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Proteiinianalyysi (2 ov)

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Esitys aiheesta: "Proteiinianalyysi (2 ov)"— Esityksen transkriptio:

1 Proteiinianalyysi 52930 (2 ov)
Liisa Holm

2 Organisaatio Luennot & Laskuharjoitukset Tentti Oheislukemisto
, ke, to 14-16, LS 2012 Tentti Bonusta aktiivisuudesta laskuharjoituksissa Oheislukemisto Lesk: Introduction to bioinformatics. Oxford University Press.

3 Aikataulu 30.3. ke Luento 31.3. to 6.4. ke Laskuharjoitus 1 7.4. to
Tentti

4 Kurssin tavoitteet miten proteiinisekvenssejä luetaan
proteiinien luokittelujärjestelmät sekvenssi – rakenne – funktio evoluutio

5 Muut kurssit Esitiedot: Soveltaminen:
Geneettinen bioinformatiikka 1-2 ov sekvenssivertailu fylogeniapuut Soveltaminen: Proteiinianalyysin harjoitustyöt 3 ov webbityökalujen käyttö

6 Johdanto

7 Proteiinien merkitys Proteiinit tekevät kaiken työn solussa ja ovat osallisina: Geenisäätelyssä Metaboliassa Signaloinnissa Tukirangassa Kuljetuksessa Solunjakautumisessa

8 Structural proteins Collagen 1K6F

9 Actin and muscles

10 Enzymes Catalytic triad: Asp, Ser, His 1CHO

11 Transcription factors
Ligand DNA 1L3L

12

13 Mistä proteiinit tulevat?
DNA > RNA > proteiini geneettinen koodi DNAn emäskolmikko koodaa yhtä aminohappoa 20 aminohappoa lineaarinen sekvenssi tyypillinen pituus aminohappoa keskimäärin noin 150 aminohappoa

14 Suuri yllätys … DNA:n rakenne on hyvin säännölinen
Watson & Crick (1953)

15 Myoglobiini Proteiinin rakenteesta puuttuu symmetria
Kendrew & Perutz (1957) 1mbn

16 Proteiinit ovat erikoislaatuisia polymeerejä:
Tietyllä proteiinilla on aina sama aminohapposekvenssi Proteiinin sekvenssi määräytyy DNA-sekvenssin perusteella Tietyllä proteiinilla on aina uniikki kolmiulotteinen rakenne. Proteiinin rakenne määräytyy aminohapposekvenssin perusteella. aina = biologinen aina (poikkeuksia löytyy)

17 Ei funktiota ilman rakennetta
Luonnon proteiinit laskostuvat spesifiseksi kolmiulotteiseksi rakenteeksi komplementaarinen interaktiopartnerille Denaturaatio tuhoaa funktion

18 Evoluutio Sekvenssi – Rakenne - Funktio
Luonnonvalinta DNA-sekvenssi Proteiinin funktio Proteiinin sekvenssi Proteiinin rakenne

19 Sekvenssi

20 proteiinien identifiointi
klassinen biokemia proteiinin puhdistus molekyylipaino isoelektrinen piste CD- ym. spektroskopia jne. laskennallinen analyysi DNA-sekvenssi  geenintunnistus, eksonit/intronit  käännös proteiiniksi sekvenssivertailut post-genomiikka transkriptioprofilointi, proteiini-proteiini-interaktiot, ym.

21 Historiaa 1953 DNA:n rakenne 1955 Ensimmäinen proteiinisekvenssi 1957
Myoglobiinin rakenne 1975 DNA:n sekvensointimenetelmät 1977 fX-174 faagin ’genomi’ 1995 Haemophilus influenzaen genomi 1996 Hiivan genomi 1998 Sukkulamadon genomi 2000 Ihmisen genomi Rakennegenomiikkaprojekti

22 Genomit DNA-sekvensointi genomiprojektit
entsymaattinen synteesi, spesifiset terminaattorit proteiinisekvenssit johdetaan DNA-sekvenssistä ORF, open reading frame varmennus: linjaus tunnetun EST:n tai cDNA:n tai proteiinin kanssa eukaryoottien eksoni-introni-ongelma genomiprojektit noin 136 organismia eukaryootteja, arkebakteereja ja eubakteereja

23

24 Proteome coverage Organism Biological Features proteins S. cerevisiae
(yeast) Genes for existence as a single-celled organism with the basic structure and organisation of the eukaryotic cell 6231 E. coli (bacterium) Genes for growth on external sources of energy, molecular cell transport through cell membrane, metabolic pathways and replication as a single cell C. elegans (Nematode) Genes for development by a unique cell lineage, nervous system and reproduction 22515 D. melanogaster (Fruit fly) Model for developmental processes by hormones and cell-cell interactions 17341 H. sapiens (human) Duplicates many gene functions in other model organisms and in addition includes control of higher brain functions 28814 Note: C. Elegans interactome paper claims 15% of proteome coverage – this is achieved using in silico methods About 136 complete proteomes deduced from complete genomes.

25 Täydellinen proteomi varmuus ”puuttuvista” geeneistä
kaikki geenit eivät ekspressoidu samaan aikaan ja samassa paikassa vaihtoehtoinen silmukointi, post-translationaaliset modifikaatiot: yhdestä geenistä voikin tulla monta proteiinia glykosylaatio fosforylaatio

26 Tietokantoja EBI NCBI - Entrez nrdb, ’non-redundant database’
NCBI - Entrez nrdb, ’non-redundant database’ aminohappoa sekvenssiä

27 Rakenne

28 Protein structure Primary structure Secondary structure
Super-secondary structure Tertiary structure Quaternary structure

29 Secondary structure α-helix β-sheet ... backbone regular patterns
no amino acid side chains regular patterns of hydrogen-bonds backbone torsion angles types of secondary structure α-helix β-sheet ...

30 α-Helix hydrogen bond pattern: n, n+4 β-Sheet

31 β-sheet β-strands view from the side view from the top

32 Cartoon representation
2TRX 2AAC

33 Supersecondary structures
local arrangments of secondary structure elements

34 Tertiary structure 1coh

35 Quaternary structure 1coh

36 Protein structure determination
Protein expression membrane proteins aggregation X-Ray crystallography NMR (nuclear magnetic resonance) Cryo-EM (electron microscopy)

37 Structures by X-ray crystallography
Crystallize protein Collect diffraction patterns Improve iteratively: Calculate electron density map Phase problem Fit amino acid trace through map

38 X-ray crystallography
Crystallization “An art as much as a science” Charges

39 Diffraction and electron density maps
Intensities X-ray source Crystal Diffraction pattern

40 Iterative refinement Resolution Higher resolution =
more accurate positioning of atoms

41 NMR Create highly concentrated protein solution Record spectra
Assign peaks to residues Calculate constraints Compute structure

42 NMR spectra 1D 2D

43 Distance constraints from NMR
From the sequence Topology Bond angles Bond lengths From the NMR experiment Torsion angles Distance constraints R CO H Torsion angle

44 Ensemble of structures
SH3-domain 1aey

45 What is the true protein structure?
X-Ray “frozen” state of a protein crystal contacts large protein structure NMR protein in solution limited in size

46 Molecular complexes via X-ray
30 S subunit of the ribosome Protein RNA 1fjg

47 Cryo-EM Single particle image reconstruction
Bacteriophage MS2 Koning et al. (2003)

48 Fitting X-Ray structures into density maps

49 GroEL-complex Hemoglobin 1gr6

50 Protein structure databases

51 Molekulaarinen funktio

52 Post-genomic view: Function = S interactions
(From left to right, figures adapted from Olsen Group Docking Page at Scripps, Dyson NMR Group Web page at Scripps, and from Computational Chemistry Page at Cornell Theory Center).

53 Enzymes Catalytic triad: Asp, Ser, His 1CHO

54 Mechanism Enzymes speed up chemical reactions
Enzymes are not consumed by the reaction Stabilization of the transition state Charge-relay cascade

55 Convergent evolution in serine proteases
same reaction same mechanism same orientation of catalytic residues different structures Chymotrypsin: His-57, Asp-102, Ser-195 Subtilisin: Asp-32, His-64, Ser-221 1cho / 1sib

56 Substrate specificity
Perona & Craik (1997)

57 Transcription factors
Ligand DNA 1L3L

58 Hydrogen bonding pattern
Vannini (2002)

59 Funktion määritys Biokemiallinen analyysi
Geneettinen analyysi, fenotyyppi Proteiini-proteiini-interaktio Työläitä menetelmiä Määritysmenetelmä usein räätälöitävä erikseen jokaiselle funktiolle

60 Evoluutio

61 Evoluutio Sekvenssi – Rakenne - Funktio
Luonnonvalinta DNA-sekvenssi Proteiinin funktio Proteiinin sekvenssi Proteiinin rakenne

62

63 Application: Finding Homologs

64 Application: Finding Homologues
Find Similar Ones in Different Organisms Human vs. Mouse vs. Yeast Easier to do Expts. on latter! (Section from NCBI Disease Genes Database Reproduced Below.) Best Sequence Similarity Matches to Date Between Positionally Cloned Human Genes and S. cerevisiae Proteins Human Disease MIM # Human GenBank BLASTX Yeast GenBank Yeast Gene Gene Acc# for P-value Gene Acc# for Description Human cDNA Yeast cDNA Hereditary Non-polyposis Colon Cancer MSH2 U e-261 MSH2 M DNA repair protein Hereditary Non-polyposis Colon Cancer MLH1 U e-196 MLH1 U DNA repair protein Cystic Fibrosis CFTR M e-167 YCF1 L Metal resistance protein Wilson Disease WND U e-161 CCC2 L Probable copper transporter Glycerol Kinase Deficiency GK L e-129 GUT1 X Glycerol kinase Bloom Syndrome BLM U e-119 SGS1 U Helicase Adrenoleukodystrophy, X-linked ALD Z e-107 PXA1 U Peroxisomal ABC transporter Ataxia Telangiectasia ATM U e-90 TEL1 U PI3 kinase Amyotrophic Lateral Sclerosis SOD1 K e-58 SOD1 J Superoxide dismutase Myotonic Dystrophy DM L e-53 YPK1 M Serine/threonine protein kinase Lowe Syndrome OCRL M e-47 YIL002C Z Putative IPP-5-phosphatase Neurofibromatosis, Type NF1 M e-46 IRA2 M Inhibitory regulator protein Choroideremia CHM X e-42 GDI1 S GDP dissociation inhibitor Diastrophic Dysplasia DTD U e-38 SUL1 X Sulfate permease Lissencephaly LIS1 L e-34 MET30 L Methionine metabolism Thomsen Disease CLC1 Z e-31 GEF1 Z Voltage-gated chloride channel Wilms Tumor WT1 X e-20 FZF1 X Sulphite resistance protein Achondroplasia FGFR3 M e-18 IPL1 U Serine/threoinine protein kinase Menkes Syndrome MNK X e-17 CCC2 L Probable copper transporter

65 Application: Finding Homologues (cont.)
Cross-Referencing, one thing to another thing Sequence Comparison and Scoring Analogous Problems for Structure Comparison Comparison has two parts: (1) Optimally Aligning 2 entities to get a Comparison Score (2) Assessing Significance of this score in a given Context

66 Mitä hyötyä proteiinien bioinformatiikasta voisi olla?
kuvitteellinen virusepidemia DNA-sekvenssi vertailu tunnettuihin viruksiin [10] antiviruslääkkeiden kehittely virukselle spesifiset proteiinit: replikaatio- tai vaippaproteiinit [01] tietokantahaut [15] homologiamallitus [25] / ab initio [55] lääkesuunnittelu, vasta-aineterapia [50] lääkeaineen biologinen siedettävyys [75]

67 sekvenssi  rakenne

68 Aminohappojen ominaisuudet
Proteiinit ovat itseorganisoituvia lineaarisia heteropolymeerejä, joiden sekvenssi on jalostunut luonnonvalinnassa 20 aminohappoa peptidirunko sivuketju sekvenssi määrää rakenteen

69 Amino Acids with Aliphatic R-Groups Glycine Gly - G 2.4 9.8
Table of a-Amino Acids Found in Proteins Amino Acid Symbol Structure* pK1 (COOH) pK2 (NH2) pK R Group Amino Acids with Aliphatic R-Groups Glycine Gly - G                                       2.4 9.8 Alanine Ala - A                                               9.9 Valine Val - V                                                         2.2 9.7 Leucine Leu - L                                                                        2.3 Isoleucine Ile - I                                                                              Non-Aromatic Amino Acids with Hydroxyl R-Groups Serine Ser - S                                                           9.2 ~13 Threonine Thr - T 2.1 9.1 Amino Acids with Sulfur-Containing R-Groups Cysteine Cys - C                                                          1.9 10.8 8.3 Methionine Met-M                                                                             9.3 Acidic Amino Acids and their Amides Aspartic Acid Asp - D                                                                    2.0 3.9 Asparagine Asn - N                                                                     8.8 Glutamic Acid Glu - E                                                                                   9.5 4.1 Glutamine Gln - Q                                                                                      Basic Amino Acids Arginine Arg - R                                                                                         1.8 9.0 12.5 Lysine Lys - K Histidine His - H                                                                         6.0 Amino Acids with Aromatic Rings Phenylalanine Phe - F Tyrosine Tyr - Y                                                                                  10.1 Tryptophan Trp-W                                                                                9.4 Imino Acids Proline Pro - P                                        10.6 *Backbone of the amino acids is red, R-groups are black

70 Aminohappojen ominaisuuksia

71 levels of complexity in folding

72 WHAT DO WE KNOW ABOUT PROTEIN FOLDING?
water soluable proteins are "globular," tight packed, water excluded from interior, folded up. bond lengths and bond angles don't vary much from equilibrium positions. structures are stable and relatively rigid. folding possibilities are limited, both along the backbone chain and within the side chain groups. folding motifs are used repetitively. with similar proteins (say from different organisms) structure tends to be more conserved than the exact sequence of amino acids. although sequence must determine structure, it is not yet possible to predict the entire structure from sequence accurately. Net stability corresponds to a few hydrogen bonds.

73 Sekundaarirakenne > tutorial
proteiini on kuin rasvapisara vedessä peptidirungon pooliset ryhmät muodostavat vetysidoksia NH -- O=C syntyy säännönmukaisia sekundaarirakenteita sivuketju moduloi sekundaarirakennepreferenssejä

74 DSSP Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometrical Features W. Kabsch & C. Sander Biopolymers 22, (1983)

75 Hydrogen bonds +0.20e N H -0.20e O C +0.42e -0.42e E ~ q1 q2 [ 1/r(ON) + 1/r(CH) – 1/r(CN) – 1/r(OH) Ideal H-bond is co-linear, r(NO)=2.9 A and E=-3.0 kcal/mol Cutoffs in DSSP allow 2.2 A excess distance and ±60º angle

76 Elementary H-bond patterns
n-turn(i) =: Hbond(i,i+n), n=3,4,5 Parallel bridge(i,j) =: [ Hbond(i-1,j) AND Hbond(j,i+1) ] OR [ Hbond(j-1,i) AND Hbond(i,j+1) ] Antiparallel bridge(i,j) =: [ Hbond(i,j) AND Hbond(j,i) ] OR [ Hbond(i-1,j+1) AND Hbond(j-1,i+1) ]

77 N-turns -N-C-C--N-C-C--N-C-C--N-C-C- H O H O H O H O
-N-C-C--N-C-C--N-C-C--N-C-C--N-C-C- H O H O H O H O H O 4-turn -N-C-C--N-C-C--N-C-C--N-C-C-—N-C-C-—N-C-C- H O H O H O H O H O H O 5-turn

78 Parallel bridge -N-C-C--N-C-C--N-C-C--N-C-C—N-C-C- H O H O H O H O H O

79 Antiparallel bridge -N-C-C--N-C-C--N-C-C--N-C-C- H O H O H O H O
O H O H O H O H -C-C-N--C-C-N--C-C-N--C-C-N- Antiparallel beta-sheet is significantly more stable due to the well aligned H-bonds.

80 Cooperative H-bond patterns
4-helix(i,i+3) =: [4-turn(i-1) AND 4-turn(i)] 3-helix(i,i+2) =: [3-turn(i-1) AND 3-turn(i)] 5-helix(i,i+4) =: [5-turn(i-1) AND 5-turn(i)] Longer helices are defined as overlaps of minimal helices

81 Beta-ladders and beta-sheets
Ladder =: set of one or more consecutive bridges of identical type Sheet =: set of one or more ladders connected by shared residues Bulge-linked ladder =: two ladders or bridges of the same type connected by at most one extra residue on one strand and at most four extra residues on the other strand

82 3-state secondary structure
Helix Strand Loop Quoted consistency of secondary structure state definition in structures between sequence-similar proteins is ~70 % Richer descriptions possible E.g. phi-psi regions

83 Amino acid preferences for different secondary structure
Alpha helix may be considered the default state for secondary structure. Although the potential energy is not as low as for beta sheet, H-bond formation is intra-strand, so there is an entropic advantage over beta sheet, where H-bonds must form from strand to strand, with strand segments that may be quite distant in the polypeptide sequence. The main criterion for alpha helix preference is that the amino acid side chain should cover and protect the backbone H-bonds in the core of the helix. Most amino acids do this with some key exceptions. alpha-helix preference: Ala,Leu,Met,Phe,Glu,Gln,His,Lys,Arg

84 The extended structure leaves the maximum space free for the amino acid side chains: as a result, those amino acids with large bulky side chains prefer to form beta sheet structures: just plain large:Tyr, Trp, (Phe, Met) bulky and awkward due to branched beta carbon:Ile, Val, Thr large S atom on beta carbon:Cys The remaining amino acids have side chains which disrupt secondary structure, and are known as secondary structure breakers: side chain H is too small to protect backbone H-bond:Gly side chain linked to alpha N, has no N-H to H-bond; rigid structure due to ring restricts to phi = -60: Pro H-bonding side chains compete directly with backbone H-bonds: Asp, Asn, Ser Clusters of breakers give rise to regions known as loops or turns which mark the boundaries of regular secondary structure, and serve to link up secondary structure segments.


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