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K THUT PHN TCH CU TRC PROTEIN PHN 1: CC KHI NIM C BN

1. Khi Nim

1.1 Protein 1.1.1 Protein l g? Tt c cc t bo sng u cha Protein, iu c ngha l protein l cht cn cho s sng. V ngi ta cho rng Protein l thnh phn quan trng nht trong cc vt cht ca s sng, cho nn trong ting anh, protein bt ngun t ch Hy Lp c ngha l th nht. V nh ngha chnh xc Protein l g, c nhiu cch ni khc nhau, nhng t chung li, u thng nht cho rng: Protein l hp cht cao phn t gi nhiu vai tr nng ct trong c th. Hu ht chng lm vic trong t bo p ng yu cu ca cc bo quan v m trong c th v cu trc, chc nng v iu ha. Protein c cha trong tt c cc phn ca c th ca bn, ln da, c bp, tc, mu, b phn c th, i mt, ngay c mng tay v xng . 1.1.2 Cu trc chc nng ca Protein Theo cng trnh nghin cu : What is protein ca Georgia C. Lauritzen thuc i hc Utah State: Protein c cu to t cc n v nh hn c gi l cc axit amin. Hin nay pht hin ra hn 20 loi axit amin khc nhau. Mi phn t protein bao gm rt nhiu cc axit amin, c sp xp theo mt trnh t ngu nhin, t to ra hng trm, hng nghn cc phn t protein c cu trc khc nhau. Hu ht cc protein l cc phn t ln c th cha hng trm axit amin c sp xp trong cc ngnh v cc chui. Trnh t axit amin xc nh cu trc khng gian 3 chiu ca protein v chc nng chuyn bit ca chng. C 5 loi cu trc khng gian, ng vi 5 chc nng ca Protein nh sau:

Khng th (antibody)

y l cc protein c kh nng bm vo cc phn t ngoi lai nh vi khun, vi rt, sau v hiu ha chng bo v c th. Trong hnh bn l cu trc khng gian ca protein khng th: Immunoglobulin G (lg G).

Enzyme

Enzyme xc tc cho hu ht cc phn ng ha hc xy ra trong t bo. Chng cng gip hnh thnh nhng phn t mi bng cch c thng tin di truyn lu tr trong DNA. Vd hnh bn:

Phenylalanine hydroxylase.
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Thng tin - Messenger

protein thng tin, nh mt s loi hormone, truyn ti tn hiu phi hp cc qu trnh sinh hc gia cc t bo, m, c quan khc nhau. V d: hormone tng trng (Growth hormone)

Thnh phn cu

trc Nhng protein ny cung cp cu trc v nui dng t bo. Trong mt phm vi ln hn, chng cn cho php t bo di chuyn. V d: Actin
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Vn chuyn-d tr

cc protein ny bm vo nhng nguyn t v phn t nh bn trong t bo v lu thng trong c th. V d: Ferritin

1.2 Vai tr Protein trong sinh hc Nh ni trn, Protein c vai tr cc k quan trng trong i sng sinh hc ni chung cng nh con ngi ni ring. 1.2.1 Vai tr Protein l nhng i phn t c cu to theo nguyn tc a phn m cc n phn l axit amin. Chng kt hp vi nhau thnh mt mch di nh cc lin kt peptide (gi l chui polypeptide). Cc chui ny c th xon cun hoc gp theo nhiu cch to thnh cc bc cu trc khng gian khc nhau ca protein. Protein hnh thnh, duy tr v thay th cc t bo trong c th. Protein chim ti trn 50% khi lng kh ca t bo v l vt liu cu trc ca t bo. Thiu protein dn n suy dinh dng, chm ln, suy gim min dch, nh hng xu n chc nng ca cc c quan trong c th.
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Protein l tham gia vo thnh phn c bp, mu, bch huyt, hocmn, men, khng th, cc tuyn bi tit v ni tit. V vy, protein c lin quan n mi chc nng sng ca c th (tun hon, h hp, sinh dc, tiu ha, bi tit hot ng thn kinh v tinh thn...). Protein cn thit cho chuyn ha bnh thng cc cht dinh dng khc, c bit l cc vitamin v cht khong. Khi thiu protein, nhiu vitamin khng pht huy y chc nng ca chng mc d khng thiu v s lng. Protein cn l ngun nng lng cho c th, thng cung cp 10%-15% nng lng ca khu phn, 1g protein t chy trong c th cho 4 Kcal (trong khi Gluxit l 4 Kcal, Lipit l 9kcal v ru l 7kcal) Protein kch thch s thm n v v th n gi vai tr chnh tip nhn cc ch n khc nhau. Thiu protein gy ra cc ri lon quan trng trong c th nh ngng ln hoc chm pht trin, m ha gan, ri lon hot ng nhiu tuyn ni tit (gip trng, sinh dc), thay i thnh phn protein mu, gim kh nng min dch sinh hc ca c th v tng tnh cm th ca c th vi cc bnh nhim khun. 1.2.2 B sung protein cho c th Sau khi c np vo c th, trong qu trnh tiu ho thc n, protein c phn hu ti d dy bi cc enzyme. N chuyn thnh cc polypeptides, cung cp cc axit amin cn thit cho s sng. Thnh phn axit amin ca c th ngi khng thay i v c th ch tip thu mt lng cc axit amin hng nh vo mc ch xy dng v ti to t chc. C 8 axit amin c th khng th tng hp c hoc ch tng hp mt lng rt t. l Lyzin, tryptophan, phenynalaninin, l - xin, izolxin, valin, treonin, metionin. Ngi ta gi chng l cc axit amin cn thit. Cc axit amin cn thit ny c ly thng qua protein ca thc n t bn ngoi. Tuy nhin, trong t nhin khng c loi protein thc n no c thnh phn hon ton ging
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vi thnh phn axit amin ca c th. Do p ng nhu cu c th cn phi hp cc loi protein thc n c thnh phn axit amin cn i nht. Gi tr dinh dng mt loi protein cao khi thnh phn axit amin cn thit trong cn i v ngc li. Hu ht thc n c ngun gc ng vt v thc vt cha y v cn i cc thnh phn ca cc axit amin cn thit . Tuy nhin, khng c mt loi thc n no c tt c m cn phi s dng mt ch hn hp nhiu loi thc n. Thc phm ngun gc ng vt (tht, c, trng, sa) l ngun protein qu, nhiu v s lng, v cn i hn v thnh phn v m axit amin cn thit cao. Hm lng cc axit amin cn thit trong thc phm ngun gc thc vt (u tng, go, m, ng, cc loi u khc...) khng cao (tr u nnh); nhng c th vn phi b sung cn i y cc loi ny. V vy, bit phi hp cc ngun protein thc n hp l s to nn gi tr dinh dng cao ca khu phn. V d go, ng, m ngho lizin cn u tng, lc, vng hm lng lyzin cao, khi phi hp go hoc m hoc ng vi u tng, vng , lc s to nn protein khu phn c gi tr dinh dng cao hn cc protein n l

2. Phn tch cu trc Protein 2.1 Phn tch cu trc Protein l g? Nh trnh by, mi mt loi protein vi chc nng khc nhau s c mt thnh phn v trnh t axit amin khc nhau, cng nh cu trc khng gian khc nhau. Chnh v vy, vic nghin cu tm ra nhng thng tin ny l v cng cn thit trong sinh hc. T cc thng tin , chng ta c th bit c chnh xc protein no, c chc nng g, t tm cch tng hp nhn to protein, hay b sung protein cn hoc loi b cc protein c hi. Phn tch cu trc Protein chnh l nhm mc ch ny.
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Hin nay, trn th gii, cng tc nghin cu v phn tch cu trc protein ang rt c y mnh. Hng lot cc cng trnh nghin cu, cc bi bo khoa hc c cng b cho php con ngi c ci nhn su v rng hn v th gii sinh hc phn t protein. V chng cng c ng dng trong rt nhiu lnh vc: y t, nng nghip, 2.2 Cc phng php phn tch Protein, tuy c gi l mt i phn t ca th gii vi m, tuy nhin mt thng ca chng ta s khng th nhn thy chng c. Chnh v vy, nghin cu nhng i tng ny, cn s dung nhng phng php c hiu vi s tr gip ca cc phng tin khoa hc k thut hin i, cng chnh l l do vic phn tch cu trc protein ch thc s khi sc trong vi nm tr li y, theo s tin b ca khoa hc k thut. Hin nay, c nhiu phng php c s dng trong nghin cu protein, sau y xin gii thiu s lc 2 phng php hu hiu nht 2.2.1 Tinh th hc tia X (X-ray crystallography) William Henry Bragg (1862-1942) l ngi i tin phong trong vic pht trin phng php X-ray. Bragg c v s hng th nghin cu, nhng cng trnh mang li tn tui cho ng l mt nh khoa hc hng u l tin b mang tnh lch s ca ng trong tinh th hc tia X. Lm vic vi ngi con trai, William Lawrence Bragg, ng pht trin mt phng php bn ph cc tinh th vi tia X nng lng cao pht ra bi nhng ng chn khng c cu trc c bit. Khi tia X i qua n tinh th, chng b nhiu x theo mt iu kin do cha con Bragg khm ph ra v do suy lun nh lng ra ng i ca chng. Hnh nh tia X nhiu x c th c chp trn knh nh, v tia X lm phi sng cc ht bc bromide khi chng va vo. Bng cch kho st hnh nh tia X nhiu x bi nhng tinh th khc nhau, Bragg v con trai ca ng c th thit lp mt s mi lin h ton hc c bn gia mt cu trc tinh th nguyn t v hnh nh nhiu x ca n. Vi thnh tu ny, , William Henry Bragg v William Lawrence Bragg c tng Gii Nobel Vt l nm 1915.
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2.2.1.1 S lc phng php Cch thng thng xc nh hnh dng mt vt l nhn trc tip vo n. Vi cc vt rt nh, nh cc phn t, xc nh cu trc lin kt gia cc nguyn t to nn phn t , ta cn dng knh hin vi phng i chng ln hng ngn t ln. Vi kh nng hin c, knh hin v in t (electron microscopes) cng ch c th phng i ln c vi t ln. L do l do gii hn nhiu x (diffraction limit): c th nhn thy mt vt hoc phn bit c 2 vt im th kch thc ti thiu ca vt hoc khong cch ti thiu gia hai vt im ln hn 1/2 ln bc sng ang s dng. Bc sng ca nh sng nhn c vo khong vi trm nanometers cn cc nguyn t th c khong cch ch n v hay bc sng cn thit nhn thy cc nguyn t l bc sng ca tia X. Nhng ta khng th to ra knh hin vi tia X. V th i hi ta phi dng cc k thut mang tnh gin tip khc. Tia X khi chiu vo tinh th s tng tc vi cc electron ha tri (valence electron) ca cc nguyn t thnh phn c phn b trong khng gian. Cc electron ny s tn x (scatter) tia X ra cc hng, ty vo s sp xp trong khng gian ca nguyn t. [Ci ny cng ging nh dng mt n pin ln chiu vo mt cy n chm (chandelier) m ta khng c php nhn thy. D khng bit cy n chm hnh dng th no, nhng da vo bng phn chiu (cc pattern) ta cng c th d on s sp xp ca cc mnh thy tinh trn cy n chm] Mt mn hnh s pha sau lu li v tr tn x v cng ca tia X b tn x. Sau khi c cc d liu ny ri, ngi ta s dng cc cng thc tnh ton phc tp xc nh v tr cc electron bao quanh cc nguyn t v t suy ra v tr cc nguyn t. Cc mu nhiu x thu c s c mi quan h vi vt pht tn cc sng chiu ti n thng qua mt php ton bin i gi l bin i Fourier (Fourier transform). Nu mt cc electron (electron density) bao quanh mi nguyn t l mt hm ton hc, th mu nhiu x tia X thu c tng ng l bin i Fourier ca hm . Vi tnh cht c th bin i ngc ca php bin i Fourier, ta c th dng my tnh xy dng li hnh nh mt electron da vo nh mu nhiu x. PDB (Protein Data Bank) lu tr cu trc

protein v cc phn t sinh hc khc min ph s dng. hin th cu trc 3D ca chng, ta dng phn mm RasMol hay Pymol. Tuy nhin, mt kh khn gp phi l nhiu x tia X t mt phn t th s cho cng thu c trn nh rt yu v kh phn bit vi nhiu. Do , ngi ta s dng tinh th. Tinh th sp xp mt lng ln cc phn t theo mt trt t v hng ging nhau, nh th cc sng nhiu x s cng hng pha ( mt s hng) lm tng cng hin th ln my o. Hin nhin, mt s hng khc, cc nhiu x ny cng trit t nhau. l l do m mu nhiu x ch l mng cc im. V th, vic to ra mt tinh th tt ng vai tr cc k quan trng thu c hnh nh c gi tr. l khi ngun ca k thut tinh th hc tia X. 2.2.1.2 u nhc im phng php Trc khi c phng php tinh th hc tia X: Vic tm hiu tinh th l da vo c tnh hnh hc ca tinh th. bit c cu trc 3D, ngi ta o cc gc ca b mt tinh th so vi cc trc tham chiu tng tng (crystallographic axes) v t cho ra hnh nh hnh hc c tnh i xng ca tinh th. Hnh nh 3D ny c chiu ln mt phng dng php chiu lp th (stereographic projection) chiu mt cu ln mt phng. Php chiu ny bo ton gc nhng khng bo ton din tch. Phng php tinh th hc tia X th da vo my o gc (goniometer): Vic xc nh trt t ca cc nguyn t da vo s phn tch cc mu nhiu x (diffraction patterns) thu c sau khi chiu tia X vo tinh th cc cht/phn t cn phn tch dng rn tinh th. Ngoi tia X, ngi ta cn dng electron (electron diffraction) hoc neutron (neutron diffraction) cho mt s mc ch c bit. Tinh th hc tia X (X-ray crystallography) c ng dng nhiu trong sinh hc xc nh cu trc ca cc i phn t nh protein, DNA hay RNA. V cc phn t ny phi c chuyn v dng tinh th. L do s dng tia X l v ta khng th nhn thy chi tit mt vt nh hn na bc sng ang s dng. M kch thc nguyn t qu nh, nn phi dng tia X v c bc sng ngn thy c chi tit nguyn t. Tuy nhin, nng lng sng th
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t l nghch vi bc sng, ngha l bc sng cng ngn th nng lng cng cao, cng d ph hng mu phn t sinh hc. l l do m phi chuyn v dng tinh th gim s ph hoi ca tia X. Tuy nhin vic ph hoi ca tia X l vn khng th trnh khi, kt qu thu c cn c xem xt k. 2.2.2 Cng hng t ht nhn ( NMR ) Phng php o ph cng hng t ht nhn c tm ra v pht trin qua nhiu giai on, c bit nh cc ng gp ca Richard R. Ernst. Nm 1966, khi lm vic chung vi mt ng nghip ngi M, Ernst pht hin ra rng nhy ca k thut cng hng t ht nhn (cho n nay ch gii hn trong vic phn tch s t ht nhn) c th c tng ln ng k bng cch thay th cc sng v tuyn qut chm thng c dng trong quang ph cng hng t ht nhn thi bng cc xung ngn cng cao. Khm ph ca ng cho php phn tch nhiu loi ht nhn hn v cc s vt liu nh hn. ng gp quan trng th nh ca ng vo lnh vc "ph hc cng hng t ht nhn" l mt k thut cho php mt phn gii cao, nghin cu "hai chiu" cc phn t ln hn so vi trc y m quang ph cng hng t ht nhn t c. Vi cc ci tin ca Ernst, cc nh khoa hc c th xc nh cu trc 3 chiu ca cc hp cht hu c v v c v cc i phn t sinh hc nh cc protein; nghin cu s tng tc gia cc phn t sinh hc v cc cht khc nh cc ion kim loi, nc, v thuc; xc nh cc loi ha cht, v nghin cu t l ca cc phn ng ha hc. ng c trao Gii Nobel Ha hc cho cng trnh ng gp ca ng vo vic pht trin ph hc cng hng t ht nhn bin i Fourier v sau vic pht trin k thut cng hng t ht nhn a chiu. Cc ng dng nn tng ca cng hng t ht nhn c trong ha hc (ph hc cng hng t ht nhn) v y hc.(chp cng hng t). 2.2.2.1 Phng php thc hin Phng php cng hng t ht nhn s c trnh by chi tit phn 2.
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2.2.2.2 u nhc im phng php Vi cc k thut ph NMR, ngi ta c th bit c mi lin h gia cc proton v carbon trong phn t. Kt hp vi ph khi v cc thng tin khc ngi ta c th xy dng c cu trc phn t ca hp cht. Trong kh nhiu trng hp, ch bng NMR ngi ta cng c th xc nh c cu trc v cu hnh lp th ca cht cn phn tch. Do c rt nhiu thng tin c trng v cu trc phn t, vic s snh ph proton hay carbon mt chiu ca mt cht vi mt cht bit cho php xc nh mt cht mt cch tin cy. Ngy nay, ch cn lng mu mc /mg /hay thp hn c th xc nh cu trc ca mt cht. Kh nng ny gip ch rt nhiu trong nghin cc cc t nhin, khi m c cht rt kh phn lp v thng ch thu c vi mt lng nh. Ngoi vic xc nh cu trc, ph cng hng t ht nhn cn c dng trong nh lng cc cht trong phn tch nh lng nh cc phng php ph khc. Tuy nhin, do thit b t tin nn cch thc ny t c s dng. Vic kt ni cng hng t ht hn vi cc thit b sc k lng c nhiu kh khn. Tuy vy, hin c nhng h thng ghp ni HPLC-NMR phn tch v xc nh cc cht chit t dc liu. Ngoi cc ph k cng hng t ht nhn dng trong phn tch cu trc, k thut xc nh hnh nh cng hng t ht nhn cng c s dng trong chn on y khoa. Cng ngh mi ny khng nhng cung cp thng tin v cu trc khng gian ba chiu ca cc phn t sinh hc m cn cho bit c im nhit ng hc v t tnh ca n. iu nay cho thy y l mt cng c quan trng cho php i su tm hiu chc nng ca cc phn t sinh hc mc phn gii c cc nguyn t trong tng lai. PHN 2. Xc nh cu trc protein bng phng php cng hng t ht nhn ( RMNRSONANCE MAGNTIQUE NUCLAIRE ) I. Tng quan v phng php RMN : 2.1.1. Lch s nghin cu cu trc Protein : Cc nghin cu v cu trc Protein bt u vo nm 1936 khi Mirsky v Pauling xut rng cc chc nng ca mt protein c lin quan n cu to ca n (s sp xp ca cc nguyn t
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trong khng gian ba chiu) (Anfinsen , 1973). Nhng li ny c mt nh hng ln n s hiu bit v sinh hc cng nh vic nghin cu v pht trin cc loi thuc mi (Russell v Eggleston, 2000). K t , vic nghin cu cu trc ba chiu ca tt c cc protein trong mt h protein cho php ta c mt cch suy ngh mi trong lnh vc Sinh hc cu trc (Hol, 2000). Nghin cu thc nghim v cu trc ca cc protein l mt qu trnh lu di v tn km, i hi phi c thit b chuyn dng v trnh chuyn mn cao. Hai phng php chnh c s dng l phng php tinh th v phng php cng hng t ht nhn (NMR) v c trnh by trong phn sau. 2.1.2 Lch s phng php NMR : Cho n nm 1985, phng php duy nht xc nh cu trc ba chiu ca mt i phn t l tinh th hc tia X nhiu x. K thut ny i hi phi c cc tinh th protein ngn np.Mc d c nhng bc tin ln trong nghin cu nhng phng php vn gp phi rt nhiu kh khn v i khi kt qu li l nhng tinh th protein khng t yu cu. Trong nhng nm gn y, nh nhng tin b trong lnh vc quang ph cng hng t ht nhn (NMR), m chng ta c th xc nh c cu trc ba chiu ca cc protein nh, c bit l protein t hn 200 axit amin (Cavanagh et al. , 1996). iu ny c c l nh vic chng ta sn xut c cc quang ph k trng cao, s pht trin ca k thut NMR a chiu v nhng tin b trong cng ngh my tnh. NMR cho php chng ta c tnh khong cch gia cc proton v mt s gc nh din. T kin thc ca mt s lng ln cc d liu NMR, c th xc nh cu trc ca mt protein vi phn gii ca trt t thu c bi tia X. Vic mt s d n gii m gen c hon thnh gn y cung cp cho cc nh khoa hc cc thng tin v trnh t ca mt s b gen. V mi protein trong mt sinh vt c mt tm quan trng sinh ha nht nh nn xc nh cu trc ba chiu ca tt c cc protein ca mt h protein s cho php chng ta hiu su hn v cu trc cng nh chc nng ca cc protein trong c th sng. V vy gn y, mt s d n phn tch cu trc protein c bt u mt vi ni trn th gi nh Nht Bn, chu u v Bc M (Heinemann, 2000; Terwilliger, 2000; Yokoyama v cng s, 2000).

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Xc nh cu trc ba chiu ca protein mt mt thi gian. N c th mt vi thng ty thuc vo di ca protein. tng tc ca s khm ph ca cc cu trc, cc nh nghin cu phi hp vi cc phng th nghim khc p dng phng php NMR phn tch mu protein. Kt qu cho thy, 16 cu trc c gii quyt trong su phng th nghim tinh th khc nhau v 17 cu trc c gii quyt trong by phng th nghim NMR khc nhau (Yee et al, 2003). S lng cc cu trc gn nh ging ht nhau c xc nh vi tinh th v NMR cho thy NMR quang ph c th ng gp ng k cho lnh vc nghin cu cu trc protein (Yee et al, 2003). Cng hng t ht nhn (NMR) l mt k thut quang ph c s dng rng ri. S a dng tuyt vi ca quang ph cho php xc nh cu trc ba chiu ca cc protein. Phn di s cung cp mt ci nhn tng quan v cc khi nim l thuyt trong phng php NMR cng nh gii thiu cc bc xc nh cu trc ca mt protein bng phng php ny. II. Cc khi nim l thuyt c bn trong phng php NMR : 2.2.1 Nhng nguyn tc c bn ca NMR NMR phn tch da trn cc tnh cht t ca ht nhn nguyn t. Cc ht nhn ca cc nguyn t to ra s tn ti ca mt momen quay ring c th c gi l spin I . Spin c gi tr l 0, 1/2, 1, 3/2, v nh vy ( I = 0 c ngha l khng c quay). Trong thc t, c mt ht c moment quay khc khng s cung cp cho ht nhn nguyn t cc thuc tnh ca mt lng cc t. Mt ht c spin 1/2 s chu nh hng ca mt t trng c hai moment t theo hai hng khc nhau, trong cng mt phng.V d nh proton 1 H, ht nhn 13 C, 19 F hoc 31 P c spin l 1/2 v hai trng thi spin: 1/2 -1/2. Cho mt spin bng mt, chng hn nh deuterium2 H hoc lithium 6 Li, c ba s nh hng s xy ra: -1, 0 v +1 (Evans, 1995). Ni chung, c (2I+1) hng cho mt spin I. Khi mu t trong mt t trng, mi s nh hng s tng ng vi mt mc nng lng. i vi cc proton c spin l 1/2, c hai mc nng lng nht (phn phi Boltzmann) ( Hnh 2-3 ). y l qu trnh chuyn i gia cc mc v s hnh thc ha hin tng cng hng t ht nhn (Sanders v Hunter, 1987).

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Hnh 2-3: Mc nng lng v trng thi spin ht nhn ca proton khi khng c t trng. S khc bit hai mc nng lng l yu hn nhiu so vi nhng g c hin th trong hnh ny. (Hnh t www.rmn.uhp-nancy.fr/Mutzenhardt/RMNSV2CM1.pdf) quan st mt tn hiu, chng ta phi ph v s cn bng gia hai mc nng lng. Mu phi chu mt t trng th hai c sn xut bi mt ngun bc x in t c xung vung gc vi trng u tin v c to ra bi nam chm. Do hin tng cm ng in t nn mt dng in cm ng s xut hin trong my thu cun dy (Cavanagh et al, 1996.) ( Hnh 2-4 ) .

Hnh 2-4: nh hng ca t trng bn ngoi ca mt mu NMR chuyn t ha dc theo x v trc y v s quay do cm ng in t xung quanh trc z. (Sanders, 1987). Nh th hin trong Hnh 2-5 , M dn dn s tr v v tr ban u ca n v song song vi B o . ( Cavanagh et al, 1996).

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Hnh 2-5: S cn bng tr li moment t ha theo nh hng song song vi B 0 (www.med.univ-rennes1.fr/cerf/edicerf/BASES/BA004_cv_rb_9.html). Trong thi gian quay tr li trng thi cn bng, cc d liu s c ghi li bng my quang ph NMR. Ht nhn ca protein s cng hng tn s khc nhau ty thuc vo mi trng khc. Cc tn hiu c ghi nhn l mt FID (cm ng phn r t do). Hnh 2-6 l mt v d v mt FID.

Hnh 2-6: Tn hiu NMR dng tn hiu tt dn theo thi gian c tn l FID (cm ng phn r t do).
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Nhng tn hiu ny c cha tt c cc thng tin cn thit nhng rt kh gii m chng. Bin i Fourier gip chng ta c th nhn thy tn hiu ny c th dng hnh sin v l mt hm theo thi gian (y = f (t)) bng cch thay i n thnh mt quang ph NMR mang chc nng ca tn s (y = f (?)) ( Hnh 2-7 ). Cc tn s cng hng sau s c phc hi li(Cavanagh et al, 1996).

Hnh 2-7: Tn hiu FID qua php bin i Fourier. (Hnh t www.rmn.uhpnancy.fr/Mutzenhardt/RMNSV2CM1.pdf) 2.2.2 Cc bc xc nh cu trc ca protein bng NMR: Trong phn ny, chng ta s tho lun v cc bc cn thit xc nh cu trc ca mt protein. Cc bc ny c trnh by phn ngay sau y. 2.2.2.1 Biu din, nh du cc ng v v tinh ch protein: NMR phn tch yu cu s lng ln cc protein tinh khit. Bng cch s dng cc k thut tiu chun , gen quan tm c nhn bn v tnh trong mt plasmid DNA v c a vo cc vi khun vi s lng rt ln. Mt s ht nhn khng quan st c bng NMR do thiu spin ht nhn (I = 0). Nhng trong nghin cu cc thnh phn c bn ct li ca ha hc hu c, chng hn nh 12 C v 16 O, chng ta c th quan st c hin tng NMR. l do cc nguyn t ny lun xut hin cc ng v vi spin ht nhn khc khng. 13 C quay 1/2 c tm thy mc 1,11% trong t nhin. Ngoi ra, ng v ny c mt t l gyromagnetic thp hn bn ln ca proton. V vy, hai yu t ny gy ra nhy rt thp cho 13 C khi phn tch NMR quang ph. 17 O c spin 5/2 tn ti t hn nhiu so vi13 C trong t nhin, dn n s quan st ca cc vch ph rt rng v gn nh khng th s dng phng php phn tch NMR.
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Ht nhn 14 N c spin 1, c th phn tch bng NMR nh du lm gim s lng cc mc nng lng. 15 N quay 1/2 l c t hn 13 C trong t nhin, c tm thy 0,37% (Cavanagh et al, 1996; VOET v VOET, 1995). N cung cp tn hiu NMR tt. T l gyromagnetic ca n nh hn 10 ln so vi proton. khc phc nhng kh khn, c th thng nht nh du cc nguyn t cacbon v nit l 13 C v 15 N. nh du amoni clorua ( 15 NH 4 Cl) v glucose ( 13 C-glucose) cch biu din duy nht ca nit v carbon trong mi trng nui cy. nh du ng v ca cc protein ny l iu cn thit phn tch v s cc th nghim NMR a chiu. i khi ngi ta thay th cc proton bi cc deuterium gim bt quang ph ca protein ln. sn xut mt protein deuterated, s tng trng ca vi khun c thc hin trong D 2 O thay v H 2 O. Cc protein ti t hp sau c tinh ch v cu trc ca chng s c c trng bi NMR. 2.2.2.2. Chun b mu Cc protein c cu trc ba chiu c phn tch bi NMR phi c nng v mi trng thch hp. Nng ca protein tinh khit trong mt mu cn phi khc nhau t 0,5 n 2.0 mM. Cc mu c th cha tp cht, nhng khng c cha cc proton tp cht v s gy nh hng ti cc proton ca protein. Mui v ion c th c tm thy trong dung dch. Mt nng ti thiu ca cc mui s cung cp cho protein mt mi trng tng t nh mi trng t nhin ca n, nhng nng qu cao s lm tn thng n, v mui l nhng yu t dn in v c th lm nng mu. Protein c th dng monomer hoc polymer v phi c n nh c th duy tr hot ng v gi vng cu trc ca n cho mt vi tun trong iu kin nhit cao. pH ca mu l rt quan trng quan st tt c cc proton ca protein. pH thng dao ng t 5 n 7. pH cao gip tng tc trao i proton vi dung mi. Dung mi c s dng thng l nc. 10% D 2 O c thm vo mu gi cho ph k ch mt gi tr tn s duy nht (Cavanagh et al 1996). Tht vy, mc cho t trng thay i lin tc t mt gi tr nh, xung c a ra lin tc trn D 2 O iu chnh tn s tham chiu ca
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cc ht nhn ca protein. Tn s cng hng c tham chiu bi mt lng nh (0,5 mM) DSS (natri 2,2-dimetyl 2 silapentane-5-sulfonate) c thm vo mu. 2.2.2.3. Ly NMR Vic c c mt s quang ph NMR to iu kin cho vic xc nh tn s cng hng ca tt c cc ht nhn ca mt hp cht. Tuy nhin, tn s cng hng ca mt ht nhn li t l thun vi t trng. Vic xc nh tn s chuyn ha cht khng phi d dng v cc thit b cng bin php c s dng phi c chnh xc n ppm (phn triu) (Cavanagh et al, 1996). Nh mt v d n gin, mt proton trong mt t trng 14 Tesla (T) 600MHz trong khi trong mt t trng mi ln nh hn (1,4092 T), 60 MHz. Tnh ton c th c thc hin t cc phng trnh 1 .

Phng trnh 1: Phng trnh ca NMR c bn. i din cho Lamor tu sai tn s (MHz), B z l t trng. Cc ht nhn ca hp cht khng cng hng cng mt tn s. Proton cng hng thng thng t 1 n 12 ppm ( Hnh 2-9 ), cc nguyn t cacbon bo thng cng hng gia 17 v 75 ppm v nit amit thng gia 100 v 132 ppm. Tnh cht ha hc b nh hng bi mi trng xung quang, chnh xc hn na l bi cc ht nhn v cc electron gn .

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Hnh 2-9: S thay i ha tnh i vi cc loi proton khc nhau. Ph 1D c ghi li trong nc. FID c bin i bi bin i Fourier (Cavanagh et al, 1996). Trong quang ph cng hng t ht nhn nhiu hn mt chiu, cc cc i c hnh thnh trong s chuyn i ha hc ca s tng quan gia hai ht nhn. Mi tng quan c ghi nhn gia cc ht nhn ca protein c th ch l s khc nhau ca mt quang ph. H c th to lin kt gia mt amino acid ring l hoc thng qua khng gian. Mt s NMR gip phn tch cu trc mch, tc l xc nh nhng thay i ha hc ca cc ht nhn trn chui chnh, v cc loi quang ph khc c tnh s cn nhiu hn xc nh s thay i ha tnh ca cc chui bn.

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Th nghim NMR Homonuclear c da trn mt loi ht duy nht ca nguyn t: proton. Hai loi th nghim c s dng l COSY v TOCSY (Braunschweiler v Ernst 1983) (Cavanagh et al, 1996). Mt s axit amin c mt spin ca ring mnh. Ly v d cn alanine. D lng ny c c trng bi nhm methyl nm H. Trong thc t, cao im ny c cng ln hn gp ba ln so vi H. Do , c th kt hp mt vi nhm dch chuyn ha hc cho mt loi axit amin, sau n s lin kt cc axit amin vi nhau. Ph NOESY cng l mt th nghim NMR homonuclear. Cng c ch nh mt chui l cc NOEs v s c trnh by chi tit hn phn sau na. Trong qu trnh phn tch cc protein ln hn, ni n l cn thit nh du d lng, mt s th nghim NMR heteronuclear c quang ph c th ln n bn ln kch thc m ta c th lc trc. Nh cp trn, xc nh cu trc ca mt protein th ta phi gii m c tt c cc h thng spin ca cc axit amin. Tng tc Noe l nhng tng tc lng cc gia cc proton gn trong khng gian (Cavanagh et al, 1996). Khong cch gia hai nguyn t hn km nhau v cng trong tng tc NOE l thp v thng khng vt qu 5A. Mi quan h ny l t l nghch v cp n mi quan h sau y.

Phng trnh 2: Mi quan h gia tng tc Noe v khong cch gia hai proton 2.2.2.4. Chuyn i quang ph phn tch Tn hiu MNR pht ra phi tri qua mt s bin i trc khi c phn tch. Vic trch xut dung mi l cch loi b cc tn hiu t cc dung mi. Nhn tn hiu fid bng mt php ton rt nhiu li ch ph. D on tuyn tnh lm tng thm cc im c d on t cc im th nghim. Ngoi cc phng php nu, bn c th thm s khng vo cui ca tn hiu ci thin phn gii ca quang ph.

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Bin i Fourier chuyn i cc tn hiu fid trong ph tn s ( Hnh 2-7 ). Chc nng ny c thc hin trn ton kch thc ca mt quang ph. u im ca phng php ny l cc tn hiu c thm vo khng gy nhiu lon. Ngoi ra cn nhiu phng php khc c s dng nng cao ph NMR. 2.2.2.5. Kt cu gii hn : Cc gii hn khc nhau v cu trc nh NOE v gc nh din rt cn xc nh cu trc ca protein bng NMR. Cc gii hn ca lin kt hydro v cc gii hn khc khng c trnh by trong bi vit ny cng c th c s dng xc nh cu trc ca protein. a. Gii hn khong cch NOE hn ch cung cp thng tin v khong cch gia cc nguyn t ln cn. Cc khong cch ny t hn 5A c th l d lng trong ni b hoc lin d lng (tun t, tm trung, tm xa). Th nghim cho 15 N-HSQC-NOESY to iu kin tm hiu tng tc gia proton v amit gn trong khng gian. Th nghim 13 C-NOESY-HSQC li lm r nhng tng tc gia tt c cc proton thuc carbon. Mi proton lin kt vi carbon ca mi lng d u c tng tc vi tt c cc proton gn lin vi cc nguyn t carbon gn trong khng gian. Quang ph ny thng khng c quy hon ton bi v vi nh thng chng cho ln nhau. S tng tc gia hai nguyn t, o c cng tng tc gip ta c th nh gi c khong cch ca cc lin kt trc cp t phn trc( phng trnh 2 ). Vic hiu chun NOE l cn thit cho vic tnh ton kt cu. b. Gii hn ca gc nh din Trong mt protein, cc lin kt tip gip vi cc peptit CN c kh nng xoay xung quanh trc ca chng. Nh vy, qu o ca mt protein trong khng gian c xc nh bi mt tp hp mt phng khp ni vi nhau ti cacbon ( Hnh 2-16 ).

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Hnh 2-16: i din ca mt chui polypeptide. Cc gc quay v N-C l gc v lin kt xung quanh 'Ca-C l nh hng ca hai mt phng ny c xc nh bi v cc gc . Cng nh khong cch Noe, nhng gc ny c th c s dng nh cc gii hn cu trc.Hai phng php thng c s dng trong NMR xc nh cc gc nh din. S dng mi quan h gia s thay i ha hc v cc cu trc th cp (v d nh cch tip cn Talos) c hin th trong hnh 2-17 v cng vi nhng hng s khp ni.

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Hnh 2-17: Mi quan h gia s thay i ha hc C C v cu trc th cp. Con s ny c ly t http://spin.niddk.nih.gov/bax/software/TALOS/ Talos (Torsion Angle Likelihood Obtained from Shift and sequence similarity) Talos l mt h thng cung cp cc c s d liu nh gi cc gc , t s chuyn i ha hc ca nm nguyn t: H, C, C, CO, N (Cornilescu et al, 1999.). Talos s dng mt chui ba axit amin trong mt protein v so snh cc bin th ca s dch chuyn ha hc (tun t gp mu) ca nm nguyn t cui cng vi cc bin th ca cc tripeptit tng t nh mt c s d liu. C s d liu ny hin cha thng tin khong 20 protein ca cc cu trc ni ting i din cho khong 3000 b ba. Talos l mt phng php thc nghim, cc gi tr gc l gi tr khng chnh xc v khng chc chn i vi cc i lng quan trng. Talos cho kt qu tt hn khi c s dng kt hp vi cc gi tr hng s khp ni. c. Khp ni cc hng s Hng s khp ni l s tch cc nh proton kp ra bng hertz . phn tch protein theo ba chiu khng gian, vic khp ni cc hng s c s dng thng xuyn nht l trong lin kt gia HN v H. Th nghim NMR HNHA (Geerten W. Vuister, 1993) c s dng xc nh cc hng s khp ni ( 3 J HNHa ). HNHA quang ph gm c ba chiu (F 1 ( 15 N), F 2 ( 1 H), F 3 ( 1 HN) (Cavanagh et al, 1996). Trn quang ph ny, t l cc tng tc gia cc nh ng honh v cc nh ng cho cho mt lng d nht nh v t l thun vi 3 J HNHa . Ta xc nh gi tr ca 3 J HNHa bng phng trnh di y :

Phng trnh 3: Phng trnh cho ta bit c khp ni lin tc 3 J HNHa Mt khi cc hng s khp ni thu c cho tt c cc axit amin ca protein m ta ang nghin cu, th nh c mi quan h Karplus (Harbison, 1993) th hin trong Hnh 2-18 , chng ta c th chuyn cc hng s khp ni vo trong cc gii hn ca gc nh din .
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Hnh 2-18: Biu Karplus m t s bin i ca cc khp ni lin tc 3 J HNHa F vi gc nh din ca chui mnh. Khi cc khp ni lin nh hn 6 Hz, gc c tm thy l khong 60 trong mt chui xon cu trc th cp. Khi ln hn 8 Hz, gc l khong 120 (-sheet). Khi n l hng s t 6 n 8 Hz th khp ni khng c s dng nh l mt rng buc cu trc bi (Cavanagh et al, 1996). Nhng rng buc nh din gip ch cho vic tnh ton cc cu trc protein. Ty thuc vo gc, mt khu vc s to thnh mt cu trc th cp trong tm hoc xon . S Ramachandran cho thy cc khu vc v m ti thun li cho vic to thnh xon v cc tm

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Hnh 2-19: Biu Ramachandran biu din nhng vng gi tr gc nh din v . (Branden v Tooze, 1991).
d. Gii hn lin kt hydro :

Lin kt hydro l tng tc tnh in gia mt nguyn t hydro gn lin vi mt nguyn t c m in cao v mt cp electron t do nm trn mt nguyn t (O, S hoc N) ca mt nhm khc. Khong cch ca cc lin kt hydro thay i t 2,7 n 3,1 (Vn Holde v Mathews, 1990). Kh khn ca cc lin kt hydro c th c bit n trong vic o vn tc trao i ca amit khi protein c ha tan trong deuterium. Deuterium khng hin th trong NMR quang ph 1 H do cn nhiu thi gian hn trc khi s bin mt ca nh, proton trao i d dng hn vi cc dung mi nn lin kt hydro cng d hnh thnh hn.

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C nhiu loi rng buc cu trc khc c th c s dng xc nh cu trc ba chiu ca protein, nhng chng khng c s dng trong nghin cu ny. 2.2.2.6. Protein ng lc Trng thi ngh l qu trnh m cc spin ca cc ht nhn trong mu tr li trng thi cn bng sau khi c kch thch. Trng thi cn bng l trng thi m mc nng lng tng ng vi mt phn b Boltzmann. T l ngh chu nh hng bi tnh cht vt l v tnh nng ng ca phn t . Do , mt nghin cu ca qu trnh ngh s cho ta bit cc thuc tnh nng ng ca cc phn t. m t cc c tnh ny, ta cn o mt s thng s Th nht, thi gian ngh T1 (i khi c gi l theo chiu dc hoc spin mng) c trng cho s cn bng nng lng ht nhn sau khi kch thch. Trng thi ngh ny l do tng tc vi mi trng. Thi gian ngh ngang T 2 (cn gi l spin-spin) cng cho php quay tr li trng thi cn bng, nhng dc theo trc x v y (Levitt, nm 2001). Thng thng, 15 NT 1 v 15 NT 2 c phn tch cho s nng ng ca mch. 2.2.2.7. Tnh ton ca cc cu trc ba chiu S m phng cu trc ba chiu c thc hin bng cch s dng tt c cc thng tin c bit cho n nay trn protein. Trnh t protein v cc rng buc cu trc cp trn l nhng thnh phn thit yu c s dng trong cc phn mm tnh ton. Mc ch l to ra mt tp hp ca cc cu trc ph hp vi cc gii hn th nghim, trong khi vn gi c cu trc hnh hc ca cc axit amin v cc peptit. ARIA l mt chng trnh c pht trin trong nhng nm gn y nh chng trnh t ng phn tch cc d liu NMR. N lm tng tc vic phn tch cc d liu thc nghim khc bng cch quy v NOESY quang ph. ARIA cng l mt h thng hiu chun ca Noe (Linge et al, 2001). S m phng cc cu trc nhit cao cho php nghin cu cc cu trc m khng cn a c h n nhit yu cu. Phng php ny cho php to ra cu trc n nh trc mi s thay i nhit . Cc cu trc ng vi nng lng thp nht c lu gi cho phin bn k tip.
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Hnh 2-20: S i din cho tnh ton ng hc phn t ca cc cu trc m phng luyn kim (MDSA) Hnh t www.bip.bham.ac.uk/osmart/bcm311_pef/slide27.html. Trong mt tnh ton ca cu trc ba chiu NMR, cc cu trc c to ra c chng ln xc nh cht lng v chnh xc. Cu trc khc cng tng t, cng vi gi tr ca RMSD. RMSD c th c tnh trn mch, cc nguyn t nng (C, O, N, S) hoc cc phn khc nhau ca chui protein. RMSD c tnh ton bng cch s dng phng trnh sau y.

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Phng trnh 4: Phng trnh ca RMSD .N l s lng mu. r i v r i i din cho cu trc. Cc vng N-cui v C-cui ca protein thng c cu cho RMSD cao hn. Loops (vng gia hai cu trc th cp) thng c mt khu vc cao hn RMSD v c cu trc nh xon v cc tm beta.

Ti liu tham kho : http://archimede.bibl.ulaval.ca/archimede/fichiers/21738/ http://fr.wikipedia.org/wiki/R%C3%A9sonance_magn%C3%A9tique_nucl%C3%A9aire

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http://glycobase.univ-lille1.fr/documents/livreRMN.PDF http://www.cours-medecine.info/biochimie/structure-proteines.html Cng c h tr: https://www.google.com.vn/ http://translate.google.com.vn/

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