白应激蛋白70家族家族（stress-70 family）/热休克蛋白热休克蛋白70家族家族（Hsp 70 family）v应激蛋白应激蛋白90家族家族（stress-90 family）/热休克蛋白热休克蛋白90家族家族（Hsp 90 family）伴侣素家族伴侣素家族（chaperonin, Cpn）T

21、he structure and function of the hsp60 family of molecular chaperones. (A) The catalysis of protein refolding. As indicated, a misfolded protein is initially captured by hydrophobic interactions along one rim of the barrel. The subsequent binding of ATP plus a protein cap increases the diameter of t

22、he barrel rim, which may transiently stretch (partly unfold) the client protein. This also confines the protein in an enclosed space, where it has a new opportunity to fold. After about 15 seconds, ATP hydrolysis ejects the protein, whether folded or not, and the cycle repeats. This type of molecula

23、r chaperone is also known as a chaperonin; it is designated as hsp60 in mitochondria, TCP-1 in the cytosol of vertebrate cells, and GroEL in bacteria. As indicated, only half of the symmetrical barrel operates on a client protein at any one time. (B) The structure of GroEL bound to its GroES cap, as

24、 determined by x-ray crystallography. On the left is shown the outside of the barrel-like structure and on the right a cross section through its center. The hsp70 family of molecular chaperones. These proteins act early, recognizing a small stretch of hydrophobic amino acids on a proteins surface. A

25、ided by a set of smaller hsp40 proteins, an hsp70 monomer binds to its target protein and then hydrolyzes a molecule of ATP to ADP, undergoing a conformational change that causes the hsp70 to clamp down very tightly on the target. After the hsp40 dissociates, the dissociation of the hsp70 protein is

26、 induced by the rapid re-binding of ATP after ADP release. Repeated cycles of hsp protein binding and release help the target protein to refold, 应激蛋白应激蛋白70家族家族(Stress-70 family）v一级结构是空间结构的基础一级结构是空间结构的基础v一级结构不同，功能不同；一级结构相一级结构不同，功能不同；一级结构相似，空间构象和功能也相似似，空间构象和功能也相似activityno activity分子病分子病 DNA TGT GGG C

27、TT CTT TTT 正常正常 mRNA ACA CCC GAA GAA AAA HbA（ 亚基）亚基） N Thr Pro Glu GluLys DNA TGT GGG CTT TTT 异常异常 mRNA ACA CCC GUA GAA AAA Hbs （ 亚基）亚基）N Thr ProVal GluLys镰刀形红细胞贫血现是由于血红蛋白发生了遗传突变引起的镰刀形红细胞贫血现是由于血红蛋白发生了遗传突变引起的 链第链第6位的位的aa线基由正常的线基由正常的Glu变成了疏水性的变成了疏水性的Val细胞色素细胞色素C的一级结构与物的一级结构与物种进化间的关系种进化间的关系-物种进物种进化树（化树

28、（phylogenetic tree）Normal folding pattern of PrP Alternative folding pattern of PrP 朊病毒蛋白朊病毒蛋白?核核 酸酸组成单位组成单位种种 类类连接方式连接方式一级结构一级结构空间结构空间结构功功 能能蛋白质蛋白质氨基酸氨基酸核苷酸核苷酸20种种A、C 、G 、T (DNA) A、C 、G 、U (RNA)肽键肽键磷酸二酯键磷酸二酯键AA排列顺序排列顺序碱基序列碱基序列二、三、四级二、三、四级结构结构 螺旋、超螺旋、蛋螺旋、超螺旋、蛋白白-核酸非共价结合核酸非共价结合生命活动直生命活动直接执行者接执行者 遗传信息

29、贮存、传代、遗传信息贮存、传代、表达表达,决定蛋白结构决定蛋白结构v变性本质变性本质: : 空间构象的改变或破坏空间构象的改变或破坏, , 不涉及一级结构的改不涉及一级结构的改变和肽键断裂变和肽键断裂稳稳定定蛋蛋白白质质分分子子结结构构的的作作用用力力氢键氢键盐键盐键二硫键二硫键Food vs Protein角蛋白变性？角蛋白变性？烫发原理烫发原理应用：测定生物样品中蛋白质的含量应用：测定生物样品中蛋白质的含量 差速离心、差速离心、 差速离心差速离心(differential centrifugation)(differential centrifugation) 溶液在不同的速度下离心，从而

30、分离和收集大小不同的溶液在不同的速度下离心，从而分离和收集大小不同的蛋白质颗粒。蛋白质颗粒。lowhighsucrosecocktail蔗糖蔗糖密度密度梯度梯度PNH2COO-NH3+NH3+COOHCOO-PP+ OH-+ H+ H+ OH-电场电场电场电场Electrophoresis-SDS-PAGE(a) Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric fie

31、ld is applied. The gel minimizes convection currents caused by small temperature gradients, and it minimizes protein movements other than those induced by the electric field.(b) Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie blue, which binds to t

32、he proteins but not to the gel itself Each band on the gel represents a different protein (or protein subunit); smaller proteins are found nearer the bottom of the gel. This gel illustrates the purification of the enzyme RNA polymerase from the bacterium E. coli. The first lane shows the proteins present in the crude cellular extract. Successive lanes show the proteins present after each purification step. The purified protein contains four