新編第03章蛋白質(zhì)的合成與修飾課件

1、第三章 蛋白質(zhì)的合成與修飾 Translation，Artificial synthesis and modification本章的主要內(nèi)容：1. 翻譯的生物學(xué)意義；2. 遺傳密碼的特性和線粒體密碼的例外；3. 翻譯體系的主要組成及其功能；4. 原核生物和真核生物蛋白質(zhì)生物合成的異同點；5. 蛋白質(zhì)合成后的加工、運輸與定位；6. 蛋白質(zhì)的化學(xué)合成與人工修飾。第一節(jié) 蛋白質(zhì)的生物合成翻譯 The Biosynthesis of Protein -Translation 在細(xì)胞質(zhì)中，以mRNA為模板，在核糖體、tRNA和多種蛋白因子等的共同作用下，將mRNA中由核苷酸排列順序決定的遺傳信息轉(zhuǎn)變成由

2、20種氨基酸組成的蛋白質(zhì)的過程。 這一過程猶如電報的翻譯過程，故又將蛋白質(zhì)的生物合成稱為翻譯（translation）。 翻譯（translation）:轉(zhuǎn)錄和翻譯統(tǒng)稱為基因表達(dá)(gene expression)。前 言翻譯的生物學(xué)意義？可以說，沒有蛋白質(zhì)，就沒有生命! 概括起來講，蛋白質(zhì)具有以下幾方面的功能： 1）催化：酶蛋白； 5）調(diào)節(jié)：激素、組蛋白； 2）防御：Ab、ABP、IFN等； 6）支持：膜系統(tǒng)； 3）運動：肌球（動）蛋白； 7）運輸：ApoE、血紅素； 4）營養(yǎng)貯存：卵清蛋白； 8）參與遺傳過程 9）其他：信號轉(zhuǎn)導(dǎo)等。 蛋白質(zhì)是生命活動的物質(zhì)基礎(chǔ)，幾乎所有生物的生命活動過程都離

3、不開蛋白質(zhì)。蛋白質(zhì)的種類成千上萬，其功能千差萬別。例如：酶蛋白；激素；膜蛋白；抗體；受體等。3-1-1 參與翻譯過程的物質(zhì)與功能 參與翻譯過程的物質(zhì)共包括：mRNA (Messenger RNA)tRNA (Transfer RNA)RibosomeAmino acids(AA)Aminoacyl-tRNA synthetase(氨?；?tRNA合成酶)Kinds of Translation Factors3-1-1 Components of Translation System and Their Functions一、Structure and Function of mRNA mRN

4、A(Messenger RNA) is the intermediate that represents one strand of a gene coding for protein. Its coding region is related to the protein sequence by the triplet genetic code. （一）mRNA的結(jié)構(gòu)特點An mRNA contains a series of codons that interact with the anticodons of aminoacyl-tRNAs so that a corresponding

5、 series of amino acids is incorporated into a polypeptide chain.Figure 6.16 Ribosome-binding sites on mRNA can be recovered from initiation complexes. They include the upstream Shine-Dalgarno sequence and the initiation codon.原核生物mRNA的結(jié)構(gòu)特點:原核生物mRNA的結(jié)構(gòu)特點The Shine-Dalgarno sequence(SD序列) is the polypu

6、rine sequence AGGAGG centered about 10 bp before the AUG initiation codon on bacterial mRNA. It is complementary to the sequence at the 3 end of 16S rRNA.真核生物mRNA的結(jié)構(gòu)特點:Functions of Cap：Protection of the mRNA from degradation.Enhancement of the mRNAs translatability.Transport of the mRNA out of the n

7、ucleus.Proper splicing of the pre-mRNA.Function of Polyadenylation : Most eukaryotic mRNAs and their precursors have a chain of AMP residues about 250 nucleotides long at their 3-ends.This poly(A) is added post-transcriptionally by poly(A) polymerase.Polyadenylation signals: AAUAAAFunctions : Poly(A

8、) enhances both the lifetime and translatability of mRNA. The only three sequences needed for splicing are : short consensus sequences at the 5 and 3splicing sites and at the branch site.The ends of nuclear introns are defined by the GU-AG rule. Splicing signals：真核生物與原核生物mRNA結(jié)構(gòu)特點比較：1. 真核生物的mRNA初始轉(zhuǎn)錄物

9、由內(nèi)含子和外顯子組成，需要進(jìn)行轉(zhuǎn)錄后加工： 1)將內(nèi)含子剪掉，將外顯子重新連接起來-剪接(splicing)， 2)在5-端加帽，3-端加poly(A)尾，才能形成成熟mRNA； 而原核生物的初始轉(zhuǎn)錄物即為成熟的mRNA，不需要轉(zhuǎn)錄后的加工。2. 真核生物的成熟mRNA需要從細(xì)胞核中進(jìn)入細(xì)胞質(zhì)，才能參與蛋白質(zhì)的翻譯；而原核生物的轉(zhuǎn)錄與翻譯偶聯(lián)在一起。真核生物mRNA為單順反子；原核生物mRNA為多順反子。原核生物的mRNA的5-端有SD序列。許多真核mRNA的AUG上游存在Kozak序列（CCACC）。（二）Genetic Codons(遺傳密碼) Seleno-Cys-tRNA UGA; P

10、yrrolysine UAG（三） Properties of Genetic Codons 三聯(lián)體，連續(xù)性，不重疊，兼并性，兼職，通用性等。但存在例外。1. ORF(開放讀框)2. 兼并性（degeneracy） 多種密碼子編碼一種氨基酸。3. 密碼子的例外 1）在支原體：UGA Trp(色)；2）在纖毛蟲：UAA和UAG Glu（谷）；3）在人的線粒體： UGA（終止密碼子） Trp(色) ； AGA，AGG（精） 終止密碼子。另加UAA、UAG， 線粒體共有4個終止密碼子； 內(nèi)部蛋氨酸的密碼子有2個：AUG和AUA；起始密碼子 有4個：AUN；4）在酵母線粒體：除上述3點外，還有CUA亮

11、 Thr(蘇)。Transfer RNA (tRNA) is the intermediate in protein synthesis that interprets the genetic code. Each tRNA can be linked to an amino acid. The tRNA has an anticodon sequence that comple-mentary to a triplet codon representing the amino acid.二、 Structure and Function of tRNAA tRNA has a sequenc

12、e of 74-95 bases that folds into a clover-leaf secondary structure with four constant arms (and an additional arm in the longer tRNAs). （一）Secondary Structure of tRNAThe cloverleaf: the structure of tRNA drawn in two dimensions, forming four distinct arm-loops.The acceptor arm consists of a base-pai

13、red stem that ends in an unpaired sequence whose free 2- or 3-OH group can be linked to an amino acid. The TC arm is named for the presence of this triplet sequence. ( stands for pseudouridine, a modified base). The anticodon arm always contains the anticodon triplet in the center of the loop.The D

14、arm is named for its content of the base Dihydrouridine (another of the modified bases in tRNA). The extra arm lies between the TC and anticodon arms and varies from 321 bases. tRNAs are called class I（35bases） if they lack it, and class II （1321 bases including 5bp in stem） if they have it. This tR

15、NA is used only for initiation. It recognizes the codons AUG or GUG(occasionally UUG). AUG GUG(1/2) UUG(1/4).Formylation is not strictly necessary, because nonformylated Met-tRNAf can function as an initiator, but it improves the efficiency with which the Met-tRNAf is used, because it is one of the

16、features recognized by the factor IF-2 that binds the initiator tRNA.The bases that face one another at the last position of the stem to which the amino acid is connected are paired in all tRNAs except tRNAf-Met. Mutations that create a base pair in this position of tRNAf-Met allow it to function in

17、 elongation. The absence of this pair is therefore important in preventing tRNAf-Met from being used in elongation. It is also needed for the formylation reaction.A series of 3 GC pairs in the stem that precedes the loop containing the anticodon is unique to tRNAf-Met. These base pairs are required

18、to allow the fMet-tRNAf to be inserted directly into the P site.（二）tRNA的三級結(jié)構(gòu) 所有的tRNA的三級結(jié)構(gòu)經(jīng)X-ray 衍射發(fā)現(xiàn)，都呈倒L形，見下圖。 A molecular model of the structure of yeast tRNAPhe is shown in Figure 5.5.（三）Functions of tRNA AA + ATP AA-AMP + PPi (氨基酸活化) AA-AMP + tRNA 氨酰基-tRNA + AMP tRNA正確識別和荷載氨基酸的原因：首先與氨?；?tRNA合成酶的

19、結(jié)構(gòu)有關(guān)；其次與反密碼子有關(guān)；副密碼子的作用非常重要。 副密碼子（paracodon）：tRNA分子上一些與氨?；?tRNA正確形成有關(guān)的堿基，如G3-U70等。（四）tRNA的豐富度與密碼子的使用頻率 同功tRNA(isoaccepting tRNA) ：識別并攜帶同一種氨基酸的不同tRNA。同功tRNA由同一種氨?；?tRNA合成酶催化合成氨酰基-tRNA。 密碼子的使用頻率: 在同一種細(xì)胞中，不同tRNA的數(shù)量多少不同，導(dǎo)致了編碼同一種AA的不同密碼子的使用頻率不同。 例如：大腸桿菌核糖體蛋白1209個密碼子，其中蘇氨酸的密碼子：ACU 38次、ACC 26次、ACA 3次、ACG 0次

20、。 密碼子優(yōu)化:Codon usage tabulated from the GenBank Genetic Sequence Data Volume 16, Supplement, Nucleic Acids Research.P315402 搖擺假說（wobble hypothesis）：編碼一種氨基酸的不同密碼子是否需要不同的tRNA來識別呢？ 不是的。一種tRNA的反密碼子可識別具有兼并性的密碼子。 為什么？原因就是反密碼子的5-端堿基具有搖擺性，一方面它的自由度大；另一方面它很少是U，幾乎不是A，如是A，則被修飾成I(次黃嘌呤)，I可與U、C、A配對；最后，在RNA中，G可和U配對。

21、所以，一種tRNA的反密碼子可識別幾種不同的密碼子。（五） Wobble hypothesis(搖擺假說)The wobble hypothesis accounts for the ability of a tRNA to recognize more than one codon by unusual (non-GC, non-AT) pairing with the third base of a codon.三、 Aminoacyl-tRNA Synthetases In spite of their common function, synthetases are a rather

22、diverse group of proteins.The individual subunits vary from 40-110 kDa, and the enzymes may be monomeric, dimeric, or tetrameric. Homologies between them are rare. Aminoacyl-tRNA synthetases are divided into the class I and class II groups by sequence and structural similarities.四、 核糖體的結(jié)構(gòu)與功能 （一）核糖體的

23、組成與結(jié)構(gòu)RNA的特異序列和功能含CGAAC與GTCG互補CCUCCU與SD序列互補有GAUC和TCG互補和Capm7G結(jié)合Electron micrographs of subunits and complete bacterial ribosomes are shown in Figure 6.2. Together with models in the corresponding orientation. The complete 70S ribosome has an asymmetric construction. The partition between the head and

24、 body of the small subunit is aligned with the notch of the large subunit, so that the platform of the small subunit fits into the large subunit. There is a cavity between the subunits which contains some of the important sites.3-1-2 Process of Protein Bio-Synthesis The main cellular components invo

25、lved in the process of protein synthesis: mRNA (Messenger RNA): Template of Translatiom tRNA (Transfer RNA): Carrier of Amino acids(adaptor) Ribosome: A Small migrating factory of Translation Aminoacyl-tRNA synthetase(氨酰基-tRNA合成酶) Kinds of Translation Factors: 蛋白質(zhì)合成的原料是細(xì)胞中的20種氨基酸(來源？)，反應(yīng)所需的能量由ATP與GT

26、P提供。 TAB. Components Required for the Five Major Stages of ProteinSynthesis in E. coliFour Stages are involved in the Process of Protein Biosynthesis:Stage 1: Activation of Amino Acids Stage 2: Initiation Stage 3: Elongation Stage 4: Termination and Release 一、 Activation of Amino Acids and Synthesis

27、 of Aminoacyl-tRNA1. 氨基酸的激活與氨?；?tRNA的合成過程 氨基酸不能直接與模板相結(jié)合，必須首先與相應(yīng)的tRNA結(jié)合，形成氨酰基tRNA。這一過程就是氨基酸的激活。 將氨基酸接合于tRNA以形成氨?；鵷RNA的激活反應(yīng)是在氨酰基-tRNA合成酶的催化作用下進(jìn)行的，需要ATP提供能量 。這個反應(yīng)是不可逆轉(zhuǎn)的 。 AA + ATP AA-AMP + PPi (氨基酸活化) AA-AMP + tRNA 氨酰基-tRNA + AMP AA + ATP AA-AMP + PPi (氨基酸活化)Synthetase Class I Synthetase Class II 氨?；?t

28、RNA是蛋白質(zhì)合成過程中的一個關(guān)鍵性物質(zhì)，它們的合成不僅僅是一個攜帶氨基酸的過程。其重要意義在于： （1）它為肽鍵的形成提供能量（-30.51kJ）； （2）tRNA上的反密碼子與mRNA上的密碼子識別，執(zhí)行遺傳信息的解讀過程； （3）保證了蛋白質(zhì)合成的準(zhǔn)確性。 2. 氨酰基-tRNA合成過程中的校正（proofreading）機制Protein biosynthesis is generally accurate. But it is thought to lie in the range of 1 error for every 104 - 105 amino acids incorpor

29、ated. 如何保證翻譯的正確性？ （1）氨?；?tRNA合成酶能夠正確識別其底物氨基酸的側(cè)鏈。有些是高度專一的，有些則專一性不高。 （2）對專一性不高的氨?；?tRNA合成酶的校正。在2個階段進(jìn)行校正：對AA-AMP的水解；對氨酰基-tRNA的水解。tRNA發(fā)揮了重要作用。 （3）氨?；?tRNA合成酶也必須識別正確的tRNA。 另外，還與tRNA的反密碼子有關(guān)；副密碼子的作用也非常重要。 二、原核生物蛋白質(zhì)翻譯過程（一）原核生物蛋白質(zhì)翻譯的起始1. 起始復(fù)合物的形成 (1) 起始因子、GTP、mRNA與30S小亞基結(jié)合: 原核起始因子有3種：IF1，IF2，和IF3。 IF1：IF1是一個

30、小的堿性蛋白，它能增加IF2和IF3的活性。IF1與16S rRNA的結(jié)合位點在A位點 。另外，IF1具有活化GTP酶的作用。 IF2：具有很強的GTP酶活性，在肽鏈合成起始時催化GTP水解。功能是生成IF2GTPfMet-tRNAMetf三元復(fù)合物，在IF3存在下，使起始tRNA與核糖體小亞基結(jié)合。 IF3：IF3與16S rRNA相互作用位點在P位點附近。IF3能通過促使mRNA的S-D序列與16S rRNA的3-端堿基配對，讓核糖體識別mRNA上的特異啟動信號，又能刺激fMet-tRNAf與核糖體結(jié)合在AUG上。 mRNA 和fMet-tRNA 結(jié)合于IF30SGTP聚合體上。在結(jié)合時，

31、fMet-tRNA與IF2-GTP復(fù)合物緊密接觸。 在核糖體小亞基上的16S rRNA 3-端有一段順序： 16S rRNA 3-UCCUCCPyA-5 (Py可以是任何嘧啶核苷酸) mRNA 5-AGGAGG-.AUG.3 正是由這樣的配對將AUG（或GUG，UUG）密碼子帶到核糖體的起始位置上 。 fMet-tRNA 與小亞基上的A位點結(jié)合。 2. 70S起始復(fù)合物的形成 30S起始復(fù)合物一旦完全形成后，IF3即釋放出來。50S大亞基參加進(jìn)來，并引起GTP水解和釋放其它兩個起始因子，最后的復(fù)合物稱為70S起始復(fù)合物。IF1：IF1是一個小的堿性蛋白，它能增加IF2和IF3的活性。IF1與1

32、6S rRNA的結(jié)合位點在A位點 。另外，IF1具有活化GTP酶的作用。IF2：具有很強的GTP酶活性，在肽鏈合成起始時催化GTP水解。功能是生成IF2GTPfMet-tRNAMetf三元復(fù)合物，在IF3存在下，使起始tRNA與核糖體小亞基結(jié)合。IF3：與16S rRNA相互作用位點在P位點附近。IF3能通過促使mRNA的S-D序列與16S rRNA的3-端堿基配對，讓核糖體識別mRNA上的特異啟動信號，又能刺激fMet-tRNAf與核糖體結(jié)合在AUG上。 （二）多肽鏈的延伸、移位循環(huán) 從起始階段形成的起始復(fù)合物可以接受第二個氨酰-tRNA，以形成蛋白質(zhì)第一個肽鍵。在第二個氨?；?tRNA進(jìn)入

33、A位點之后，便形成一個肽鍵，并產(chǎn)生出一個連接于第二個氨基酸的tRNA上的二肽。然后便發(fā)生移位，肽酰tRNA和與之結(jié)合的mRNA密碼子協(xié)同轉(zhuǎn)移至P位點。這個氨基酸加成過程一再重復(fù)，每次加上一個氨基酸，直至形成一條完整的多肽鏈。 肽鏈的延伸要求有延伸因子EF-TU和EF-TS參與。 Figure 6.20 EF-Tu-GTP places aminoacyl-tRNA on the ribosome and then is released as EF-Tu-GDP. EF-Ts is required to mediate the replacement of GDP by GTP. The r

34、eaction consumes GTP and releases GDP. The only aminoacyl-tRNA that cannot be recognized by EF-Tu-GTP is fMet-tRNAf, whose failure to bind prevents it from responding to internal AUG or GUG codons. Figure 6.24 Binding of factors EF-Tu and EF-G alternates as ribosomes accept new aminoacyl-tRNA, form

35、peptide bonds, and translocate. Figure 6.21 Peptide bond formation takes place by reaction between the polypeptide of peptidyl-tRNA in the P site and the amino acid of aminoacyl-tRNA in the A site. Peptidyl transferase is the activity of the ribosomal 50S subunit that synthesizes a peptide bond when

36、 an amino acid is added to a growing polypeptide chain. The actual catalytic activity is a propery of the rRNA.Figure 6.23 Models for translocation involve two stages. First, at peptide bond formation the aminoacyl end of the tRNA in the A site becomes located in the P site. Second, the anticodon en

37、d of the tRNA becomes located in the P site. Second, the anticodon end of the tRNA becomes located in the P site.（三）多肽鏈合成的終止Figure 6.27 Molecular mimicry enables the elongation factor Tu-tRNA complex, the translocation factor EF-G, and the release factors RF1/2-RF3 to bind to the same ribosomal site

38、.Figure 6.8 Initiation requires free ribosome subunits. When ribosomes are released at termination, they dissociate to generate free subunits. Initiation factors are present only on dissociated 30S subunits. When subunits reaassociate to give a functional ribosome at initiation, they release the fac

39、tors. 三、真核生物蛋白質(zhì)的生物合成 真核生物蛋白質(zhì)合成與原核生物兩者相比，密碼相同，各種組分相似，亦有核糖體、tRNA及各種蛋白質(zhì)因子?？偟暮铣赏緩揭蚕嗨疲衅鹗?、延伸及終止階段，但也有不同之處。（一）真核生物蛋白質(zhì)合成的起始 真核生物的起始氨基酸是蛋氨酸。參與翻譯起始反應(yīng)的起始因子已發(fā)現(xiàn)有10幾種。這個過程可分為3個步驟： 1. 43S前起始復(fù)合物的形成 起始因子eIF-2與GTP形成穩(wěn)定復(fù)合物，后者與Met-tRNAMetI形成三元復(fù)合物，再與40S亞基形成43S前起始復(fù)合物。 2. 48S前起始復(fù)合物的形成 在起始因子eIF-4A，eIF-4B，eIF-4E和ATP的參與下，43S

40、前起始復(fù)合物與mRNA結(jié)合。eIF-4A有使mRNA二級結(jié)構(gòu)解旋的作用。eIF-4B則有結(jié)合mRNA并識別起始密碼子AUG的作用。形成48S前起始復(fù)合物。 據(jù)Kozak等的研究，大多數(shù)起始密碼子的上游存在CCACC（稱為 Kozak序列 ）AUGG。在43S前起始復(fù)合物沿mRNA向3端方向移動時，遇到CCACC序列時，即停止移動。起始密碼子AUG的識別可能是通過與tRNA上的反密碼子的作用。eIF-2也參與了這個識別過程。 3. 80S起始復(fù)合物的形成 48S前起始復(fù)合物與核糖體60S大亞基結(jié)合，便形成了80S起始復(fù)合物。 這一過程由GTP水解提供能量。各種起始因子釋放出來，參與下一輪的起始復(fù)

41、合物形成。Figure 6.18 In eukaryotic initiation, eIF-2 forms a ternary complex with Met-tRNAf. The ternary complex binds to free 40S subunits, which attach to the 5 end of mRNA. Later in the reaction, GTP is hydrolyzed when eIF-2 is released in the form of eIF2-GDP. eIF-2B regenerates the active form. Fig

42、ure 6.19 Several eukaryotic initiation factors are required to unwind mRNA, bind the subunit initiation complex, and support joining with the large subunit. 1.肽鏈的延伸 真核生物的肽鏈延伸與原核相似，只是延伸因子EF-TU和EF-TS被eEF-1取代，而EF-G則被eEF-2取代。在真菌中，還要求第三種因子，即eEF-3的參與，以維持其翻譯的準(zhǔn)確性。 2.肽鏈的終止 真核生物肽鏈合成的終止僅涉及一個釋放因子eRF。eRF分子量約為115

43、kD。它可識別3種終止密碼子：UAA，UAG，UGA。eRF在活化了肽酰轉(zhuǎn)移酶釋放新生的肽鏈后，即從核糖體上解離。解離要求GTP的水解。故肽鏈合成的終止需要消耗能量。（二）肽鏈的延伸與終止四、原核生物與真核生物翻譯的比較 原核生物的翻譯與轉(zhuǎn)錄偶聯(lián)在一起，即邊轉(zhuǎn)錄邊翻譯；而真核生物的翻譯與轉(zhuǎn)錄不偶聯(lián)。真核mRNA前體需經(jīng)加工修飾成為成熟mRNA后，從核內(nèi)輸入細(xì)胞質(zhì)，然后進(jìn)行翻譯。 真核生物蛋白質(zhì)合成機構(gòu)比原核生物復(fù)雜，起始步驟涉及起始因子眾多，過程復(fù)雜。如起始氨基酸；核糖體組成；起始因子的種類等等。 真核生物蛋白質(zhì)合成的調(diào)控復(fù)雜。 真核生物與原核生物的蛋白質(zhì)合成可為不同的抑制劑所抑制。3-1-3

44、 蛋白質(zhì)翻譯后的加工 蛋白質(zhì)的結(jié)構(gòu)：一級、二級、三級、四級結(jié)構(gòu)。蛋白質(zhì)變性： 天然態(tài)（折疊態(tài)） 變成變性態(tài)（伸展態(tài)）體外蛋白質(zhì)的復(fù)性： U I N變性因素快慢一、蛋白質(zhì)的折疊 （一）體外蛋白質(zhì)折疊的機制 總之，體外蛋白質(zhì)的折疊可能是始于疏水坍塌，或始于轉(zhuǎn)角，或始于共價鍵相互作用（如二硫鍵的形成）。 在折疊早期，可能這三種方式聯(lián)合起作用。之后，可能沿著有限的多途徑形成中間態(tài)（熔球態(tài)）。這個過程是快速的。最后再由中間態(tài)進(jìn)入天然態(tài)，此過程比較慢，是折疊反應(yīng)的限速步驟。（二）體內(nèi)蛋白質(zhì)的折疊 體內(nèi)環(huán)境復(fù)雜；需要助折疊蛋白（folding helper）的參與，從而降低了折疊的錯誤，提高了效率(95%)

45、；在翻譯結(jié)束之前即開始（鄒氏學(xué)說）。助折疊蛋白： 1. 酶 ：蛋白質(zhì)二硫鍵異構(gòu)酶；肽酰脯氨酰順反異構(gòu)酶。與新生肽鏈的折疊密切相關(guān)，加速蛋白質(zhì)折疊過程。 2. 分子伴侶：細(xì)胞內(nèi)幫助新生肽鏈正確組裝，成為成熟蛋白質(zhì)，而本身卻不是最終功能蛋白質(zhì)分子的組成成分的分子，都稱為分子伴侶（molecular chaperone）。 Figure 8.5 Chaperone families have eukaryotic and bacterial counterparts (named in parentheses).脅迫-70（stress-70）家族： 分子伴侶蛋白：脅迫70（stress70 ）家族

46、；分子伴侶（chaperonin）家族。廣泛存在于原核和真核生物細(xì)胞中。Figure 8.6 DnaJ assists the binding of DnaK (Hsp70), which assists the folding of nascent proteins. ATP hydrolysis drives conformational change. GrpE displaces the ADP; this causes the chaperones to be released. Multiple cycles of association and dissociation may

47、occur during the folding of a substrate protein。分子伴侶（chaperonin）家族：Figure 8.8 GroEL forms an oligomer of two rings, each comprising a hollow cylinder made of 7 subunits.Figure 8.9 Two rings of GroEL associate back to back to form a hollow cylinder. GroES forms a dome that covers the central cavity o

48、n one side. Protein substrates bind to the cavity in the distal ring.二、蛋白質(zhì)的修飾 （一）末端氨基的脫甲?；蚇端甲硫氨酸的切除 對起始氨基酸的修飾。 （二）多肽鏈的水解斷裂: 胰島素的修飾過程。Prepro-insulin Pro-insulin insulin Pre-peptideC-peptide圖5. 人胰島素原的分子結(jié)構(gòu)模式圖Fig.5 Sketch of the Structure of Human Proinsulin圖6. 由胰島素原轉(zhuǎn)變?yōu)橐葝u素Fig.6 Proinsulin Changing into

49、 Insulin （三）氨基酸側(cè)鏈的修飾 二硫鍵的形成；羥化作用；氨基酸殘基的交聯(lián)；羧化作用；甲基化等。詳細(xì)內(nèi)容參見下表。 （四）糖基化：生成糖蛋白。膜蛋白和分泌蛋白多為糖蛋白。 （五）脂類對蛋白質(zhì)的共價修飾 (1)在翻譯中，連接肉豆寇酸于N端甘氨酸； (2)在翻譯后，脂肪酸與半胱氨酸、絲氨酸或蘇氨酸側(cè)鏈酯化，以脂酰CoA為供體； (3)在翻譯后，通過乙醇胺將糖基磷脂酰肌醇（GPI）連接于多肽前體的接近C端的氨基酸殘基上，生成與膜結(jié)合的GPI錨定蛋白（GPIanchored protein）。 表. 蛋白質(zhì)生物合成中氨基酸殘基的修飾氨基酸 修飾方式精氨酸 ADP核糖基化；氨基未端甲基化天冬酰胺

50、 ADP核糖基化；糖基化；氨基未端甲基化；羥化作用天冬氨酸 在GPI錨錠蛋白中以酰胺連接于乙醇胺；羥化作用半胱氨酸 二硫鍵形成；脂肪?；饔霉劝彼?羥基化作用；甲基化作用谷氨酰胺 賴氨酸氨基交聯(lián)；氨基末端甲基化；內(nèi)部環(huán)化成氨基末端焦谷氨酸甘氨酸 轉(zhuǎn)變成羥基未端酰胺；氨基未端的肉豆寇?；M氨酸 形成白喉酰胺（dipbthamide），ADP核糖基化；氨基末端甲基化賴氨酸 羥化作用后5羥賴氨酸糖基化；交聯(lián)形成；乙酰化作用甲硫氨酸 氨基未端甲?；鶊F(tuán)脫甲?；饔?；氨基未端甲基化苯丙氨酸 氨基未端甲基化脯氨酸 羥化作用形成3或4羥脯氨酸；氨基未端甲基化絲氨酸 磷酸化；糖基化；脂肪?；辉趖RNA水平上硒

51、代半胱氨酸的形成蘇氨酸 磷酸化作用；糖基化作用；脂肪?；饔美野彼?磷酸化作用；哺乳動物-微管蛋白中羥基未端殘基的交換 （六）ADP核糖基化 （七）乙?；?乙酰化普遍存在于原核和真核生物中。有二種:(1)由結(jié)合于核糖體的乙酰基轉(zhuǎn)移酶催化，將乙酰CoA的乙酰基轉(zhuǎn)移至正在合成的多肽鏈上；（2）翻譯后由細(xì)胞質(zhì)的酶催化發(fā)生乙酰化。 （八）磷酸化 酶、受體、介體及調(diào)節(jié)因子等蛋白質(zhì)的普遍修飾方式。在細(xì)胞生長和代謝調(diào)節(jié)中有重要功能。發(fā)生在翻譯后，由各種蛋白激酶催化進(jìn)行。 （九）C 端酰胺基的引入 （十）酪氨基的硫酸化 真核生物蛋白質(zhì)的酪氨酸硫酸化。 Protein translocation describ

52、es the movement of a protein across a membrane. This occurs across the membranes of organelles in eukaryotes, or across the plasma membrane in bacteria. Each membrane across which proteins are translocated has a channel specialized for the purpose. Two types of Ribosome in cells：Free ribosome and me

53、mbranous ribosome The leader of a protein is a short N-terminal sequence responsible for initiating passage into or through a membrane. Signal sequences are most often leaders that are located at the N-terminus. N-terminal signal sequences are usually cleaved off the protein during the insertion pro

54、cess. 3-1-4 蛋白質(zhì)的跨膜運輸與定位 Figure 8.1 Proteins that are localized post-translationally are released into the cytosol after synthesis on free ribosomes. Proteins that are localized cotranslationally associate with the ER membrane during synthesis. 一、蛋白質(zhì)轉(zhuǎn)位的途徑1. 共翻譯轉(zhuǎn)位（co-translational translocation） Prote

55、ins that are localized cotranslationally associate with the ER membrane during synthesis, so their ribosomes are membrane-bound. The proteins pass into the endoplasmic reticulum, along to the Golgi, and then through the plasma membrane, unless they have signals that cause retention at one of the ste

56、ps on the pathway. They may also be directed to other organelles, such as endosomes or lysosomes. 2. 翻譯后轉(zhuǎn)位（post-translational translocation ） Proteins that are localized post-translationally are released into the cytosol after synthesis on free ribosomes. Some have signals for targeting to organelle

57、s such as the nucleus or mitochondria. Figure 8.3 Membrane-bound ribosomes have proteins with N-terminal sequences that enter the ER during synthesis. The proteins may flow through to the plasma membrane or may be diverted to other destinations by specific signals.（一）Co-translational translocation1.

58、 進(jìn)入內(nèi)質(zhì)網(wǎng)The signal recognition particle(SRP) is a ribonucleo-protein complex that recognizes and binds to signal sequences during translation and guides the ribosome to the translocation channel. SRPs from different organisms may have different compositions, but all contain related proteins and RNAs.

59、Function: It can bind to the signal sequence of a nascent secretory protein. And it can bind to a protein (the SRP receptor) located in the membrane. Signal-SRP binding causes protein synthesis to pause. But protein synthesis resumes when the SRP binds to the SRP receptor in the membrane. Signal pep

60、tidase is an enzyme within the membrane of the ER that specifically removes the signal sequences from proteins as they are translocated. Analogous activities are present in bacteria, archae-bacteria, and in each organelle in a eukaryotic cell into which proteins are targeted and translocated by mean