Protein foldingProtein folding

James Dewey WasonFrancis Harry Compton Crick

DNA RNA Proein

?

中国启动人类肝脏蛋白质组计划国际人类蛋白质组计划的２０％以上的任务。

proteomics

为什么要开展蛋白质折叠的研究 ?

研究的源动力

disease

免疫 病毒

Protein folding & live

蛋白质研究先驱

吴 宪 1893,11,24---1959,8,8

１９１２年到美国麻省理工学院。因愤于我国海军落后，初学造船工程。因受赫胥黎“生命的物理基础”一文的影响，２年后改习化学。１９１６年毕业，获理学学士学位，留校任化学系助教。１９１７年进哈佛大学医学院生物系，成为美国著名生物化学家 Folin 教授的研究生，进行血液化学研究。1924 年起用各种方法使蛋白质变性，1931 年得出如下理论：蛋白质的变性是由于蛋白质分子由折叠而变为舒展。

变性剂巯基乙醇

复性

ribonuclease

Some denatured proteins

can be renatured

denatured molecule

Anfinsen 原理 1961

Probability that correct folding would occur in ribonuclease

given that there are 8 cysteine residues 1/7 x 1/5 x 1/3 x 1 = 1/105

expected activity ~ 1%

observed activity was 100%

• if one conformation is explored every 0.1 psec, then time to refold (t) = 1087 sec

• 2n torsion angles can have 32n ~ 10n

possible conformations

• directed pathways of folding must exists

• if n =100, then number of conformations, 10100

Mechanisms to explain re-folding Factors driving protein folding

Unfolded state

Formation of elements of 2-stru.

Framework Framework modelmodel

Folded con.

Assembly of 2-stru.

gabcdefgabcdefgabcdefgabcdefgabcdSTHMKQLEDKVEELLSKNYHLENEVARLKKLVGER

GCN4 leucine zipper

CD spectra of GCN4 leucine zipper in the presence of different concentrations of SDS

SDS

4 M GuHCl

Changes of ellipticity at 222 nm in the presence of different concentrations of SDS

0

0. 2

0. 4

0. 6

0. 8

1

0 0. 2 0. 4 0. 6 0. 8 1[SDS] (mM)

Native gel electrophoresis of leucine zipper treated with SDS of different concentrations

Lane 1 was the native leucine zipper peptide (control); lanes 2- 6 were samples treated with 0.1, 0.2, 0.3, 0.6, and 1.0 mM SDS

some lead straight downhill

Energy surfaces to visualize protein folding pathwaysA a more realistic energy landscape

the protein is funneled towards a native statemany pathways are possible

others may lead to energyminima that delay proper folding

Changes of fluorescence emission spectra of Tg denatured in various concent

rations of GuHCl

0

10

20

30

40

50

60

310 320 330 340 350 360 370 380Wavel ength (nm)

Inte

nsit

y

1

6

5

4

32

0.0 1.0 2.0 3.0 4.0 5.0 6.0[Gnd-HCl] (M)

0.0

0.2

0.4

0.6

0.8

1.0

Cha

nges

of e

miss

ion

max

imum

Thyroglobulin

ANS binding characteristics of Tg in various GuHCl concentrations

0

5

10

15

20

400 450 500 550 600Wavel ength (nm)

Inte

nsity

1

234

5

6

78

0.0 1.0 2.0 3.0 4.0 5.0 6.0[Gnd-HCl] ( M)

0.0

1.0

2.0

3.0

4.0

Rela

tive

inte

nsity

蛋白质功能区

肌酸激酶活性部位荧光探针暴露的速度常数盐酸胍 （ M ）

荧光OPTA 内源荧光

失活k1 k2 k1 k2

0.3 0.38 0.049 0.0150.5 1.18 0.11 0.0038 3.6 0.0031.0 2.9 0.04 4.3

酶活性部位的柔性学说邹承鲁

Protein–protein interface design

erythropoietin

EPO-EPOR ERPH1-EPOR

LiuS,LiuSY,ZhuXL, LiangHH,CaoAN,ChangZJandLaiLH*. Nonnaturalprotein-proteininteraction-pairdesignbykeyresiduesgrafting.PNAS,2007,104,5330

药物研究

抗氧化剂对 A1-40结构的影响庾照学等 , 中国病理生理杂志 ,2000,16

FT - IR spectra of A1 - 40 in PBS(pH7. 4) for 30min

FT - IR spectra of A1 - 40 in PBS(pH7. 4) for 7 days

FT - IR spectra of A1 - 40 in PBS(pH7. 4) for 7 days

FT- IR spectra of A1 - 40 in PBS(pH7. 4) with TA9901 for 7 days (percent ratio : A1 – 40 :TA9901 = 1 1)∶

衰 减 全 反 射 红 外 光 谱 研究人乳腺癌组织

A1635/A1652

A1625/A1652

A1645/A1652

A1662/A1652

A1682/A1652

benign 0.98 0.65 0.64 0.49 0.17

malignant 0.43 0.23 0.56 0.37 0.09

开阔思路

记忆合金棒矫正脊柱侧凸

卢世璧( 院士 )

HSP 肿瘤疫苗

分子伴侣molecular chaperones

新生肽链的折叠

Misfolding & Protein Misfolding & Protein Conformational DisorConformational Disor

dersders

疯牛病带给生命科学界的思考疯牛病带给生命科学界的思考Mad cow

TSETSE

Alzheimer’s D. Amyloid Protein & Tau protein

Familial visceral Amyloidosis Lysozyme

….

Parkinson D. α-synuclein

Huntington D. Glutamine-repeatPrion D. Prion protein

Sickle cell anaemia Haemoglobin

Conformational BrainsDisorders

ProteinConforma-tionalDisorders (PCD)

Human Prion Diseases• Sporadic form

Creutzfeldt-Jakob disease (CJD)

• Familial (inherited) formFamilial CJDFatal familial insomniaGerstmann-Straussler-Scheinker syndrome

• Acquired (transmitted) formIatrogenic CJDKuruNew Variant CJD (related to Mad Cow Disease)

Animal Prion Diseases

– ScrapieSheep and goat

– Bovine spongiform encephalopathy (Mad Cow Disease)Cattle

– Feline spongiform encephalopathyCat (domestic cats, cheetahs, pumas)

– Transmissible mink encephalopathy Mink

– Chronic wasting diseaseMule deer, elk

朊病毒 (Prion) 病的共同特征

临床表现 :痴呆、共剂失调、震颤等症状

病理学上的特点 :大脑皮层的神经原细胞退化、空泡变性、死亡、消失，星状胶质细胞增生，蛋白酶抗性的 PrP 积聚，有时产生淀粉样斑

Prion Protein Gene (PRNP)

---Located on chromosome 20 in humans, chromosome 2 in mouse

---Encodes a glycoprotein with two sites for N-linked oligosaccharites and a C-terminal GPI anchor

---High expression in brain. Lower expression in peripheral tissues

---~10-15% of all cases are familial. About 20 mutations are linked to familial disease.

The Nobel Prize in Physiology or Medicine 1976

for their discoveries concerning new mechanisms for the origin and dissemination of infectious diseases

Baruch S. Blumberg

D. Carleton Gajdusek

Stanley B. PrusinerThe Nobel Prize in Physiology

or Medicine 1997for his discovery of Prions - a new biological principle of infe

ction

DNA RNA ProteinDNA RNA Proteintranscriptiontranscription translationtranslation

Sequence structure functionSequence structure functionfoldingfolding

二个中心法则

1. Genetics:

2. Protein:中心法则?

Conformational transition: from alpha-helix rich to beta-sheet rich

PrPc PrPsc

PrPc PrPsc PrP27-30

References

• Roger H.Pain ， Mechanisms of Protein Folding• Bengt Nölting ， Proein Folding Kinetics –Biophysical Methods• Leninger, Principles of Biochemistry,Worth Publishing, • Mathews and Van Holde, Biochemistry, Benjamin Cummings

思考问题：1. 蛋白质折叠中的”中心法则”？2. 联系生物物理技术部分所学内容， 哪些技术可以用来进行蛋白质折叠 研究，其根据是什么？