Anna Adell, Mariona Bartrolí, Marina Brasó i Abel Eraso MEMBRANE PROTEINS.

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Transcript of Anna Adell, Mariona Bartrolí, Marina Brasó i Abel Eraso MEMBRANE PROTEINS.

Diapositiva 1

Anna Adell, Mariona Bartrol, Marina Bras i Abel ErasoMEMBRANEPROTEINS1Index IntroductionWhat is cell membrane?Integral ProteinsCrystallization

Transmembrane Proteins - Barrels-Helical

GPCRsWhat are G protein coupled receptors?ClassificationSequence conservationClass ASCOPChronologyAlignments2-Adrenergic ReceptorExtracellular Loop 2 (ECL2)Ligand binding siteIonic LockActivation

ConclusionsBibliography

21. Introduction What is cell membrane ?

Composed of a phospholipid bilayer with a collage of many different proteins, lipids and carbohydrates

It has many functions

They can be classified into two main categories: peripheral and integral proteins Fig2. Cell membrane with integral proteins embedded.Fig1. Cell membrane with peripheral proteins.Membrane Proteins:3

Extracellular regionIntracellular regionTransmembrane regionTransmembrane proteinsDomain Functions Extracellular Involved in cell cell signalingLet interactions with other molecules Transmembrane Forms channels and poresIntracellular Activates intracellular signaling pathways1. Introduction Fig3. Cell membrane with a transmembrane protein.4CRYSTALLIZATION. Why is it so complicated?Membrane protein structural biology:The most challenging targets in structural biology1. Introduction Difficulties in handling and crystallization of integral membrane proteins reside in the amphiphatic nature of their surface

They have hydrophobic transmembrane regions and two hydrophillic regions, one of each side of the membrane

As a result, membrane proteins are not soluble in aqueous buffers or in organic solvents

5CRYSTALLIZATION. Why is it so complicated?Trying to solubilize and crystalize transmembrane proteins:1. Introduction

Addition of detergents doesnt guarantee stably solubilized membrane proteinsNowadays, crystallization techniques try to add lipids to the detergents to improve the solubilization process To solubilize transmembrane proteins is necessary to add amphipathic molecules such as detergents to cover the transmembrane region6Index IntroductionWhat is cell membrane?Integral ProteinsCrystallization

Transmembrane Proteins - Barrels-Helical

GPCRsWhat are G protein coupled receptors?ClassificationSequence conservationClass ASCOPChronologyAlignments2-Adrenergic ReceptorExtracellular Loop 2 (ECL2)Ligand binding siteIonic LockActivation

ConclusionsBibliography

72. Types of transmembrane proteins - Barrels Are found in the outer membranes of Gram negative bacteria It is formed by an array of beta strands arranged in an antiparallel mannerFig4. NalP proteinFig5. OmpA proteinExtracellular Intracellular Fig6. NalP proteinFig7. OmpA proteinExtracellular Intracellular

82. Types of transmembrane proteins - Helical

Extracellular Fig9. Rodhopsin

Intracellular Fig8. PufX proteinFig10. Bitopic proteins and Polytopic proteins9Index IntroductionWhat is cell membrane?Integral ProteinsCrystallization

Transmembrane Proteins - Barrels-Helical

GPCRsWhat are G protein coupled receptors?ClassificationSequence conservationClass ASCOPChronologyAlignments2-Adrenergic ReceptorExtracellular Loop 2 (ECL2)Ligand binding siteIonic LockActivation

ConclusionsBibliography

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3. GPCRs What are G protein-coupled receptors ? Mediate the effect of numerous ligandsActivate different signaling pathsOne of the most important types of receptorsGPCRThe most important family of membrane proteins113. GPCRs Classification Fig13. Scheme of structure of the class C

Fig12. Scheme of structure of the class B

Fig11. Scheme of structure of the class A123. GPCRsSequence ConservationA_adenosine P..FAI..TI.........---.........STGFCA..ACHGCLF.IACFVLVLTQSSIFSLLA..IA..IDRY..IA.....I--RIPL-RY-NGLVT A_beta-1 P..FGA..TI.........VVW.........GRW-EY..GSFFCEL.WTSVDVLCVTASIETLCV..IA..LDRY..LA.....I--TSPF-RY-QSLLT A_beta P..FGA..AH.........ILM.........KMW-TF..GNFWCEF.WTSIDVLCVTASIETLCV..IA..VDRY..FA.....I--TSPF-KY-QSLLT A_rhodopsin T..STL..YT.........SLH.........GYF-VF..GPTGCNL.EGFFATLGGEIALWSLVV..LA..IERY..VV.....VCKPMSN---F--RFG B_Glucagon R..YSQ..KIg......ddLSV.........STW-LSdgAVAGCRV.AAVFMQYGIVANYCWLLVegLY..LHNL..LG.....L-ATLPERSFF----- B_Pituitary A..ISV..FI.........---.........KDWILY..AEQDSN-.HCFISTVECKAVMVFFHY..CV..VSNY..FW.....L-FIEGL--YLFTLLV B_Parathyroid Y..SGA..TLdeaerlteeELRaiaqappppATA-AA..GYAGCRVaVTFFLYFLATNYYWILVE..--..-GLY..LH.....SLIFMAF---FS---E B_Vasoactive IkdLAL..FD.........SGE.........SDQCSE..GSVGCKA.AMVFFQYCVMANFFWLLV..EG..LYLYtlLA.....V-SFFSERKYF----- C_Gamma-aminob P..LGLdgYH.........IGR.........NQF---..-PFVCQ-.-ARLWLLGLGFSLG----..--..---Y..GS.....MFTKIWW---VHTVFT C_Metabotropic P..FTL..IA.........--K.........PTT---..--TSCYL.QRLLVGLSSAMCYSALVT..KTnrIARI..LAgskkkICTRKP-------RFM C_Metabotropic P..STA..VC.........TLR.........RLG---..--LGTAF.SVCYSALLTKTN------..-R..IARI..FG.....-GAREGA---QRPRFI C_Taste P..TRP..AC.........LLR.........QALFAL..G-----F.TIFLSCLTVRS--FQLII..I-..----..FK.....FSTKVPTF--YHAWVQ

A_adenosine GTRAKGIIAIC...WVL.SFAIG-.LTPM....LGW-N----NCGQPKEGKNHSQGCGEGQVAC.LF-EDV--.-----VPMNYMVYFN..FFA.CVLVP A_beta-1 RARARGLVCTV...WAI.SALVSF.LPIL....MHWWRAESDEA--RR--CYNDPKC------C.D-F--VT-.------NRAY-AIAS..SVV.SFYVP A_beta KNKARVIILMV...WIV.SGLTSF.LPIQ....MHWYRATHQEA--IN--CYANETC------C.D-F--FT-.------NQAY-AIAS..SIV.SFYVP A_rhodopsin ENHAIMGVAFT...WVM.ALACAA.-PPL....AGWS-------------R---YIPEGLQCSC.GID--YYT.LKPEVNNESF-VIYM..FVV.HFTIP B_Glucagon -----------...---.SLYLG-.----....IGW------GAPMLFVVPWAVVK-------C.LF-ENV-Q.CWTSNDNMGFWWILR..FPV.FLAI- B_Pituitary ETFFPERRYFY...WYT.IIGWGT.PTVC....VTVW------A-TLR--LYFDDTG------CwDM-NDST-.------ALWW-VIKG..PVVgSIMVN B_Parathyroid KKYLWGFTVFG...WGLpAVFVAV.----....--WV---SVRA------TLANTG-------C.W-D--LS-.----SGNKKW-IIQV..PIL.ASIV- B_Vasoactive ----WGYILIG...WGV.PSTFT-.----....MVWT-----IA-RIH---FEDYG-------C.W-D---T-.-----INSSLWWIIKgpILT.SILVN C_Gamma-aminob KKEEKKEWRKTlepWKL.YATVGL.LVGMdvltLAIWQIVDPLHRTIE--TFAKEEP-----KE.DID--VSIlPQLEHCSSRKMNTWL..GIF.YGYKG C_Metabotropic SAWAQVIIA--...---.SILISVqLTLV....VTLI---------IM--E--PPMP-------.-IL--SY-.---PSIKEVYLICNT..SNL.GVVAP C_Metabotropic SPASQVAIC--...--L.ALISGQ.LLIV....VAWL---VVEA----PGTGKETAP-------.ER-REVV-.----TLRCNH-RDAS..MLG.SLAYN C_Taste NHGAGLFVMIS...SAA.QLLIC-.LTWL....VVW---TPLPA---R--EYQR-FP------H.LVMLECT-.---ETNSLGF-----..-IL.AFLYNFig14. Fragment of sequence alignment of the three main classes of GPCRs 133. GPCRs Class A Fig17. Scheme of structure of the class C

Fig16. Scheme of structure of the class B

Fig15. Scheme of structure of the class A14Class A. SCOP

Fig 18. Class A classification from SCOP (Structural Classification of Proteins)3. GPCRs15Class A. ChronologyBefore 20002000200720082008-2012 RhodopsinPalczewski et al. Aug. 20001 -Adrenergic Receptor Warne et al. July 2008

2-Adenosine Receptor Jaakola et al. Oct. 20082-Adrenergic ReceptorD1-RH1-RSPS13. GPCRsCherezov et al. Nov. 200716TM7TM6TM5TM4TM3TM2TM1adenosine AVLAILGNVLVCWAVWL..NSNLQNV...TNYFVVSLAAADIAVGVLAIP.FAITI---STGFCAACHGCLFIACFVLVLTQSSIFSLLAIAIDRYIAI--RIPL-RY-NGLV.TGTR.A.KGIIAICWVLSFAIG-LTPMLGW-N---- beta-1 VLLIVAGNVLVIVAIAK..TPRLQTL...TNLFIMSLASADLVMGLLVVP.FGATIVVWGRW-EYGSFFCELWTSVDVLCVTASIETLCVIALDRYLAI--TSPF-RY-QSLL.TRAR.A.RGLVCTVWAISALVSFLPILMHWWRAESD beta VLAIVFGNVLVITAIAK..FERLQTV...TNYFITSLACADLVMGLAVVP.FGAAHILMKMW-TFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAI--TSPF-KY-QSLL.TKNK.A.RVIILMVWIVSGLTSFLPIQMHWYRATHQ chemokine_r FLTGIVGNGLVILVMGY..QKKLRSM...TDKYRLHLSVADLLFVIT-LP.FWAVDAVANWY--FGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAI-VHATN-S-Q--RP.RKLL.AeKVVYVGVWIPALLLT-IPDFI--F-ANVSD2 IAVIVFGNVLVCMAVSR..EKALQTT...TNYLIVSLAVADLLVATLVMP.WVVYLEVVGEW-KFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAV--AMPML--YNTRYsSKRR.V.TVMISIVWVLSFTISC-PLLFGL-N---- galanin FLLGTVGNGLVLAVLLQpgPSAWQEPgstTDLFILNLAVADLCFILCCVP.FQATIYTLDAW-LFGALVCKAVHLLIYLTMYASSFTLAAVSVDRYLAV--RHPL-R-SRALR.TPRN.A.RAAVGLVWLLAALFSA--PYLSYY----- histamine ILITVAGNVVVCLAVGL..NRRLRNL...TNCFIVSLAITDLLLGLLVLP.FSAIYQLSCKW-SFGKVFCNIYTSLDVMLCTASILNLFMISLDRYCAV--MDPL-RY-PVLV.TPVR.V.AISLVLIWVISITLSFLSIHLGW-NSRNE melatonin TAVDVVGNLLVILSVLR..NRKLRNA...GNLFLVSLALADLVVAFYPYP.LILVAIFYDGW-ALGEEHCKASAFVMGLSVIGSVFNITAIAINRYCYICHSMAYHRIYR---.-RWH.T.PLHICLIWLLTV-VALLPNFFVGSL----muscarinic SLVTIIGNILVMVSIKV..NRHLQTV...NNYFLFSLACADLIIGVFSMNlYTLYT-VIGYW-PLGPVVCDLWLALDYVVSNASVMNLLIISFDRYFCV--TKPL-TY-PVKR.TTKM.A.GMMIAAAWVLSFILWA-PAILFW------delta CAVGLLGNVLVMFGIVR..YTKMKTA...TNIYIFNLALAD-ALATSTLP.FQSAKYLMETW-PFGELLCKAVLSIDYYNMFTSIFTLTMMSVDRYIAVCHPVKALD-FR---.TPAK.A.KLINICIWVLASGVGV-PIMVMAV--TRPkappa FVVGLVGNSLVMFVIIR..YTKMKTA...TNIYIFNLALAD-ALVTTTMP.FQSTVYLMNSW-PFGDVLCKIVISIDYYNMFTSIFTLTMMSVDRYIAVCHPVKALD-FR---.TPLK.A.KIINICIWLLSSSVGISAIVLGG-TKVREMu-type CVVGLFGNFLVMYVIVR..YTKMKTA...TNIYIFNLALAD-ALATSTLP.FQSVNYLMGTW-PFGTILCKIVISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALD-FR---.TPRN.A.KIINVCNWILSSAIG-LPVMFMAT-----orexin FVVALVGNTLVCLAVWR..NHHMRTV...TNYFIVNLSLADVLVTAICLP.ASLLVDITESW-LFGHALCKVIPYLQAVSVSVAVLTLSFIALDRWYAICHPLLF-K--S---.TARR.A.RGSILGIWAVSLAIMV--PQAAVM-----rhodopsin IVLGFPINFLTLYVTVQ..HKKLRTP...LNYILLNLAVADLFMVLGGFT.STLYTSLHGYF-VFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSN---F--RF.GENH.A.IMGVAFTWVMALACAA-PPLAGWS-----5-hydroxytrypIFCAVLGNACVVAAIAL..ERSLQNV...ANYLIGSLAVTDLMVSVLVLP.MAALYQVLNKW-TLGQVTCDLFIALDVLCCTSSILHLCAIALDRYWAI--TDPI-DYVN-KR.TPRR.A.AALISLTWLIGFLIS-IPPMLGW-R--TP somatostatin CAAGLGGNTLVIYVVLR..FAKMKTV...TNIYILNLAVAD-VLYMLGLP.FLATQNAASFW-PFGPVLCRLVMTLDGVNQFTSVFCLTVMSVDRYLAV--VHPL---SSARW.RRPRvA.KLASAAAWVLSLCMS-LPLLV--F-ADVQ

adenosine NCGQP.KEGKNHSQGCGEGQVACLF-EDV-------VPMNYMVYFNF...FACVLVPLLLMLGVYLR....I...FLAAR...HAA....K...SLAIIVGLFALCWLPLHI...IN....CFT...F.FC-Pbeta-1 EA--R.R--CYNDPKC------CD-F--VT-------NRAY-AIASS...VVSFYVPLCIMAFVYLR....V...FREAQ..