第二章 蛋白質的結構與功能

1、Chapter 3Chapter 3Protein Structure & FunctionProtein Structure & 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).肽鏈：肽鏈：主鏈（主鏈（backbonebackbone）+ + 側鏈（側鏈（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多肽鏈可以看成由多肽鏈可以看成由C串聯(lián)起來的無數(shù)個肽平面組成串聯(lián)起來的無數(shù)個肽平面組成一級結構級結構（初級結構）（初級結構）v 指多肽鏈中指多肽鏈中氨基酸的排列順序氨基酸的排列順序稱為蛋白質的稱為蛋白質的一級結構。一級結構。v 維系一級結構的化學鍵：維系一級結構的化學鍵：肽鍵肽鍵（主鍵）（主鍵）Primary structurePrimary structure牛胰核糖核酸牛胰核糖核酸酶一級結構：酶一級結構：一條肽鏈一條肽鏈124個氨基個氨基酸殘基，缺色酸殘基，缺色氨酸氨酸四個二硫鍵四個二硫鍵胰島素的一級結構：

4、胰島素的一級結構：兩條肽鏈，兩條肽鏈，A A鏈和鏈和B B鏈鏈5151個氨基酸殘基個氨基酸殘基3 3 個二硫鍵個二硫鍵Protein space structureProtein space structure空間結構的表示方法：空間結構的表示方法：金屬絲模型金屬絲模型主鏈金屬絲模型主鏈金屬絲模型Backbone wire model 飄帶模型飄帶模型Ribbon model香腸模型香腸模型Sausage model空間填充模型空間填充模型 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定義定義：蛋白質分子中局部肽鏈的肽：蛋白質分子中局部肽鏈的肽平面通過平面通過碳原子碳原子旋轉，使碳鏈主鏈旋轉，使碳鏈主鏈沿中心軸盤曲成穩(wěn)定的沿中心軸盤曲成穩(wěn)定的右手螺旋右手螺旋構象構象n螺旋是主要的二級結構形式，存螺旋是主要的二級結構形式，存在于大多數(shù)的蛋白質中，如在于大多數(shù)的蛋白質中，如 角蛋角蛋白（皮膚、毛發(fā)、指甲、和角）全部白（皮膚、毛發(fā)、指甲、和角）全部是是螺旋螺旋 - -螺旋（螺旋（ -helix)-helix) -螺旋

7、（螺旋（ -helix)模型圖模型圖5.4 A 3.6個殘基個殘基氫鍵氫鍵側鏈基團側鏈基團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. (膠原質）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定義定義：多肽鏈的局部肽段，以肽多肽鏈的局部肽段，以肽鍵平面為單位折疊成鋸齒狀的一種鍵平面為單位折疊成鋸齒狀的一種較伸展結構較伸展結構. . - -折折疊疊 （ -pleated sheet)-pleated sheet)模型模型圖圖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.片層并非是平直而是扭曲的片層并非是平直而是扭曲的-折疊的要點：折疊的要點： - -轉角轉角 （ -turn)-turn)n定義定義：多肽鏈中的某局部出現(xiàn)多肽鏈中的某局部出現(xiàn)180度回折度回折處形成處形成的特定構象。的特定構象。n一般由四個氨基酸組成，常見氨基酸：一般由四個氨基酸組成，常見氨基酸：Pro,Gly,Asp,Asn,Trp。 以氫鍵維持穩(wěn)定，以氫鍵維持穩(wěn)定，第第1個個氨基酸殘基的氨基酸殘基的C=O與與第第4個個殘基的殘基的N-H之間形成之間形成氫鍵。氫鍵。n 賦予蛋白質較大的構象柔性，常位于三維結構賦予蛋白質較大的構象柔性，常位于三維結構的表面，有些具有特定功能。的表面，有些具有

14、特定功能。-轉角轉角 (-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.無規(guī)則卷曲（無規(guī)則卷曲（ random coil ）n定義定義：沒有明確規(guī)律性的那部分肽段的結構。：沒有明確規(guī)律性的那部分肽段的結構。但這些但這些“無規(guī)則卷曲無規(guī)則卷曲”也象其他二級結構那樣是也

15、象其他二級結構那樣是明確而穩(wěn)定的結構。即蛋白質在其多肽鏈的不同明確而穩(wěn)定的結構。即蛋白質在其多肽鏈的不同區(qū)段可形成不同的二級結構。區(qū)段可形成不同的二級結構。n作用作用：賦予蛋白質較大的構象柔性，常構成蛋：賦予蛋白質較大的構象柔性，常構成蛋白質的特定功能部位。白質的特定功能部位。超二級結構超二級結構Motif 的常見組合的常見組合結構域結構域結構域的特征：結構域的特征：v定義定義：指在二級結構的基礎上，多肽鏈進一步卷：指在二級結構的基礎上，多肽鏈進一步卷曲、折疊而形成具有一定規(guī)律的三維空間結構。三曲、折疊而形成具有一定規(guī)律的三維空間結構。三級結構是一條多肽鏈的級結構是一條多肽鏈的完整的構象完整的

16、構象，包括全部的主，包括全部的主鏈和側鏈的專一性的空間排布。鏈和側鏈的專一性的空間排布。v維系三級結構的化學鍵維系三級結構的化學鍵：氫鍵、疏水鍵、鹽鍵和氫鍵、疏水鍵、鹽鍵和范德華力范德華力等次級鍵以及等次級鍵以及二硫鍵。二硫鍵。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.蚯蚓肌紅蛋白三級結構蚯蚓肌紅蛋白三級結構（ -螺旋螺旋）激酶相關蛋白激酶相關蛋白半乳糖苷變旋酶半乳糖苷變旋酶三級結構三級結構（-折疊折疊）氧化鐵還原蛋白氧化鐵還原蛋白三級結構三級結構半乳糖苷激酶三級結構半乳糖苷激酶三級結構（ -螺旋、螺旋、-折疊折疊）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. 八聚體組蛋白

19、核心八聚體組蛋白核心2(H2A,H2B,H3,H4)蛋白質空間結構的總結蛋白質空間結構的總結l細胞內高稠密介質，以及狹窄空間使得伸展的和部細胞內高稠密介質，以及狹窄空間使得伸展的和部分折疊的蛋白質在高濃度時傾向于聚集；無法自我分折疊的蛋白質在高濃度時傾向于聚集；無法自我迅速完成正確折疊；迅速完成正確折疊；l“輔助性組裝學說輔助性組裝學說”（assisted assembling）：新生肽：新生肽鏈折疊并組裝成有功能的蛋白質并非都能自發(fā)完成，鏈折疊并組裝成有功能的蛋白質并非都能自發(fā)完成，在相當多的情況下是需要其他蛋白質幫助的，這類在相當多的情況下是需要其他蛋白質幫助的，這類幫助蛋白包括幫助蛋白包

20、括分子伴侶分子伴侶（molecular chaperones）和和折折疊酶疊酶（folding enzymes, foldases）。1987,Lasky: molecular chaperone分子伴侶主要的分類分子伴侶主要的分類：v伴侶素家族伴侶素家族（chaperonin,Cpn）v應激蛋白應激蛋白70家族家族（stress-70 family）/熱休克蛋白熱休克蛋白70家族家族（Hsp 70 family）v應激蛋白應激蛋白90家族家族（stress-90 family）/熱休克蛋白熱休克蛋白90家族家族（Hsp 90 family）伴侶素家族伴侶素家族（chaperonin, Cpn

21、）The 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

22、 the 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 molecu

23、lar 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,

24、as 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.

25、 Aided 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

26、is 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, 應激蛋白應激蛋白70家族家族(Stress-70 family）v一級結構是空間結構的基礎一級結構是空間結構的基礎v一級結構不同，功能不同；一級結構相一級結構不同，功能不同；一級結構相似，空間構象和功能也相似似，空間構象和功能也相似activityno activity分子病分子病 DNA TGT GGG

27、 CTT CTT TTT 正常正常 mRNA ACA CCC GAA GAA AAA HbA（ 亞基）亞基） N Thr Pro Glu GluLys DNA TGT GGG CTT TTT 異常異常 mRNA ACA CCC GUA GAA AAA Hbs （ 亞基）亞基）N Thr ProVal GluLys鐮刀形紅細胞貧血現(xiàn)是由于血紅蛋白發(fā)生了遺傳突變引起的鐮刀形紅細胞貧血現(xiàn)是由于血紅蛋白發(fā)生了遺傳突變引起的 鏈第鏈第6位的位的aa線基由正常的線基由正常的Glu變成了疏水性的變成了疏水性的Val細胞色素細胞色素C的一級結構與物的一級結構與物種進化間的關系種進化間的關系-物種進物種進化樹（

28、化樹（phylogenetic tree）Normal folding pattern of PrP Alternative folding pattern of PrP 朊病毒蛋白朊病毒蛋白?核核 酸酸組成單位組成單位種種 類類連接方式連接方式一級結構一級結構空間結構空間結構功功 能能蛋白質蛋白質氨基酸氨基酸核苷酸核苷酸20種種A、C 、G 、T (DNA) A、C 、G 、U (RNA)肽鍵肽鍵磷酸二酯鍵磷酸二酯鍵AA排列順序排列順序堿基序列堿基序列二、三、四級二、三、四級結構結構 螺旋、超螺旋、蛋螺旋、超螺旋、蛋白白-核酸非共價結合核酸非共價結合生命活動直生命活動直接執(zhí)行者接執(zhí)行者 遺傳

29、信息貯存、傳代、遺傳信息貯存、傳代、表達表達,決定蛋白結構決定蛋白結構v變性本質變性本質: : 空間構象的改變或破壞空間構象的改變或破壞, , 不涉及一級結構的改不涉及一級結構的改變和肽鍵斷裂變和肽鍵斷裂穩(wěn)穩(wěn)定定蛋蛋白白質質分分子子結結構構的的作作用用力力氫鍵氫鍵鹽鍵鹽鍵二硫鍵二硫鍵Food vs Protein角蛋白變性？角蛋白變性？燙發(fā)原理燙發(fā)原理應用：測定生物樣品中蛋白質的含量應用：測定生物樣品中蛋白質的含量 差速離心、差速離心、 差速離心差速離心(differential centrifugation)(differential centrifugation) 溶液在不同的速度下離心，

30、從而分離和收集大小不同的溶液在不同的速度下離心，從而分離和收集大小不同的蛋白質顆粒。蛋白質顆粒。lowhighsucrosecocktail蔗糖蔗糖密度密度梯度梯度PNH2COO-NH3+NH3+COOHCOO-PP+ OH-+ H+ H+ OH-電場電場電場電場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 f

31、ield 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

32、 the 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