Chapter 4 Three-Dimensional Structure of Protein

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1 Chap. 4 단백질의입체구조 (Three-Dimensional Structure of Protein)

2 The twenty amino acids found in biological systems are: All amino acids have the same general formula

3 단백질의분류 1) 생물학적기능에의한분류 a) enzyme : 가장종류다양, 생화학적촉매, catalytic activity b) transport protein : 예 ) 혈액 (hemoglobin, O 2 운반 ; lipoprotein, 지방운반 ) c) nutrient & storage : 예 ) ferritin (Fe 저장 ), ovalbumin( 개체부화 ) d) contractile or motile protein : actin or myosin ( 근육 ) e) structural protein : 구조강화및보호 (collagen, keratin...) f) defense : antibody or immunoglobulin, antimicrobial peptides... g) regulatory protein : peptide hormons, 유전자조절관련단백질... h) other proteins : stress proteins (heat shock proteins), anti-freeze... 2) 모양 (shape) 에의한분류 : globular & fibrous proteins 3) 아미노산이외의성분을포함하는단백질 (conjugated proteins) a) glycoprotein : sugar group b) phosphoylated protein : phosphate group b) lipoprotein : lipid c) heme protein : hemoglobin d) metalloprotein : Zn, Cu, Fe...

4 단백질의성질 ⑴ 용해성 : 막단백질을제외한대부분단백질은물과묽은염용액에녹는다. ⑵ 등전점 : 단백질은고유의등전점을가진다. 등전점부근에서가장녹기어렵다. ⑶ 염석 : 단백질의용해도는염류의농도에따라변화한다. 저농도의전해질에서는용해도가커지고 (salting-in), 고농도에서는작아져침전이생긴다 (salting-out, 즉염석 ). ⑷ 유기용매 : 에탄올 아세톤등유기용매에의해침전한다. ⑸ 자외선흡수 : 단백질용액은분자내방향족아미노산잔기의특이적흡수에따라 280nm 부근에서흡수대를나타낸다. ⑹ 발색반응 : 펩티드결합에또는구성아미노산의곁사슬인작용기의반응성에의거하여, 여러발색반응을일으킨다. 이것은단백질의검출이나정량에이용된다. ⑺ 가수분해 : 강한산 (6N 염산 ) 에서가열하면가수분해되어구성성분인 L-α- 아미노산이된다. 아미노산의조성을측정. ⑻ 변성 : 1 차구조는변화되지않고고차구조만이파괴. 천연단백질과는다른물성. 변성의원인 : 극단적인알칼리성, 산성, 유기용매 중금속염 요소염산구아니딘과같은변성제 계면활성제, 가열 동결 교반 희석 흡착 자외선 X 선 고압 초음파등의물리적인자. 생물활성의감소또는상실. ⑼ 면역 : 천연단백질은항원성을갖는다. 다른종의동물조직에주입하면, 동물은면역되어그단백질 ( 항원 ) 에대해특이적항체를형성한다. 항체는항원이외의단백질과는반응하지않으므로단백질의미량검출에이용한다.

5 겸상적혈구빈혈증 (sickle-cell anemia) : 비정상단백질에의한질병의예 정상 비정상 단백질일차구조의변화 : 악성빈혈 b chain mutation : E6 V6 (Hb 의중합 distortion)

6 Peptide Bonds amino group side chain carboxyl group

7 Levels of Description

8 Levels of Description

9 Secondary Structure secondary structure refers to certain common repeating structures it is a local description of structure 2 common secondary structures a helices b strands a 3rd category, called coil or loop, refers to everything else

10 단백질의구조 : 1,2,3 & 4 차구조 1 차 : 단백질에서아미노산의배열순서 N- 말단에서 C- 말단으로읽음 2 차 : 단백질의일부아미노산의반복적구조. 수소결합에의한공간적배열. ( 알파나선, 베타병풍, 턴 ); 초이차구조 : 이차구조의조합 3 차 : 단백질구성아미노산전체의삼차원구조 ( 단량체 ); 4 차 : 중합체구조 ( 다량체 )

11 단백질의이차구조 (Secondary Structures): a-helix -pitch (0.54 nm=5.4å) : the distant to complete one turn -rise (0.15 nm= 1.5 Å): the distant between one aa to another -약 3.6 residues/ one turn (5.4Å/3.6aa=1.5Å/aa)

12 비교 : DNA helix p-0.54 nm R-0.15 nm DNA helix -pitch (3.40 nm) -rise (0.33 nm) -diameter of helix (2.37 nm) -major groove & minor groove DNA helix >> protein helix DNA protein

단백질의이차구조 (Secondary Structures): b-sheet -anti-parallel: The hydrogen bonds are essentially perpendicular to the beta strands, and the space between hydrogen-bonded pairs is alternately wide and

13 단백질의이차구조 (Secondary Structures): b-sheet -anti-parallel: The hydrogen bonds are essentially perpendicular to the beta strands, and the space between hydrogen-bonded pairs is alternately wide and narrow 14.0Å / 2 residues = 7.0Å / residue -parallel : The hydrogen bonds are evenly spaced but slanted 13.0Å / 2 residues = 6.5Å / residue N C N N C N C C Anti-parallel ( 역평행병풍 ) Parallel( 평행병풍 )

14 a-helix 나 b-sheet 구조에서나타나는아미노산 2차와 3차구조예측가능 a-helix : E, M, A b-sheet : V, Y, I b-turn : P, G, N, D, S (P : helix breaker)

15 Secondary Structure Prediction given: an amino-acid sequence do:predict a secondary-structure state (a, b, coil) for each residue in the sequence KELVLALYDYQEKSPREVTMKKGDILTLLM... cccbbbbcccccccccccccbbbbccccccbbbbbb...

16 Secondary Structure Prediction one common approach: make prediction for a given residue by considering a window of n (typically 13-21) neighboring residues learn model that performs mapping from window of residues to secondary structure state KELVLALYDYQ EKSPREVTMKKGD ILTLLM... b

17 Ramachandran plot : phi and psi angles 로존재가능한 2 차구조예측 f Y - phi ( f ) : rotation about Ca-N bond ; psi ( Y ) : rotation about Ca-C bond

18 b-turn : compact 한구조형성에중요 Turns : 4개 AAs (AA1-AA2-AA3-AA4) AA1--AA4 : H-bonded -type I : Pro can not be at 3rd position -type IIa : 2, 3번아미노산이주로 Gly -type IIb : 2(Pro), 3(gly) -type III : any aa Loop : 7 개 AAs

19 단백질의구조적세부단위 : motif 초이차구조 (supersecondary structure) motif 는주로다음구조들에의해형성 - helix-loop-helix (ala) - bab unit - hair-pin - Greek key Supersecondary structure (motifs, domain): small, discrete, commonly observed aggregates of secondary structures

20 단백질의기능적세부단위 : domain (motif 의조합 ) Domain : independently folded globular units (DNA 결합, 단백질결합 ) - 25 AAs ~ 300 AAs ( 평균적으로약 100 아미노산 ) 예 ) DNA binding, leucine zipper, transcriptional activaiton domains... Leucine Zipper domain : GCN4

21 단백질삼차구조와삼차구조를안정시키는힘 삼차구조 : 단백질의곁사슬 (sidechain) 과보결원자단 (prosthetic group) 을포함하는모든원자의삼차원적배열 ( 단량체 ).

22 단백질의 4 차구조 : 중합구조 subunit의중합체형성방법에따라분류 a) reversible vs irreversible association irreversible : A+A..+ A (n개) ----> An 비가역적이어서해리되지않는경우. ex) TNV reversible : A+A+A+...(n개 ) <---> An ( 가역적 ) (Keq = Ka/Kd) Go = - RT ln Keq b) homogeneous vs heterogeneous association b1) homogeneous : A+A+A+...(n개 ) <---> An b2) heterogeneous : A+B+C+D <--->ABCD

23 Quaternary (4 ) structure : examples Globular Protein Subunits Alcohol de hydrogenase 2 Triose phosphate isome rase 2 Aldolase 3 He moglobin Lactate dehydrogenas e 4 Pyruvate kinase 4 Insulin 6

24 단백질의형태상분류 섬유상단백질 (Fibrous protein) :asymmetric (extended st.) 폴리펩티드사슬이긴섬유상또는평면상으로배열된단백질. 예 ) a-keratins : a-helix b-keratins : b-sheet Collagen : triple-stranded a-helix 구형단백질 (Globular Protein) : compact 예 : Hb, Mb

S, A rich-- G 가사이사이배열 ) b-sheet (paralled & anti-parallel ) Collagen :(tendons, connective

25 Fibrous protein a-keratins ( 모발, scales, horns, nails, claw...) single-helix : R-handed multiple(triple)-helix : L-handed twist b-keratins : ( silk : G, S, A rich-- G 가사이사이배열 ) b-sheet (paralled & anti-parallel ) Collagen :(tendons, connective tissue ; Gly-X-Y 반복구조 (X=Pro, Y=HO-proline)) triple-stranded a-helix (R-handed twist (inter-strand H-bond ) single-helix: L-handed helix

26 Hydroxyproline 합성 : 비타민 C 의역활 약한콜라겐 괴혈병 강한콜라겐 ( 수소결합 ) 정상인

27 괴혈병환자의치아

28 이 : 구조 1. 치관 : 잇몸에서돌출한보이는부분의이 2. 뿌리 : 잇몸에묻혀있는부분의이 3. 목 : 치관과잇뿌리사이의부분 4. 법랑질 ( 에나멜질 ) : 치관의상아질을싸고있는백색의치밀하고단단한물질 5. 상아질 : 뼈와유사한이의주된부분 ( 콜라겐 ) 6. 백악질 : 치근을피복한골질과같은경도의결합조직 7. 치수 : 혈관과신경을포함하는부드러운조직

29 비타민 C 결핍증 : Scorbutic Pose

30 비타민 C 결핍증 : Hemorrhages

31 Vitamin C 공급원

32 Vitamin C 공급원

Linus Carl Pauling 28. Feb. 1901-19. August 1994 Linus Pauling, Ph.D.

33 Linus Carl Pauling 28. Feb August 1994 Linus Pauling, Ph.D., was the only person ever to win two unshared Nobel prizes. He received these awards for chemistry in 1954 and for peace in 말년엔비타민 C 가감기와암등을예방한다는신념으로연구함.

34 Globular Protein - compact - primary structure가 3 구조결정 (tertiary structure) **Cris Anfinsen 실험 ** - Hydrophobic interaction 이중요 - a-helix등의소수성면은물에노출을줄여 2 layer 이상형성경향 - secondary valence bond 가중요 ( 공유결합이아닌다른결합들 ) -- 정전기적결합, 이온-이온, 이온-D, D-D 결합, -- van der waals 인력과척력, H-bond. - b-turn :compact한구조형성에중요 - 아미노산의 chirality가 folding pattern 결정 - a-helix : 주로 R-handed helix

NH2-CO-NH2 : 수소결합파괴 Guanidine hydrochloride( 구아니딘염산염 ) : NH2-C=NH2(+)-NH2

35 단백질변성 (protein denaturation) b-mercaptoethanol (HS-CH 2 -CH 2 -OH), DTT(dithiothreitol ), TCEP : 환원제 (tris(2-carboxyethyl)phosphine) Urea ( 요소 ): NH2-CO-NH2 : 수소결합파괴 Guanidine hydrochloride( 구아니딘염산염 ) : NH2-C=NH2(+)-NH2 (Cl-) : 수소결합파괴 SDS (sodium dodecyl sulfate, H3C-(CH2)10-CH2OSO3-Na+ ) : anionic detergent

36 Anfinsen 의실험 : 단백질의변성과복구 가설 : 단백질의 folding( 접힘 ) 은 1 차구조의정보에의해 자발적으로일어나며 cooperative 하게일어난다. Anfinsen 실험 (1972 노벨상 ) ME(mercaptoethanol - 단백질의 s- s 결합제거 ) 처리후 - unfolding, 다시 folding 과정을거칠때 1) 요소가있을때 2) 요소가없을때 서로다른결과를얻었음. 요소에의해단백질의 folding 과정이방해받아원래대로 folding 하지못해활성이심하게작아짐 ribonuclease A (124 AA) Christian B. Anfinsen

37 Anfinsen s thermodynamic hypothesis The 3D structure of a protein in its native environment is the one in which the Gibbs free energy of the whole system is the lowest. sequence determines structure : But this is not always true! Anfinsen, C.B. Principles that govern the folding of protein chains. Science 181, (1973).

38 Anfinsen 이후 : 열충격단백질에의한 active refolding 단백질접힘도우미 단백질의 folding( 접힘 ) 은자발적뿐아니라경우에따라선 chaperone 단백질의도움으로일어난다. -ATP dependent active refolding -GroEL, hsp 70 (heat shock protein 70)... - 열충격단백질 / 열충격전사활성인자

$diffraction 방법에의한단백질구조분석 ( 결정-고체구조 ) 스펙트럼의최대 spaceal$ resolution은입사광파장의약 1/2 이다.

39 단백질의구조결정기술 : NMR and X-ray crystallography a) X-ray diffraction 방법에의한단백질구조분석 ( 결정-고체구조 ) 스펙트럼의최대 spaceal resolution은입사광파장의약 1/2 이다. - light microscopy : 가시광영역 source, 5000 Å - X-ray source (cupper line) : 1.54 Å 비교 : 수소결합의거리 : 약 2.7 Å b) NMR : 용액상

40 Allosteric property Allosteric property ( 다른자리입체성 ) : Binding of substrate to one site stimulates the binding of substrate to another site on multi-subunit Protein. - 주로 oligomeric structure 에서나타남. 예 ) 헤모글로빈 (hemoglobin, Hb, 4 량체 ) 마이오그로빈 ( 단량체 ) no allosteric p.

156 AAs < Stereo view of the hemoglobin tetramer.

41 hemoglobin (Hb) 과 myoglobin (Mb) a) hemoglobin (Hb) : 적혈구내존재, 산소 / 이산화탄소운반, allosteric 사량체 (4X) : (α1 β1 α2 β2); α = 141 AAs, β = 146 AAs b) myoglobin (Mb) : 근육에존재, 산소저장, 단량체, non allosteric, 156 AAs < Stereo view of the hemoglobin tetramer.> Color key: alpha1, purple ; alpha2, green ; beta1, yellow ; beta2, blue; heme groups, red Stereo view of myoglobin. > The heme group is red

42 헴기구조 헴기결합산소 / 이산화탄소 25,000x 100x

43 Hb : deoxy vs oxy forms deoxy Hb oxy Hb

44 Hb 와 Mb 의 O 2 결합곡선 Hb: -sigmoidal shape (s 자곡선형 ), Hb + no 2 <--> HbO 2 K a = [HbO 2 ]/[Hb][O 2 ] n ; Y = K a [O 2 ] n /(1+K a [O 2 ] n ) ** Y : 평균결합수 - cooperative (allosteric, Hill 상수, n >1), - 적은농도변화 (DC) 에결합력이민감히변화함.

45 Hb 와 Mb 의 O 2 결합곡선 Mb: -hyperbolic shape( 쌍곡선형 ) Mb + O 2 <--> MbO 2 K a = [MbO 2 ]/[Mb][O 2 ]' Y = K a [O 2 ]/(1 + K a [O 2 ]) = [O 2 ]/(k d + [O 2 ]) -non cooperative (non allosteric, n=1) - 모든산소압력에서 Hb 에비해산소에대한결합은강하나, 농도변화에민감하지않음.

46 Hb 에의한산소와이산화탄소의전달

47 Hb / O 2 결합에영향을주는인자 1) Bohr effect ; The negative influence of H + and CO 2 on O 2 binding CO 2 ph ph 효과 : ph 7.6 : 40 mmhg O 2 : HbO 2 80% ; ph 6.8 : 40 mmhg O 2 : HbO 2 45% CO 2 효과 : 체내 : CO 2 + H 2 O <-> H 2 CO 3 (slow process ; t 1/2 ~ 10 초 ) 적혈구 : (carbonic anhydrase 에의해반응속도가빨라진다.) CO 2 + H 2 O <--> H 2 CO 3 ---> H+ + HCO 3-2) BPG- 2,3-bisphosphoglycerate ( 적혈구내약 5mM 존재 ) GBP 의 Hb 결합력 a) HbO2 : K d = 10-3 M -1, K a =10 3 b) HHb+ : K d = 10-5 M -1, K a =10 5 : BPG 구조 - deoxy Hb form 에 GBP 결합시, O 2 의 Hb 결합방해 :

48 Hb pocket BPG (electrostatic interaction) BPG Effects

49 태아 (HbF) 와모체 Hb 의산소결합력곡선 α1 β1 α2 β2 모체 (Hb) α1 g1 α2 g2 태아 (HbF) -BPG : 덜강하게결합 (O 2 결합력큼 ) - 구조적이유 o (α1 g1 α2 g2) O (+) 하전적음 - 인력작음 태아 > 모체

The Prion Diseases Prion : 28kDa protein 양 -scrapie; 소 - 광우병사람 : Creutzeldt-Jacob 병 Prions, once dismissed as an impossibility, have now gained wide recognition as extraordinary agents

50 The Prion Diseases Prion : 28kDa protein 양 -scrapie; 소 - 광우병사람 : Creutzeldt-Jacob 병 Prions, once dismissed as an impossibility, have now gained wide recognition as extraordinary agents that cause a number of infectious, genetic and spontaneous disorders. Stanley B. Prusiner The Nobel Prize in Physiology or Medicine 1997 UCSF 정상프라이온 (PrP : 나선구조 ) 비정상프라이온 (PrP SC : 병풍구조 )

51 4 장끝

52 4 장 Problems: 1. 다음조건에서단백질의 4차구조를예측 ( 그려보시오 ) 하시오. 용매 분자량 완충용액 200,000 6M GdnHCl 100,000 6M GdnHCl +100mM beta-mercaptoethanol 75,000+25, a)a 단백질의 280 nm 에서의흡광도는 0.80 이고농도는 0.50ug/ml 라면 A 단백질의몰흡광상수는? 1cm pathlength cell was used. 단단백질은단량체이고단백질의분자량은 40,000 이다. b) 만약단백질이수용액상에서 homodimer 라면이때의 A 단백질의몰흡광상수는?

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PowerPoint Presentation Amino Acids, Peptides, & Proteins Amino Acids Amino acid classes Biological activities Titration of Amino acids Amino acids reaction Peptides Proteins structure 단백질의 3 차원구조는 peptide bond 에의해결합된 Aa 서열에의해결정되며