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Class Schedule, Fall, 2014 Myoglobin & Hemoglobin 생체분자의구조와작용 lect ure Topic 17 화학의기본이론 Counting and Detecting Substances 18 화학의기본이론 Science of Heat, Thermochemistry Science of 19 화학의기본이론 Bonding and

1 Class Schedule, Fall, 2014 Myoglobin & Hemoglobin 생체분자의구조와작용 lect ure Topic 17 화학의기본이론 Counting and Detecting Substances 18 화학의기본이론 Science of Heat, Thermochemistry Science of 19 화학의기본이론 Bonding and Chemical Structure Chemistry 20 화학의기본이론 Concept of the Quantum Theory 21 생명현상과화학 Hemoglobin, Myoglobin, Enzymes 22 생명현상과화학 Medicines 23 생활과화학1 Food and related 24 생활과화학2 Special Materials 25 생활과화학3 TBA는이번학기에파라벤으로정하려고함. 26 우주의구성물질 Building(or unbuilding) blocks of the Universe 27 특별사항 Reading Material(TBA) 28 특별사항 Reading Material(TBA) 29 화학과미래세계 The future near and far away 30 화학이란무엇인가?-II 31 Review 32 Final Exam(45%) Title 공부할내용 아미노산 아마노산, 펩타이드결합, 단백질 단백질의구조 1 차, 2 차, 3 차, 4 차 단백질의기능 Myoglobin - 산소운반단백질 Hemoglobin 또다른차원의산소운반단백질 낫세포빈혈증 H amino group H N + H 아미노산의기본구조 Hydrogen H C R C carboxyl group a-carbon 아미노산에서처럼 (-) 전하와 (+) 전하가동시에존재하는중성분자를쯔비터이온 (zwitterion) 이라한다. 아미노산은산으로서, 동시에염기로서작용할수있으므로양쪽성이온 (amphoteric ion) 이다. R-group R= 비극성 비극성아미노산 R= 극성 ( 수소결합자리가있는 R: -H or -NH) 극성아미노산 R-group R= 음이온 (-C - ) 산성아미노산 R= 양이온 (-NH 3+ ) 염기성아미노산 비극성아미노산 극성아미노산 1

아미노산은입체이성질체 (stereoisomer) 를갖는다 원자의공간상배열의차이 다른분자 입체이성질체 (stereoisomer = optical isomer = enantiomer) 이런성질을손대칭 ( 키랄성 ) 이라한다

두개의거울상이성질체 펩타이드결합의형성 펩타이드결합 ( 축합반응의일종 ) carboxyl group 과 amino group 이결합하여 Peptide bond 가형성 물분자가떨어진다.

* H 2 : H from carboxyl group H from amino group 아미노산과펩타이드결합 Polypeptide 사슬 Gly 의 amino 기 H 2 N Gly Met Glu Cys Tyr CH N

2 아미노산은입체이성질체 (stereoisomer) 를갖는다 원자의공간상배열의차이 다른분자 입체이성질체 (stereoisomer = optical isomer = enantiomer) 이런성질을손대칭 ( 키랄성 ) 이라한다 생체내에존재하는아미노산은주로 L 형이다. Cf. 생체내에존재하는당은주로 D 형이다. 두개의거울상이성질체 펩타이드결합의형성 펩타이드결합 ( 축합반응의일종 ) carboxyl group 과 amino group 이결합하여 Peptide bond 가형성 물분자가떨어진다. D 형아미노산으로인공합성된단백질은단백질을분해하는 protease 가분해할수없다. L 형단백질만이생리활성을갖는다. * H 2 : H from carboxyl group H from amino group 아미노산과펩타이드결합 Polypeptide 사슬 Gly 의 amino 기 H 2 N Gly Met Glu Cys Tyr CH N terminal (N 말단 ) C terminal (C 말단 ) 펩타이드기술법 : N-terminal 은왼쪽, C-terminal 은오른쪽에 cf. mrna 로부터 peptide 의합성순서도 N-terminal 에서 C-terminal 순서로합성됨 예 ) Met Gly Glu ---- Val 이라면 Met 의 N 말단이 free, Val 의 C 말단이 free, 합성은 Met 부터 Val 까지순서로 Polypeptide 사슬 Polypeptide 사슬 Polypeptide Backbone R 기는교대로반대편에위치한다 2

단백질의구조 아미노산서열분석 1 차구조 (primary structure): 아미노산의서열 2 차구조 (secondary structure): polypeptide

(quaternary structure): 2 개이상의 polypeptide 들이결합해서단백질을만드는구조 Step 1: S-S 결합절단 perforic

Carboxypeptidase로 C-말단분해 분석 Step 4: Fragmentation 특별한 aa sequence를끊는효소를사용하여작은 peptide로분해예 )

Step 6: rdering of the fragment sequences 단백질구조분석 X-ray Crystallography 를이용하여특이한결정구조분석 1 차구조

Example the enzyme lysozyme: 1 2 3 4 5 126 127 128 129 Lys-Val-Phe-Gly-Arg.

3 단백질의구조 아미노산서열분석 1 차구조 (primary structure): 아미노산의서열 2 차구조 (secondary structure): polypeptide 의국부적접힘구조 (α-helix, β-sheet 구조가이에속한다 ) 3 차구조 (tertiary structure): polypeptide 의 3 차원구조 4 차구조 (quaternary structure): 2 개이상의 polypeptide 들이결합해서단백질을만드는구조 Step 1: S-S 결합절단 perforic acid로산화시킨다 Step 2: N-terminal 결정 Sanger method 사용하여 N-말단분해 분석 Step 3: C-terminal 결정 Carboxypeptidase로 C-말단분해 분석 Step 4: Fragmentation 특별한 aa sequence를끊는효소를사용하여작은 peptide로분해예 ) trypsin: Lys, Arg의 C 절단 Step 5: Sequencing of fragment Edman degradation에의해 N-말단부터하나씩절단 분석 Step 6: rdering of the fragment sequences 단백질구조분석 X-ray Crystallography 를이용하여특이한결정구조분석 1 차구조 Sequence of amino acids in a protein connected via peptide linkage. Example the enzyme lysozyme: Lys-Val-Phe-Gly-Arg...Gly-Cys-Arg-Leu Note: By convention, amino acid sequences are written starting with the amino terminus. 2 차구조 2 차구조 secondary structure Regular patterns of relatively small segments of a protein held together mainly by H-bonds a-helix 구조 Examples: -sheet 구조 α- helix -structure 수소결합 3

Supersecondary structure 단백질에서의대표적 domain

unit -barrel( 원통 ) unit Greek key ( 격자무늬 )

에결합하는단백질에존재 3 차구조 Tertiary Structure verall

Two basic types are globular and fibrous.

열역학적으로더안정하니까 예 : Globular (Pepsin) Fibrous

소수성상호작용 (2) 정전기적상호작용 (3) 수소결합 (4) 공유결합 : 예,

4 Supersecondary structure 단백질에서의대표적 domain Leu 의 R -a- unit -meander ( 꾸불꾸불 ) unit a-a unit -barrel( 원통 ) unit Greek key ( 격자무늬 ) unit EF hand Helix-loop-helix 구조 Ca binding site Zn finger Zn binding site DNA 에결합하는단백질에존재 Leucine zipper 지퍼모양 DNA 에결합하는단백질에존재 3 차구조 Tertiary Structure verall 3-D shape of a protein. Two basic types are globular and fibrous. 단백질의구조와안정성 왜 3 차원구조를갖는가? 열역학적으로더안정하니까 예 : Globular (Pepsin) Fibrous (Collagen) 3 차구조를만드는 ( 유발시키는 ) 힘 (1) 소수성상호작용 (2) 정전기적상호작용 (3) 수소결합 (4) 공유결합 : 예, S-S bond 3 차구조를유지하는상호작용 4 차구조 Quaternary Structure verall 3-D shape of a multi-subunit protein Example: Rabbit muscle glycogen phosphorylase 4

5 4 차구조 3차구조가 4차구조로발전하는것은분자간힘 cf. 1차구조 : 공유결합, 2/3차구조 : 분자내상호작용, 4차구조 : 분자간힘 4차구조의장점 (1) 긴 polypeptide를합성하는것보다짧은것을여러개합성하는것이유리 (2) 초거대분자 ( 예, collagen) 에서손상부분은일부교체가가능 (3) 복잡한소단위의상호작용을이용한효소활성제어 ( 예, allosteric enzyme) 단백질의변성 (denaturation) 단백질의 denaturation : 3 차구조의변화 (1 차구조는보통불변 ) 기능상실 Denaturation 의원인 1. 강산 / 강알칼리 : ph 에따라전하변화 정전기인력변화, pi 부근에서는침전 2. 유기용매 : 소수성상호작용파괴 3. 세제 : 세제는 amphipathic 이므로단백질을풀어지게함 4. 환원제 : S-S bond 파괴 5. 염농도 : - 소량의염 : 섬유상단백질의경우불용성 염첨가 -> 정전기적인력제거 용해도증가 ( 염용 : salting in) - 고농도의염 : 단백질주위의 solvation sphere 제거 단백질응집 ( 염석 : salting out) 6. 중금속이온 : 중금속은단백질의음전하와결합 정전기적인력변화 7. 온도변화 : 온도상승 단백질분자운동증가 H-bond, 정전기적인력등파괴 단백질풀어짐 8. 기계적스트레스 : shear stress 증가 약한결합력파괴 변형 ( 예 ) 계란의흰자를저으면? Anfinsen 실험 : 가역적변형 1. 촉매작용 : 모든생화학반응촉진 단백질의기능 2. 구조적기능 : 생체의구조체예 ) 콜라겐, 피브로인 (fibroin: 실크단백질 ), elastin ( 혈관, 피부조직 ) 3. 운동기능 : 운동에관여예 ) 세포골격 (cytoskelaton) 단백질 :actin,tubulin 세포분열, 세포내이입등에역할 4. 방어기능 : 세포보호능력예 ) keratin( 상처시조직보호 ), fibrinogen( 혈액응고 ), immunoglobin( 면역체 ) 5. 조절기능 : 조절능력예 ) 호르몬 (ex, insulin), 성장인자 (growth factor) 6. 운반기능 : 수송능력예 ) 이온 / 포도당운송세포막단백질, 헤모글로빈 ( 산소수송 ) 즉, 1 차구조 ( 아미노산서열 ) 는 3 차원구조를결정된다. 7. 저장기능 : 필수영양소저장예 ) ovalbumin( 새알의알부민 ), casein( 포유동물의유기질소공급원 ) 8. 스트레스반응 : 내스트레스기능예 ) heat shock protein(hsp, 열손상단백질의분해촉진 ), metallothionein(cys 가풍부한단백질로 Cd, Hg 와결합 무독화 ) DNA repair protein( 손상된 DNA 를수선하는단백질 ) Protein: The Machinery of Life 산소운반단백질 NH 2-Val-His-Leu-Thr-Pro-Glu-Glu- Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp- Gly-Lys-Val-Asn-Val-Asp-Glu-Val- Gly-Gly-Glu-.. Myoglobin Exhibits Michaelis-Menten properties Hemoglobin Exhibits allosteric properties 5

tissues arterial pressure Myoglobin In 1958, John Kendrew and

Myoglobin Single polypeptide with 154(human) amino acids C 774

$cardiac muscle Diffraction pattern of myoglobin by X-ray$ crystallography [high] in diving mammals like whale & seals

고래의근육에많이존재, 장시간잠수가능 ) Myoglobin has high affinity for 2.

만약, Fe 3+ (hematin) 로비가역산화되면 hematin 은산소결합불가

= 2.8 Torr Allows myoglobin to act as 2 storage reserve.

6 tissues arterial pressure Myoglobin In 1958, John Kendrew and associates determined the structure of myoglobin 마이오글로빈 Myoglobin Single polypeptide with 154(human) amino acids C 774 H 1224 N S 5 17,183.8 daltons(human) 8 a helices (A-H) Located in skeletal & cardiac muscle Diffraction pattern of myoglobin by X-ray crystallography [high] in diving mammals like whale & seals 마이오글로빈 Myoglobin 2 결합곡선 마이오글로빈은골격, 심장에존재하여산소전달역할을하는단백질 ( 예, 고래의근육에많이존재, 장시간잠수가능 ) Myoglobin has high affinity for 단일펩타이드로구성 중앙에 heme 구조 자유 Fe 2+ 는 2 에강한친화력 Cf. 만약, Fe 3+ (hematin) 로비가역산화되면 hematin 은산소결합불가 마이오글로빈은중앙에소수성그룹존재하여산소친화력약화 Fe 2+ 의산화를방지 Fe 2+ 와산소의가역적결합유도 P 50 = 2.8 Torr Allows myoglobin to act as 2 storage reserve. Releases 2 when p 2 becomes low indicating 2 deprivation. saturation with p 2 (partial pressure of 2 ) (Torr) Heme Prosthetic Group 헴보결분자단 Heme (Fe 2+ ) has affinity for 2. N N Fe N Hematin (Fe 3+ ) cannot bind 2. H N Located in crevice where it is protected from oxidation. 6

마이오글로빈의헴과리간드결합 마이오글로빈의산소결합 먼 His-N 은산소결합을안정화 2

atom. distal histidine His 93 (proximal his)

가까운 His-N 은 heme 의 Fe 2+ 와직접결합 His 64 (distal

7 마이오글로빈의헴과리간드결합 마이오글로빈의산소결합 먼 His-N 은산소결합을안정화 2 binds to only available coordination site on iron atom. distal histidine His 93 (proximal his) binds directly to iron. 가까운 His-N 은 heme 의 Fe 2+ 와직접결합 His 64 (distal his) stabilizes the 2 binding site. proximal histidine 마이오글로빈 -C 결합 적혈구 Red Blood Cell C binds tightly; linear. 2 binds less tightly, bent structure. Distal His forces bent binding of both, weakens C binding. C Fe Fe Model Molecule: Hemoglobin 헤모글로빈 (hemoglobin) 헤모글로빈은폐속의산소를전달역할 총 4 개 (α-chain 2 개, β-chain 2 개 ) 의폴리펩타이드로구성 (4 차구조 ) a 2 2 Two α (141 AA/ α)subunits and two β (146 AA/ β)subunits 7

Heme 산소결합곡선 Hb 는산소 1 개결합하면결합이더욱증가 정맥압 동맥압 Hb

polypeptide chain resembles myoglobin tertiary

Invariant residues indicate importance of those

2,3-bisphosphoglycerate (BPG) 증가 : 해당과정에서발생하는음전하분자

8 Heme 산소결합곡선 Hb 는산소 1 개결합하면결합이더욱증가 정맥압 동맥압 Hb 의산소해리를촉진하는인자 헤모글로빈구조 왕성한세포는산소많이필요 어떻게산소전달을촉진할까? 왕성 산소소모 C 2 발생?? Hb 의산소해리를촉진하는인자 1. C 2 증가 H +, HC 3- 증가 H + 가음이온그룹에결합 deoxy Hb (H + binding site 많다 ) 로전환 이과정에산소는해리됨 2. ph 감소 H + 증가 산소해리증가 (ph effect = Bohr effect) Each polypeptide chain resembles myoglobin tertiary structure but 1 sequence varies. Invariant residues indicate importance of those residues in function. 3. 2,3-bisphosphoglycerate (BPG) 증가 : 해당과정에서발생하는음전하분자 BPG 증가 deoxy Hb 전환 산소해리증가 Allosteric Transition of Hb Hb exhibits + cooperativity. 헤모글로빈 2 결합 Water bound to heme Iron Eaton et al. Nature Struct. Biol. 1999, 6, 351 8

헤모글로빈 2 결합 xygen bound to heme Iron Hb T-state deoxy Hb 변이종 HbA 2 a 2 d 2 Present in ~2% of

for 2 http://oregonstate.edu/instruction/bb450/stryer/ch10/slide27.

com/horton/medialib/media_portfolio/text_images/fg04_50.

Stabilizes T state and promotes unloading of 2 to active tissues.

9 헤모글로빈 2 결합 xygen bound to heme Iron Hb T-state deoxy Hb 변이종 HbA 2 a 2 d 2 Present in ~2% of adults Embryonic Hb a 2 e 2 Has affinity for 2 Hb R-state - oxy Fetal Hb a 2 g 2 Has affinity for 2 Bohr 효과 2, 3-Bisphosphoglycerate C 2 ph Some side groups remain protonated at lower ph. Stabilizes T state and promotes unloading of 2 to active tissues. Binding of C 2 also stabilizes T state. C 2 binds to a amino groups. Stabilizes deoxyhb (T state) Facilitates unloading of 2 in tissue. - - P - - P 2, 3-bisphosphoglycerate - saturation with BPG + BPG p 2 (partial pressure of 2 ) (Torr) 9

2,3-BPG Binding to Hb High Altitude and BPG At higher altitudes, the [BPG] increases allowing Hb to unload 2 more easily. http://oregonstate.edu/instruction/bb450/stryer/ch10/slide26.

If BPG depleted blood is used for a transfusion, the R state Hb doesn t release 2. Add inosine to stored blood to maintain BPG levels. C Poisoining C is competitive inhibitor of 2.

Sickle Cell Anemia 낫세포빈혈증 Normal red blood cells are round like doughnuts, and they move through small blood tubes in the body to deliver oxygen.

10 2,3-BPG Binding to Hb High Altitude and BPG At higher altitudes, the [BPG] increases allowing Hb to unload 2 more easily. Stored Blood & BPG 2,3-BPG becomes depleted in stored blood, so R state of Hb is stabilized. If BPG depleted blood is used for a transfusion, the R state Hb doesn t release 2. Add inosine to stored blood to maintain BPG levels. C Poisoining C is competitive inhibitor of 2. Affinity is 200X greater than that of 2. C also inhibits unloading 2 of in tissues. Sickle Cell Anemia 낫세포빈혈증 Normal red blood cells are round like doughnuts, and they move through small blood tubes in the body to deliver oxygen. The origin of the disease is a small change in the protein hemoglobin The change in cell structure arises from a change in the structure of hemoglobin. Sickle red blood cells become hard, sticky and shaped like sickles. When these hard and pointed red cells go through the small blood tube, they clog the flow and break apart. This can anemia. A single change in an amino acid causes hemoglobin to aggregate. a 10

Differences in Red Blood Cells Scanning electron microscopic image of Red

structure caused by the single mutation Normal mrna Normal protein Mutant

thr pro glu glu lys 1 2 3 4 5 6 7 8 Mutation (in DNA) GUG CAC CUG ACU CCU

(glu), a negatively charged amino acid, is replaced by valine (val), which

11 Differences in Red Blood Cells Scanning electron microscopic image of Red bllod cells Hemoglobin S Sickle Cell Hemoglobin Significant change in structure caused by the single mutation Normal mrna Normal protein Mutant mrna Mutant protein 낫세포헤모글로빈 GUG CAC CUG ACU CCU GAG GAG AAG val his leu thr pro glu glu lys Mutation (in DNA) GUG CAC CUG ACU CCU GUG GAG AAG val his leu thr pro val glu lys Glutamate (glu), a negatively charged amino acid, is replaced by valine (val), which has no charge. at 6β -CH 2 -CH 2 -CH -CH-(CH 3 ) 2 L-Glutamic acid (Glu/ E) L-Valine (Val / V) 11

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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