Transcript En7
2. Caracterizarea enzimei
2.2. Determinarea structurii primare
Mw
Structura
primara
Localizare AA
Identificarea de parti functionale
Determinarea pI
Stabilirea unor relatii de evolutie
2. Caracterizarea enzimei
2.2. Determinarea structurii primare
Structura
primara
NCBI
Estimarea Mw
http://www.ncbi.nlm.nih.gov/pubmed
Cea mai mare baza de date
UniProt
http://www.uniprot.org/
P0DJD7
Pepsina umana 1-388
MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNP
ARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGS
SNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTG
ILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGA
TPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNW
VPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANI
QSDIGAS ENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEG
SCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA
2. Caracterizarea enzimei
2.2. Determinarea structurii primare
Structura
primara
Estimarea Mw
http://web.expasy.org/peptide_mass/
Mw (masa monoizotopica): 41950.55
pI (teoretic): 4,16
http://protcalc.sourceforge.net/cgi-bin/protcalc
Mw (masa monoizotopica): 41977.06
pI (teoretic): 4,33
2. Caracterizarea enzimei
2.2. Determinarea structurii primare
Structura
primara
Locatie proteina
MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNP
ARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGS
SNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTG
ILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGA
TPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNW
VPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANI
QSDIGAS ENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEG
SCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA
Secventa semnal
http://www.signalpeptide.de/index.php
Peptida activatoare
2. Caracterizarea enzimei
2.2. Determinarea structurii primare
Structura
primara
Localizare AA
MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNP
ARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGS
SNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTG
ILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGA
TPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNW
VPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANI
QSDIGASENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEG
SCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA
6C
3 punti disulfidice
4W
fluorescenta
2. Caracterizarea enzimei
2.2. Determinarea structurii primare
Compararea secventelor
P0DJD7, C1HAC9
CLUSTAL 2.1 http://www.ebi.ac.uk/Tools/msa/clustalw2/
Se aleg secventele in format FASTA (UNIPROT)
2. Caracterizarea enzimei
2.2. Determinarea structurii primare
Compararea secventelor
P0DJD7
Q4R$P0
C1HAC9
CLUSTAL 2.1 http://www.ebi.ac.uk/Tools/msa/clustalw2/
Se aleg secventele in format FASTA (UNIPROT)
2. Caracterizarea enzimei
2.2. Determinarea structurii secundare
Structura secundara descrie modalitatile de impachetare ale
lantului polipeptidic
-helix
-pliata paralela
2. Caracterizarea enzimei
2.2. Determinarea structurii secundare
Enzima/proteina noua
Secventa primara noua
Fara omologi in bazele de date
Transmembranara?
Situsuri de fosforilare
Structura secundara
care poate fi intuita
2. Caracterizarea enzimei
2.2. Determinarea structurii secundare
Baze de date – structura secundara
Elementele de -helix
http://www-nmr.cabm.rutgers.edu/bioinformatics/
Proteomic_tools/Helical_wheel/
Elementele de -helix si -pliate - GARNIER
http://biotools.umassmed.edu/cgi-bin/biobin/garnier
Structura primara a mioglobinei umane P02144 (UniParc)
MGLSDGEWQLVLNVWGKVEADIPGHGQE
VLIRLFKGHPETLEKFDKFKHLKSEDEMKA
SE DLKKHGATVLTALGGILKKKGHHEAEIK
PLAQSHATKHKIPVKYLEFISECIIQVLQSKH
PGDFGADAQGAMNKALELFRKDMASNYKELGFQG
2. Caracterizarea enzimei
2.2. Determinarea structurii secundare
Baze de date – structura secundara
JPRED
http://www.compbio.dundee.ac.uk/www-jpred/
PSI PRED
http://bioinf.cs.ucl.ac.uk/psipred/
COILS (elemente spiralate)
http://www.ch.embnet.org/cgi-bin/COILS_form_parser
2. Caracterizarea enzimei
2.2. Determinarea structurii secundare/supersecundare
Motivele structurale – sunt segmente scurte cu regiuni similare
Majoritatea 10-20 AA
Motivele comune → lanturi de AA → domeniile transmembranare
→ situsuri de fosforilare
Motivul
Michael Rossmann
Structura butoi TIM
2. Caracterizarea enzimei
2.2. Determinarea structurii secundare/supersecundare
Alte motivele structurale
Situsul activ al aspartil proteazelor
NCBI protease, partial [Human T-lymphotropic virus 1]
PROSITE
http://prosite.expasy.org/
HPTPKKLHRGGGLTSPPTLQQVLPNQDLASILPVIPLDPARRPVIKAQV
DTQTSHPKTIEALLDTG ADMTVLPIALFSNNTPLKDTSVLGAGGQTQD
HFKLTSLPVLIRLPFRTTPIVLTSCLVDTKNNWAI IGRDALQQCQGVLYL
PEAKRPPVILPIQAPAVLGLEHLPRPPEISQFPLNQNASRPCNTWSGR
PWR QAISNPTPGQEITQYSQLKKPMEPGDSSTTCGPLTL
Protein kinaza C –situsuri de fosforilare din proteine
ST-xRK
Determinarea structurii tertiare
Puterea catalitica
Structura
primara
Structura
tertiara
Specificitatea
enzimelor
Difractie
raze X
Structura
3D
RMN
Difractia cu raze X
K
1.
2.
3.
4.
Determinarea structurii proteinelor prin difractie cu raze X:
Cristalizarea proteinei
Imprestierea radiatiei X de catre planurile cristalului
Din diagrama de difractie este modelata harta densitatii electronice
Contruirea unui model de proteina pe baza densitatii electronice
Deducerea fazei razelor imprastiate
Hg
Insertie
atomi grei
U
Pt
Densitate
electronica
mare
E-CH2-SH + CH3-Hg-NO3
catena laterala
a cisteinei
E-CH2—
—OH + I2
catena laterala
a tirozinei
Imprastierea
razelor X
E-CH2-S-Hg- CH3 + NO3- + H+
I
E-CH2—
—OH
+
HI
Cristalografie cu raze X
Harta tridimensionala
de densitate electronica
Harta densitatii electronice a complexului
Proteina talomera-ADN
Harta de densitate electronica a moleculeler
Forma
generala
unghi de refractie mic
Rezolutie modesta (5-6 Å)
Spoturi cu unghi
de refractie mai mare
Rezolutie medie (3,5 Å)
Traseu lant
polipetidic
Spoturi cu unghi
de refractie mai mare
Rezolutie medie (3 Å)
Catena
laterala
Spoturi cu unghi
de refractie mai mare
Rezolutie buna (2,5 Å)
Spoturi cu unghi
de refractie mai mare
cristal omogen
Determinarea
pozitiei atomilor
±0,4 Å
Rezolutie mare (1,9 Å)
Pozitia atomilor
±0,2 Å
Retele cristaline
Harta densitatii electronice
Structura tridimensionala
Structura primara
Legaturi
amidice
Resturi din
catena laterala
Structura quasistatica
rotatii
vibratii
Legarea ligandului la substrat
hexokinaza
Modificari
conformationale
Accesul oxigenului la hemul hemoglobinei
Structura oxi- si deoxihemoglobinei
Localizarea atomilor de H
Difractia cu raze X
Atomi de H
Pozitia atomilor de H
Densitate
de e- redusa
Difractie cu
neutroni
Imprastierea
neutronilor
Sincroton-producerea de neutroni
The beam makes about 104 orbits inside the synchrotron as it is accelerated before being kicked in a single
revolution into the extracted proton beam line, delivering 4 μC of protons in two 100 ns long pulses to a tungsten
spallation target. The entire acceleration process is repeated 50 times per second, so that a mean current of 200 μA
Determinarea structurii in solutie
Stingerea fluorescentei
Schimb izotopic (H-D)
RMN
Dicroism circular
in solutie
Structuri
flexibile
Tripsinogen
Clivare
proteolitica
Tripsina
N-terminal
Lant flexibil
Difractie cu
neutroni
Mioglobina
In 95%D2O
Modificarea
legaturilor
amidice
tirozina
RMN
fenilalanina
Modele teoretice
Structura 3D
a enzimelor
Mecanisme de reactie
Similaritati structurale
Motivul Rossman
lactate dehidrogenaza
Caseta de legare
a nucleotidelor
alcool dehidrogenaza
Motiv
Rossman
Structura cuaternara
Interactiuni dintre monomeri
Aldolaza KDPG
trimer
Neuramidaza
tetramer
Insulina hexamer
Kinaza-octamer
Structura proteinei
Primara
Secundara
Super-secundara
Tertiara
Cuaternara
Triptofan sintaza
indol-3-glicerol-fosfat
indol + serina
Aspartat carbamoilaza
R6C6
Subunitatea
Subunitatile 2
indol + 3-glicerol-fosfat
triptofan
Complexul piruvat dehidrogenazei - purtator grupe acil
Complex multienzimatic - 60 subunitati:
24 - dihidrolipoil transacetilaza
24 - piruvat dehidrogenaza
12 - dihidrolipoil dehidrogenaza
E2 dihidrolipoil transacetylase
8 trimeri aranjati dupa o simetrie cubica
Avantajele complexelor enzimatice
Difuzia rapida a intermediarului de la o enzima la alta
Canalizarea compusilor de-a lungul complexului
Protejarea intermediarilor instabili
Transfer intermolecular rapid
Baze de date cu structura tridimensionala a proteinelor
Structuri 3D determinate experimental
http://www.rcsb.org/pdb/
Expert Protein Analysis System ExPASy
http://www.expasy.org/proteomics
http://www.uniprot.org/ (pir.georgetown.edu)
5 milioane proteine
http://www.nextprot.org/
Proteine umane
http://string-db.org/
Interactiuni dintre proteine
Swiss PDB viewer