Proteiinianalyysi 52930 (2 ov) Liisa Holm

Organisaatio Luennot & Laskuharjoitukset Tentti Oheislukemisto 30.3.-28.4.2005, ke, to 14-16, LS 2012 http://www.bioinfo.biocenter.helsinki.fi:8080/downloads/teaching/spring2005/proteiinianalyysi/index.html Tentti Bonusta aktiivisuudesta laskuharjoituksissa Oheislukemisto Lesk: Introduction to bioinformatics. Oxford University Press.

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

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

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

Johdanto

Proteiinien merkitys Proteiinit tekevät kaiken työn solussa ja ovat osallisina: Geenisäätelyssä Metaboliassa Signaloinnissa Tukirangassa Kuljetuksessa Solunjakautumisessa http://www.websters-online-dictionary.org/definition/english/ce/cell.html

Structural proteins Collagen 1K6F http://www.aw-bc.com/mathews/ch06/fi6p13ad.htm

Actin and muscles

Enzymes Catalytic triad: Asp, Ser, His 1CHO

Transcription factors Ligand DNA 1L3L

Mistä proteiinit tulevat? DNA > RNA > proteiini geneettinen koodi DNAn emäskolmikko koodaa yhtä aminohappoa 20 aminohappoa lineaarinen sekvenssi tyypillinen pituus 100-400 aminohappoa keskimäärin noin 150 aminohappoa

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

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

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)

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

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

Sekvenssi

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.

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

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

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 4356 - 5333 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.

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

Tietokantoja EBI NCBI - Entrez nrdb, ’non-redundant database’ http://www.ebi.ac.uk http://www.ebi.ac.uk/proteome NCBI - Entrez http://www.ncbi.nlm.nih.gov nrdb, ’non-redundant database’ 490.374.618 aminohappoa 1.504.726 sekvenssiä

Rakenne

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

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 ...

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

β-sheet β-strands view from the side view from the top http://broccoli.mfn.ki.se/pps_course_96

Cartoon representation 2TRX 2AAC

Supersecondary structures local arrangments of secondary structure elements http://www.expasy.org/swissmod/course/text/chapter2.htm

Tertiary structure 1coh

Quaternary structure 1coh

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

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

X-ray crystallography Crystallization “An art as much as a science” Charges http://crystal.uah.edu/~carter/protein/crystal.htm

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

Iterative refinement Resolution Higher resolution = more accurate positioning of atoms http://www.sci.sdsu.edu/TFrey/Bio750/Bio750X-Ray.html

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

NMR spectra 1D 2D http://www.cryst.bbk.ac.uk/PPS2/projects/schirra/html/2dnmr.htm

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

Ensemble of structures SH3-domain 1aey

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

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

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

Fitting X-Ray structures into density maps

GroEL-complex Hemoglobin 1gr6

Protein structure databases http://www.wwpdb.org/index.html

Molekulaarinen funktio

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).

Enzymes Catalytic triad: Asp, Ser, His 1CHO

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

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

Substrate specificity Perona & Craik (1997)

Transcription factors Ligand DNA 1L3L

Hydrogen bonding pattern Vannini (2002)

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

Evoluutio

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

Application: Finding Homologs

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 120436 MSH2 U03911 9.2e-261 MSH2 M84170 DNA repair protein Hereditary Non-polyposis Colon Cancer 120436 MLH1 U07418 6.3e-196 MLH1 U07187 DNA repair protein Cystic Fibrosis 219700 CFTR M28668 1.3e-167 YCF1 L35237 Metal resistance protein Wilson Disease 277900 WND U11700 5.9e-161 CCC2 L36317 Probable copper transporter Glycerol Kinase Deficiency 307030 GK L13943 1.8e-129 GUT1 X69049 Glycerol kinase Bloom Syndrome 210900 BLM U39817 2.6e-119 SGS1 U22341 Helicase Adrenoleukodystrophy, X-linked 300100 ALD Z21876 3.4e-107 PXA1 U17065 Peroxisomal ABC transporter Ataxia Telangiectasia 208900 ATM U26455 2.8e-90 TEL1 U31331 PI3 kinase Amyotrophic Lateral Sclerosis 105400 SOD1 K00065 2.0e-58 SOD1 J03279 Superoxide dismutase Myotonic Dystrophy 160900 DM L19268 5.4e-53 YPK1 M21307 Serine/threonine protein kinase Lowe Syndrome 309000 OCRL M88162 1.2e-47 YIL002C Z47047 Putative IPP-5-phosphatase Neurofibromatosis, Type 1 162200 NF1 M89914 2.0e-46 IRA2 M33779 Inhibitory regulator protein Choroideremia 303100 CHM X78121 2.1e-42 GDI1 S69371 GDP dissociation inhibitor Diastrophic Dysplasia 222600 DTD U14528 7.2e-38 SUL1 X82013 Sulfate permease Lissencephaly 247200 LIS1 L13385 1.7e-34 MET30 L26505 Methionine metabolism Thomsen Disease 160800 CLC1 Z25884 7.9e-31 GEF1 Z23117 Voltage-gated chloride channel Wilms Tumor 194070 WT1 X51630 1.1e-20 FZF1 X67787 Sulphite resistance protein Achondroplasia 100800 FGFR3 M58051 2.0e-18 IPL1 U07163 Serine/threoinine protein kinase Menkes Syndrome 309400 MNK X69208 2.1e-17 CCC2 L36317 Probable copper transporter

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

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]

sekvenssi  rakenne

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

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

Aminohappojen ominaisuuksia

levels of complexity in folding

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.

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

DSSP Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometrical Features W. Kabsch & C. Sander Biopolymers 22, 2577-2637 (1983)

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

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) ]

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

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

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.

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

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

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

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

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.