4. Caracterizarea enzimei Mw Localizare AA · 2018-04-16 · Structura primara Locatie proteina 4....

Structura

primară

Mw

Localizare AA

Identificarea de parti functionale

Stabilirea unor relatii de evolutie

4. Caracterizarea enzimei

Determinarea pI

Structura primară Estimarea Mw

4. Caracterizarea enzimei4.1. Determinarea masei moleculare

NCBI http://www.ncbi.nlm.nih.gov/pubmed

Cea mai mare baza de date

UniProt http://www.uniprot.org/

P0DJD7.1 Pepsina umana 1-388

MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNP

ARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGS

SNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTG

ILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGA

TPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNW

VPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANI

QSDIGAS ENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEG

SCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA

http://web.expasy.org/peptide_mass/

Structura primară Estimarea Mw

4. Caracterizarea enzimei4.1. Determinarea structurii primare

Mw (masa monoizotopica): 41950.55

https://web.expasy.org/compute_pi/

Structura primară Estimarea pI


http://isoelectric.org/



Mw: 57304.81pI: 5.89

Fosfatază alcalină umană

http://protcalc.sourceforge.net/cgi-bin/protcalc



http://www.biosyn.com/PeptidePropertyCalculator/

Mw: 57307.72pI: 6.35

Fosfatază alcalină umană

pI: 6.67

www.bioinformatics.org/sms2/protein_iep.html

pI: 6.66







QSDIGAS ENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEG


Structura primara Locatie proteina


Secventa semnal

http://www.signalpeptide.de/index.php

Peptida activatoare

Structura primara Locatie proteina


Secventa semnal

Kalderon, B. & Pines, O. (2014),

Protein folding as a driving force for dual

protein targeting in eukaryotes,

Front. Mol. Biosci.,1, 1-9

Structura primara

Locatie proteina


Secventa semnal

https://www.uniprot.org/blast/

Reactii catalizate de enzime din E. coli

otsA - trehaloz fosfat sintaza (TPS)

otsB - trehaloz fosfat fosfataza (TPP)

treC - trehaloz fosfat hidrolaza (TPH)

treF - trehalaza

Schluepmann, H. & all (2003),

PNAS,100, 6849-6854

Structura

primaraLocalizare proteina

Secventa semnal

http://www.cbs.dtu.dk/services/SignalP/


Structura



Acil-CoA dehidrogenaza umană

PubMed AAA74424.1

Structura

primaraLocatie proteina

Secventa semnal Phobius

http://phobius.sbc.su.se/

Structura


Secventa semnal Phobius

http://phobius.sbc.su.se/

P0DJD7

Pepsina umana 1-388

Estimarea proteinelor din S. Cerevisiae

care contin o peptida semnal

Yofe, I. et al., Nature Methods, 13, 371-378 (2016)

Structura


Secventa semnal Signal-3L 2.0 prediction

http://www.csbio.sjtu.edu.cn/bioinf/Signal-3L/

Rezultate via Email - lent

Structura


Structura

primara

Similaritati cu alte peptide din baze de

date existente

http://www.uniprot.org/peptidesearch/


Structura

primaraPeptide search








QSDIGASENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEG


Structura

primaraLocalizare AA


6 C 3 punti disulfidice

4 W fluorescenta

Compararea secventelor 4. Caracterizarea enzimei

4.3. Determinarea structurii primare

Clustal Omega https://www.ebi.ac.uk/Tools/msa/clustalo/

P0DJD7, C1HAC9

Se aleg secventele in format FASTA (UNIPROT)



Kalign https://www.ebi.ac.uk/Tools/msa/kalign/

Compararea secventelor


CLUSTAL 2.1 http://www.ebi.ac.uk/Tools/msa/clustalw2/

P0DJD7

Q4R$P0

C1HAC9

Se aleg secventele in format FASTA (UNIPROT)

mitogen-activated protein kinase 14

(MAPKK)

atlasgeneticsoncology.org/Genes/

Compararea secventelor 4. Caracterizarea enzimei4.3. Determinarea structurii primare

Arbore

filogenetic



PIR (Protein informatic resource)

http://pir.georgetown.edu/pirwww/search/pairwise.shtml

P0DJD7, C1HAC9

Se aleg secventele in format FASTA

sau codul de acces (UNIPROT)

Tipul/Clasa?4. Caracterizarea enzimei


Pfam (Colectie baza de date familii de proteine)

http://pfam.xfam.org/

Aspartil proteaza

Tipul/Clasa4. Caracterizarea enzimei


Pfam

Cu/Zn superoxid dismutaza

Alvaro G. Estévez, Ed., Mental and Behavioural

Disorders and Diseases of the Nervous System »

"Current Advances in Amyotrophic Lateral

Sclerosis„ (2013), Chapter 5. Superoxide

Dismutase and Oxidative Stress in

Amyotrophic Lateral Sclerosis

PhosphoELM

4. Caracterizarea enzimei4.3. Determinarea situsurilor de fosforilare

PhosphoELM phospho.elm.eu.org

4.3. Determinarea situsurilor de fosforilare

Motivele structurale – sunt segmente scurte cu regiuni similare

Majoritatea 10-20 AA

4. Caracterizarea enzimei4.4. Exemple de motive structurale

Motiv structural catalitic in endonucleaze

PD.....D/ExK la C-terminal Nidhanapati, K. R. et al. Frontiers in Bioscience 17, 1094-1107, 2012

Motivele structurale

4. Caracterizarea enzimei4.4. Determinarea structurii secundare/supersecundare

Motivele Walker

Caracteristice enzimelor ce scindeaza ATP

Secvente conservate de tipul

GxGKS/T

DExx

DEAH

DEPH

Fosfat (ATP-Mg)

Coordinare Mg2+Esential clivare ADN



Motivele Walker

Ohtsu, I. et al., PLOSONE, 2015Ec, Escherichia coli;

Bs Bacillus subtilis;

Lr, Lactobacillus reuteri;

Lf, Lactobacillus fermentum. Dassa, E.

Essays In Biochemistry

2011, 50, 19-42.



Motivele Walker (buclă P)

Proteina H-Ras

interacțiune cu GTPKinază

interacțiune cu ATP

Alte motivele structurale


Tirozin-fosfatazele

http://wmpeople.wm.edu/

Situs activ conservat

C(X)5R

http://mol-biol4masters.masters.grkraj.org/



Serin/treonin-fosfatazele

Frank Rusnak, and Pamela Mertz

Physiol Rev 2000;80:1483-1521 A. Calcineurina umana

B. Fosfataza acida (fasole violeta)



Serin/treonin-fosfatazele

Nuclear factor of activated T cells (NFAT)

NFAT substrat al calcineurinei

Musson, R. E.A., Cobbaert C. M., Smit N.P.M.

Clinical Chemistry Mar 2012, 58 (3) 511-522

Calcineurin (PDB 1AUI)

rosu – Zn2+, roz –Fe3+ albastru Ca2+

Ke, H., Huai, Q., BBRC

2013, 331, 1095-1102

http://clincancerres.aacrjournals.org/

Alte motivele structurale – motivul HPN in enzimele E2 ubiquitin ligazei

(calea metabolica a ubiquitinarii)


Cook,B.W. & Shaw, G. S.

Biochemical Journal, 445(2),

167-174, 2012.



Scop

http://scop2.mrc-lmb.cam.ac.uk/0.html

Relatiile dintre proteine

Clase structurale

Tipuri de proteine

Elemente de evolutie structurala

Clase structurale

Impachetarea proteinelor in

functie de continutul

structurii secundare



Scop

Protein Relationships

Other Relationships

Motif

CGxCxxCx(n)C motiv legare ion metalic

N - terminal ADH

Motivul Rosmann element de conexiune în buclă

GX[GS]XX[GSTA]

GS - contact cu pirofosfat (nucleotidă)



MOTIF Search

http://www.genome.jp/tools/motif/


MOTIF Search http://www.genome.jp/tools/motif/

pepsinogen A

MKWLLLLGLVALSECIMYKVPLIRKKSFRRTLSERGLLKDFLKKHNLNPARKY

FPQWKAPTLVDEQPLEN YLDMEYFGTIGIGTPAQDFTVLFDTGSSNLWVPSV

YCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTG SMTGILGYDTVQVGGISD

TNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWNQGLVSQ DL

FSVYLSADDQSGSVVIFGGIDSSYYTGSLNWVPVTVEGYWQITVDSITMNGEA

IACAEGCQAIVDTGT SLLTGPTSPIANIQSDIGASENSDGDMVVSCSAISSLPDI

VFTINGVQYPVPPSAYILQSEGSCISGFQG MNLPTESGELWILGDVFIRQYFTV

FERANNQVGLAPVA


MOTIF Search

HUMAN Tyrosine-protein phosphatase non-receptor type 6 Homo sapiens MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDF

YDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQA

ETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGG

LETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTA

KAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSD

YINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNK

CVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDH

GVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDC

DIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNIT

YPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKR

Motivele structurale-domenii/situsuri


InterPro

http://www.ebi.ac.uk/interpro/

IPR011895

Domeniul catalitic

Motiv de legare al

Fosfatului de tiamina

Domeniul EKR (bacterii,

eucariote inferioare)



https://www.cellsignal.com/


4. Caracterizarea enzimei4. 4. Determinarea structurii secundare/supersecundare

CATH

http://www.cathdb.info/

95 milioane de domenii proteice

6,199 superfamilii

Superfamilia

3.30.70.330Domeniul


4. Caracterizarea enzimei4. 4. Determinarea structurii secundare/supersecundare

CATH

Structuri 3D determinate experimental

https://www.rcsb.org/

4. Caracterizarea enzimei4.5. Baze de date cu structura tridimensionala a proteinelor

Structuri 3D determinate experimental

RCSB PDB


http://www.expasy.org/proteomics

Expert Protein Analysis System ExPASy


https://www.proteinmodelportal.org/

http://www.uniprot.org/ (pir.georgetown.edu)

5 milioane proteine – conexiune cu alte str 3D

UniProt


http://www.uniprot.org/

http://www.nextprot.org/

Proteine umane

NextProt

4. Caracterizarea enzimei4.5. Baze de date cu proteine

http://www.nextprot.org/

STRING http://string-db.org/

Interactiuni dintre proteine

4. Caracterizarea enzimei4.5. Baze de date cu interacțiuni între proteine

Comparari structuri

3D proteine

Dali

http://ekhidna.biocenter.

helsinki.fi/dali/

4. Caracterizarea

enzimei

Entrez Databases

http://www.ncbi.nlm.nih.gov/Class/MLACourse/Original8Hour/Entrez/


4.5. Baze de date cu structura tridimensionala a

proteinelor


4.6. Pachete de vizualizare 3D - Swiss PDB viewer

File Open 1HEW (baza de date)

Ferestre

de lucru Fereastră grafică

Control Panel Catenele laterale

Catena polipeptidică

Etichetări atomi

Reprezentare schematică

îngroşatăBara de control

Bara de aliniere

(Alignment)Ferestre

specifice

Wind/Alignment

Structura primara

Swiss PDB viewer

Selectare secvente scurte

Resturi marcate cu rosu

in Control PanelMutarea cursorului

mouse-ului

Localizare aminoacid individual

In structura tridimensionala

Graficul RamachandranFerestre

specifice

Wind / Ramachandran Plot

Swiss PDB viewer

Restrictiile conformaţionale

ale resturilor din proteină

Fiecare punct

Unghiurile φ şi θ

(fiecare rest din proteina)

DisplayInteriorul

proteinei

Slab

Swiss PDB viewer

Vizualizarea

atomilor

C-alb

S-galben

O-rosu

P-portocaliu

N-albastru

H-cian

Vizualizarea

3D

faţă Alt+5

sus Alt+8

bază Alt+2

stanga Alt+ 4

dreapta Alt+6

initial Alt+ 7

Swiss PDB viewer

Vizualizarea atomilor de O

din legaturile peptidice

Display / Show backbone

Catena laterala

aminoacid

Control Panel / side

Selectare aminoacid

Vizualizarea pozitiei

unui aminoacid

Control Panel / Label

Vizualizarea densitatii

electronice a unui

aminoacid

Control Panel / ::v

Baze de date cu nomenclatura enzimelor

UniProtKB/Swiss-Prot http://enzyme.expasy.org/

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4. Caracterizarea enzimei Mw Localizare AA · 2018-04-16 · Structura primara Locatie proteina 4....

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