Autumn Semester 2024 Proteins, Karel Kubíček 1 F1190: Proteiny doc. Mgr. Karel Kubíček, Ph.D. Proteiny I] Doporučená literatura 1)Whitford, D.: Proteins – Structure and Function, John Wiley & Sons, Ltd. 2005 2)Cotterill, R.: Biophysics: An Introduction, John Wiley & Sons, Ltd. 2002 3)Voet, D, and Voetová, J.G.: Biochemie, Victoria Publishing 4)Murray, R.K., Granner, D. K., Mayes, P., A., Rodwell, V., W.: Harper’s Illustrated Biochemistry, Lange Medical Books, 2003 5)Schuenemann, V.: Biophysik: Eine Einfuehrung, Springer, 2005 6)Garrett, R.H., Grisham, C.M.: Biochemistry, 2nd ed., 1999 II] Aminokyseliny 1)Tvoří monomerní jednotky peptidů a proteinů 2)20 L-a-aminokyselin 3)Všechny aminokyseliny složeny ze tří částí – NH2- (amino) skupina, -COOH (karboxylová) skupina, -Ca-R (alfa uhlík s postranním řetězcem) 4) Esenciální a neesenciální kyseliny – organismus si je umí syntetizovat nebo je potřeba je přijímat v potravě 5) Označujeme třípísmennými nebo jednopísmennými zkratkami (zápis Val-Thr-Ile-Pro nebo VTIP – u jednopísmenného zápisu bez pomlčky) 1) 2 Proteins, Karel Kubíček Autumn Semester 2024 Levels of Protein Structure Autumn Semester 2024 3 Proteins, Karel Kubíček 6) Aminokyseliny s alifatickým postranním řetězcem C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\ala.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\gly.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\ile.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\leu.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\val.gif 4 Proteins, Karel Kubíček Autumn Semester 2024 7) Aminokyseliny s hydroxylovou (OH) skupinou C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\ser.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\thr.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\tyr.gif 8) Aminokyseliny s atomem síry C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\cys.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\met.gif 5 Proteins, Karel Kubíček Autumn Semester 2024 9) Aminokyseliny s acidickými skupinami nebo jejich amidy C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\asn.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\asp.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\gln.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\glu.gif 10) Imino kyselina C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\pro.gif 6 Proteins, Karel Kubíček Autumn Semester 2024 11) Aminokyseliny s basickými skupinami C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\arg.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\his.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\lys.gif 12) Aminokyseliny s aromatickými kruhy C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\phe.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\trp.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\tyr.gif C:\Documents and Settings\lelli\Documenti\Immagini\AMMINOACIDI\his.gif 7 Proteins, Karel Kubíček Autumn Semester 2024 A R N D C Q E G H I L K M F P S T W Y V 8 Autumn Semester 2024 Proteins, Karel Kubíček Proteins, Karel Kubíček 9 III] Peptidy, proteiny – vznik polymerů polymerizační reakcí polymerizace.png Autumn Semester 2024 peptidova_vazba_2.png Proteins, Karel Kubíček 10 IV] Peptidová vazba – pseudo dvojitá vazba => amidová rovina peptidova_vazba.png peptidova_vazba_1.png peptidova_vazba_3.png Autumn Semester 2024 Proteins, Karel Kubíček 11 parametry_polypept_reterze.png V] Proteinová páteř, primární struktura, číslování od N-konce (terminu) směrem k C-konci Autumn Semester 2024 Autumn Semester 2024 Proteins, Karel Kubíček 12 Informace o 3D (proteinové) struktuře jsou zapsány v kartézských souřadnicích Nejrozšířenější formát PDB (ProteinDataBank, www.pdb.org ) ATOM 128 N HIS O 18 20.321 6.124 17.761 1.00 11.40 ATOM 129 CA HIS O 18 21.097 5.169 18.563 1.00 13.62 ATOM 130 C HIS O 18 22.581 5.413 18.454 1.00 17.00 ATOM 131 O HIS O 18 23.031 5.592 17.321 1.00 15.45 ATOM 132 CB HIS O 18 20.883 3.747 18.034 1.00 16.68 ATOM 133 CG HIS O 18 19.557 3.103 18.437 1.00 11.72 ATOM 134 ND1 HIS O 18 19.252 2.806 19.725 1.00 11.66 ATOM 135 CD2 HIS O 18 18.479 2.751 17.657 1.00 16.32 ATOM 136 CE1 HIS O 18 18.021 2.238 19.730 1.00 14.91 ATOM 137 NE2 HIS O 18 17.552 2.185 18.473 1.00 17.58 HETATM 1633 NC HEM O 104 15.182 3.191 16.831 1.00 21.22 HETATM 1634 C1C HEM O 104 15.433 3.334 15.488 1.00 17.49 HETATM 1635 C2C HEM O 104 15.046 4.605 15.145 1.00 31.21 HETATM 1636 C3C HEM O 104 14.623 5.242 16.323 1.00 14.38 HETATM 1637 C4C HEM O 104 14.661 4.346 17.360 1.00 14.50 HETATM 1638 CMC HEM O 104 15.299 5.349 13.850 1.00 15.54 HETATM 1639 CAC HEM O 104 14.314 6.707 16.409 1.00 31.67 HETATM 1640 CBC HEM O 104 13.170 7.262 15.615 1.00 10.23 HETATM 1609 FE HEM O 104 15.801 1.483 17.954 1.00 9.37 ftp://ftp.wwpdb.org/pub/pdb/doc/format_descriptions/Format_v33_A4.pdf Autumn Semester 2024 Proteins, Karel Kubíček 13 vazba.png Autumn Semester 2024 Proteins, Karel Kubíček 14 porovnani_delky_vazeb.png Autumn Semester 2024 Proteins, Karel Kubíček 15 histogram_vazebnych_delek.png Autumn Semester 2024 Proteins, Karel Kubíček 16 uhly.png Autumn Semester 2024 Proteins, Karel Kubíček 17 hodnoty_uhlu.png Autumn Semester 2024 Proteins, Karel Kubíček 18 dihedralni_uhel.png Autumn Semester 2024 Proteins, Karel Kubíček 19 vypocet_dihedralnich_uhlu.png Autumn Semester 2024 Proteins, Karel Kubíček 20 dihedralni_uhly.png amide_plane.png Proteins, Karel Kubíček 21 AEKGYA.png f - CO, N, Ca, CO (CO někdy značeno C’) y - N, Ca, CO, N Autumn Semester 2024 Autumn Semester 2024 Proteins, Karel Kubíček 22 Structural element f y n d p a-helix -57 -47 3.6 1.5 5.5 310-helix -49 -26 3.0 2.0 6.0 b-helix -57 -70 4.4 1.1 5.0 Polyproline II helix -79 +149 3.0 3.1 9.4 Parallel b-strand -119 +113 2.0 3.2 6.4 Antiparallel b-strand -139 +135 2.0 3.4 6.8 Structural parameters for protein secondary structures fand y are the conformational angles of the mainchain, with w ~180°(trans conformation) n = number of residues per turn d = displacement between successive residues along the helix/strand axis p = the pitch of helix/strand, the distance along the helix/strand axis of a complete sec. struct. element. Note that p=n x d (equation is exact, error is in rounding of n and d) Proteins, Karel Kubíček 23 ramachandran.png VI] Ramachandranův diagram Autumn Semester 2024 Autumn Semester 2024 Proteins, Karel Kubíček 24 ramachandran_proteins_2003_50_437.png ramachandran_proteins_2003_50_437B.png Ramachandranův diagram – komplet Autumn Semester 2024 Proteins, Karel Kubíček 25 https://proteopedia.org/wiki/index.php/Tutorial:Ramachandran_principle_and_phi_psi_angles Text Description automatically generated Autumn Semester 2024 Proteins, Karel Kubíček 26 Autumn Semester 2024 Proteins, Karel Kubíček 27 helix_3.png Proteins, Karel Kubíček 28 VII] Sekundární struktura 1)a-šroubovice (a-helix) 2)b-skládaný list (b-sheet) 3)Ohyb, smyčka (loop/turn) helix_1.png helix_2.png helix_zobr.png Autumn Semester 2024 Autumn Semester 2024 Proteins, Karel Kubíček 29 Proteins, Karel Kubíček 30 VII] Sekundární struktura 1)a-šroubovice (a-helix) 2)b-skládaný list (b-sheet) 3)Ohyb, smyčka (loop/turn) beta_sheet.png antiparalelní uspořádání paralelní uspořádání beta_sheet_zobr.png beta_sheet_zobr.png beta_sheet_zobr.png Autumn Semester 2024 Proteins, Karel Kubíček 31 VII] Sekundární struktura 1)a-šroubovice (a-helix) 2)b-skládaný list (b-sheet) 3)Ohyb, smyčka (loop/turn) beta_smycka.png gama_beta_smycka.png b-smyčka/ohyb (4 residua) g-smyčka/ohyb (3 residua) Autumn Semester 2024 1 2 3 4 5 6 8 7 9 10 Autumn Semester 2024 Proteins, Karel Kubíček 32 Ostatní motivy sekundární struktury : 1.polyprolinová šroubovice I & II – levotočivá, 3.3 nebo 3 (PPI, PPII, v uvedeném pořadí) aminokyseliny/otočku 2.šroubovice 310 (srovnej s 3.613)– pravotočivá, 3 aminokyseliny/otáčku, 10 atomů vytváří kruh uzavřený vodíkovou vazbou, např. poly-Ala 3.p-šroubovice – pravotočivá, 4.1 aminokyseliny/otáčku 4.b-šroubovice – vzniká uspořádáním b-skládaných listů do pravo- i levotočivé šroubovice. Autumn Semester 2024 Proteins, Karel Kubíček 33 Primary sequence reveals important clues about a protein One sequence keeps silent about its three-dimensional structure Two aligned sequences whisper But tables of many aligned sequences shout out loud Primary sequence reveals important clues about a protein • Evolution conserves amino acids that are important to protein structure and function across species. Sequence comparison of multiple “homologs” of a particular protein reveals highly conserved regions that are important for function. • Clusters of conserved residues are called “motifs” -- motifs carry out a particular function or form a particular structure that is important for the conserved protein. DnaG E. coli ...EPNRLLVVEGYMDVVAL... DnaG S. typ ...EPQRLLVVEGYMDVVAL... DnaG B. subt ...KQERAVLFEGFADVYTA... gp4 T3 ...GGKKIVVTEGEIDMLTV... gp4 T7 ...GGKKIVVTEGEIDALTV... small hydrophobic large hydrophobic polar positive charge negative charge motif Autumn Semester 2024 34 Proteins, Karel Kubíček :::: ** :* . Autumn Semester 2024 Proteins, Karel Kubíček 35 Structure of function identified Motif Nucleotide-binding site G*G**G N-glycosylation site N*S or N*T Nuclear protein transit sequence KKKRKV Factor IX proteinase cleavage site IEGR Serine proteinase active site GDSGG Acid proteinase active site FDTGS Fibronectin cell adhesion sequence RGDS Copper binding site H***H…H or H****H…H * = any single amino acid (AA) *** = several AAs ... = any number of AAs Some motifs in protein sequences Charged and polar R-groups tend to map to protein surfaces Autumn Semester 2024 36 Proteins, Karel Kubíček Proteins, Karel Kubíček 37 Protein folding The Levinthal paradox states that if an averaged sized protein would sample all possible conformations before finding the one with the lowest energy, the whole process would take billions of years. Proteins typically fold within 0.1 and 1000 seconds, therefore the protein folding process must be directed some way through a specific folding pathway. Autumn Semester 2024 Protein folding Anfinsen's Classic Experiment: The "Protein Folding Problem" asks a very simple question: "How does the primary structure of a protein determine its 2ｰ and 3ｰ structure?". We have known for many decades that proteins fold into their correct 3-D structures inside the cell. But correct folding during synthesis on the ribosome or later with assistance from unknown cellular factors could explain the in vivo results. In the 1960's, Anfinsen and his coworkers performed a series of seminal experiments in vitro that answered a key part of the problem. The original work led Anfinsen to propose his "Thermodynamic Hypothesis", which states that the native conformation of a protein is adopted spontaneously. In other words, there is sufficient information contained in the protein sequence to guarantee correct folding from any of a large number of unfolded states. A schematic diagram of Anfinsen's experiment is shown below in two parts: Autumn Semester 2024 38 Proteins, Karel Kubíček anfinsen.png Autumn Semester 2024 39 Proteins, Karel Kubíček Anfin Autumn Semester 2024 40 Proteins, Karel Kubíček Proteins, Karel Kubíček 41 VII] Terciární struktura proteinů primar-sek-terciar_strukt.png 1) Kolagen Primární struktura: Sekundární struktura: Terciární struktura: Autumn Semester 2024 Proteins, Karel Kubíček 42 2) Disulfidický můstek: 2-SH (z cysteinu)-> -S-S- IgG.png H2L2_IgG.png Disulfidické můstky v imunoglobulinu G Autumn Semester 2024 Proteins, Karel Kubíček 43 3) Strukturní motivy v proteinech motivy_proteinu.png b-meandr Řecký klíč Swiss/Jelly roll Autumn Semester 2024 Proteins, Karel Kubíček 44 3D-struktury.png Autumn Semester 2024 Proteins, Karel Kubíček 45 VIII] Kvartérní struktura proteinů 1)Multimery 2)Homo-/hetero- -mery kvarterni_struktura.png Autumn Semester 2024 Examples of other quaternary structures Tetramer Hexamer Filament SSB DNA helicase Recombinase Allows coordinated Allows coordinated DNA binding Allows complete DNA binding and ATP hydrolysis coverage of an extended molecule Autumn Semester 2024 46 Proteins, Karel Kubíček Autumn Semester 2024 Proteins, Karel Kubíček 47 Grigoryan and Keating, 2008 Autumn Semester 2024 Proteins, Karel Kubíček 48 Autumn Semester 2024 Proteins, Karel Kubíček 49 Autumn Semester 2024 Proteins, Karel Kubíček 50 Autumn Semester 2024 Proteins, Karel Kubíček 51 Autumn Semester 2024 Proteins, Karel Kubíček 52 Confidence in structural features of proteins determined by X-ray crystallography (estimates are very rough and strongly depend on the quality of the data) Structural feature Resolution 5 Å 3 Å 2.5 Å 2 Å 1.5 Å Chain tracing - Fair Good Good Good Secondary structure Helices fair Fair Good Good Good Sidechain conformations - - Fair Good Good Orientation of peptide planes - - Fair Good Good Protein hydrogen atoms visible - - - - Good Proteins, Karel Kubíček 53 XII] Metody pro určování třídimenzionální struktury (bio)molekul na atomární úrovni 1)NMR – nukleární (=jaderná) magnetická rezonance – měření (také) v kapalném prostředí 2)Rentgenová krystalografie - (měření především v krystalu) 3)Kryo-elektronová mikroskopie Autumn Semester 2024 Autumn Semester 2024 Proteins, Karel Kubíček Screen Shot 2015-09-29 at 11.15.37.png J Mol Graph Model 19, 2001, 26-59 54 Autumn Semester 2024 Proteins, Karel Kubíček Screen Shot 2015-09-29 at 11.11.31.png BiochimBiophysActa 1804, 2010, 1231-64 55 Proteins, Karel Kubíček 56 IX] Funkce proteinů: 1)Enzymy – katalyzátory biologických reakcí 2)Transportní proteiny (hemoglobin) 3)Regulační proteiny (např. hormon insulin) 4)Skladovací (storage) – např. ovalbumin – zdroj dusíku pro vyvíjející se ptačí embryo 5)“Pohybové” proteiny – actin, myosin, tubulin, dynein, kinesin 6)Strukturní proteiny – zajišťujicí vytvoření a udržení biologické struktury – a-keratin, kolagen, elastin, fibroin etc. 7)Ochranné – imunoglobuliny, fibrinogen, thrombin 8) 8) Autumn Semester 2024 Proteins, Karel Kubíček 57 X] Proteinová databáze – PDB WWW.RCSB.ORG XI] Vizualizační programy 1)PyMol 2)Chimera 3)VMD 4)MolMol 5)RasMol 6)Insight II Autumn Semester 2024