biola(protein)

8/13/2019 Biola(Protein)

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PROTEIN

struktur dan fungsiLaboratorium Biokimia

Fakultas Biologi UGM


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KUIS

Apa dasarnya anda mengambil matakuliah

Biokimia Lanjut

Dengan mengikuti kuliah Biokimia Lanjut,

harapan anda mendakitkan


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Central dogma


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Translasiproses pembacaan kodon menjadi

asam amino yang saling bergabung melaluiikatan peptida

Komponen dalam translasi

1.mRNA tersusun dari kode genetik

2.Ribosom

3.tRNA pembawa a.a

4.enzim


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Translation process consists of 3 main stages

Initiation

Elongation

Termination

InitiationActivation of amino acids forincorporation intoproteins.


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Activation of amino acids for

incorporation into proteins.


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Kode genetikTiga nukleotida - kodonkode untuk

asam amino pada protein


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Tidak semuakodon digunakan

dengan frekuensiyang sama.

Terdapat variasikodon yangdigunakan antarspesies yang

berbeda.


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Wobble Hypothesis


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Relationships of DNA to mRNA to

polypeptide chain.


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Transfer RNA (tRNA)

Terdiri dari asam nukleat

dan asam amino spesifik

berfungsi untukmenghubungkan sekuenasam nukleatprovide thelink between the nucleicacid mRNA dengan asamamino yang dikodenya

Antikodonsekuen yang

terdiri dari 3 nukeotidapada tRNA yangkomplemen dengankodon

Struktur tRNA


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Two initiation factors (IF1&IF3) bind to a 70S

ribosome.promote the dissociationof 70S ribosomes into free30S and 50S subunits.

mRNA and IF2, whichcarries- GTP- the charged tRNA

bind to a free 30S subunit.

After these have allbound, the 30S initiationcomplex is complete.

Only tRNAfMetis accepted toform the initiation complex.

All further charged tRNAsrequire fully assembled (i.e.,70S) ribosomes

The Shine-Dalgarno

sequence

help ribosomesand mRNA aligns correctly forthe start of translation.

Ribosome consists of- A site aminoacyl

- P site peptidyl- E site exit


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Peptide bondformation

catalyzed by anenzyme complexcalledpeptidyltransferase

Peptidyltransferaseconsists of someribosomal proteins andthe ribosomal RNA acts as a ribozyme.

The processis repeated until atermination signal isreached.


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Termination oftranslationoccurs whenone of the stop codons (UAA,

UAG, or UGA) appears in theA site of the ribosome.

No tRNAs correspond to thosesequences, so no tRNA

is bound during termination.

Proteins called release factorsparticipate in termination


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Posttranslational Processing of

Proteins

Folding

Amino acid modification (some proteins)

Proteolytic cleavage

FOLDING Before a newly translated polypeptidecan be active, it must be folded into

the proper 3-D structure and it mayhave to associate with other subunits.


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Enzymes/protein involve in folding process

1. Cis-trans isomerase for proline

Proline is the only amino acid in proteins forms peptide bondsin which the trans isomer is only slightly favored (4 to 1 versus1000 to 1 for other residues).

Thus, during folding, there is a significant chance that thewrong proline isomer will form first. Cells have enzymesto catalyze the cis-trans isomerization necessary tospeed correct folding.

2. disulfide bond making enzymes3. Chaperonins (molecular chaperones)

a protein to help keep it properly folded and non-aggregated.


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Insulin is synthesized singlepolypeptidepreproinsulin

has leader sequence(help it be transported through thecell membrane)

Specific protease cleaves leader

sequenceproinsulin.

Proinsulin folds into specific 3Dstructure and disulfide bondsform

Another protease cuts moleculeinsulin2 polypeptidechains


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Chaperones

Function to keep a newlysynthesized protein from

either improperly folding oraggregating

After synthesized, protein

needs to fold in order to haveits function

The folding pattern is dictatedin the amino acid sequence of

the protein.

a. Some proteins capable tofold into its proper 3-Dstructure by itself withoutany help of other molecules

b. Some proteins needchaperonesto fold(example in human hsp 70)

c. Some proteins need biggerproteinchaperoninsto

be able to fold correctly.

Chaperoninsa polysubunitprotein form a cage likeshape give micro

environment to protein


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Fig. 1. The consequences of 14-3-3 interaction with target proteins can be classified into several generic modes-of-action. These are represented

here with 14-3-3 protein dimers shown in orange and interacting proteins in shades of blue, green and purple. (a) First, interaction can directly alter

the activity of the target. This can occur through changes to the specific activity or the half-life of an enzyme. (b) A scaffold function, in which 14-3-3

brings together two other proteins, is also a popular suggestion in the literature, although relatively few bona-fideexamples are knownall in

animal cells [44]. (c) Conformational change brought about by interactions at multiple sites on a target protein is also known in animals [45]. (d)

Binding of 14-3-3s to cleavable signal peptides (light-blue region) of nuclear-encoded animal mitochondrial and plant chloroplast proteins stimulate

import into these organelles [46]. (e) Modulation of nuclear import and export is increasingly being revealed as a common mode-of-action of 14-3-

3s. The best current model is that 14-3-3 can variously either mask or expose nuclear import and/or export signals on target proteins [47and 48]. (f)

A further general role in protein transport in the endomembrane system has recently been uncovered in animal cells. 14-3-3s promote the forward

transport of proteins arriving at the Golgi apparatus (pale greygreen) in COPII-coated vesicles derived from the endoplasmic reticulum (greygreen). Interaction with 14-3-3 masks -COP binding sites, preventing entry into the retrograde transport system [49and 50].
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Protein Targeting

Nascent proteins

contain signal sequence

determinetheir ultimate destination.

Bacterianewly synthesized protein can: stay in thecytosol, send to the plasma membran, outer

membrane, periplasmic, extracellular.Eukaryotescan direct proteins to internal sites

lysosomes, mitochondria etc.

Nascent polypeptideE.R and glycosylated golgicomplex and modified sorted for deliveryto lysosomes, secretory vesicle and plasmamembrane.


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Translokasi Protein ditranslokasi( disebut pro-

protein)) membentuk kompleks

dengan chaperon di sitosol

Kompleks ini melindungi protein agar

tidak mengalami folding,

The complex keeps the protein from

folding prematurely, which would

prevent it from passing through the

secretory porean ATPase that helps

drive the translocation

after the pro-protein is translocated,

the leader peptide is cleaved by a

membrane-bound protease and the

protein can fold into its active 3-d

form.


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Signal recognition particle (SRP) detects signalsequence and brings ribosome to the ER membrane


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Banyak protein

mitokondrial

disintesis di

sitosol dan

diimport ke

organel


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Bagaimana mempelajari fungsi

protein?


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This molecular model of the Hd3a protein was built using the Swiss-Protautomated comparative protein modeling server, based on its sequence homologyto two members of the RKIP protein family whose structures have beendetermined by x-ray crystallographic methods :Arabidopsis thalianaFT and TFL1(Protein databank accession numbers 1WKP and 1WKO, respectively).

Molecular Model of the Hd3a Protein

Hd3a protein

contains a largecentral b -sheet(yellow ribbon)which is flanked onone side by asmaller b-sheet andon the other by an

a-helix (red ribbon).


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. . . .10 . . . .20 . . . .30 . . . .40 . . . .50 . . . .60 . . . .70CEN_Anthirrinum_1:...MAAKVSSDPLVIGRVIGDVVDHFTSTVKMSVIYNSNNSIKHVYNGHELFPSAVTSTPRVEVHGGDMR: 67SP_Tomato_ 1:...MASKMC.EPLVIGRVIGEVVDYFCPSVKMSVVYNNN...KHVYNGHEFFPSSVTSKPRVEVHGGDLR: 63TFL1_Arabidopsis_1:MENMGTRVI.EPLIMGRVVGDVLDFFTPTTKMNVSYNKK....QVSNGHELFPSSVSSKPRVEIHGGDLR: 65FT_Arabidopsis_ 1:..MSIN..IRDPLIVSRVVGDVLDPFNRSITLKVTYGQR....EVTNGLDLRPSQVQNKPRVEIGGEDLR: 62Hd3a_Rice_ 1:..MAGSGRDRDPLVVGRVVGDVLDAFVRSTNLKVTYGSK....TVSNGCELKPSMVTHQPRVEVGGNDMR: 64consensus 1:---*****--*!!***!!*!*!*!-!--****-!*!*-*-----!*!!****!!-!***!!!!**!*!*!: 70

. . . .80 . . . .90 . . . 100 . . . 110 . . . 120 . . . 130 . . . 140CEN_Anthirrinum_68:SFFTLIMTDPDVPGPSDPYLREHLHWIVTDIPGTTDSSFGKEVVSYEMP.....................:116SP_Tomato_ 64:SFFTLIMIDPDVPGPSDPYLREHLHWIVTDIPGTTDCSFGREVVGYEMPRPNIGIHRFVFLLFKQKKRQT:133TFL1_Arabidopsis_66:SFFTLVMIDPDVPGPSDPFLKEHLHWIVTNIPGTTDATFGKEVVSYELPRPSIGIHRFVFVLFRQKQRRV:135FT_Arabidopsis_63:NFYTLVMVDPDVPSPSNPHLREYLHWLVTDIPATTGTTFGNEIVCYENPSPTAGIHRVVFILFRQLGRQT:132

Hd3a_Rice_ 65:TFYTLVMVDPDAPSPSDPNLREYLHWLVTDIPGTTAASFGQEVMCYESPRPTMGIHRLVFVLFQQLGRQT:134consensus 71:*!*!!*!*!!!*!*!!*!*!*!*!!!*!!*!!*!!*-*!!*!**-!!*!********-*******--***:140

. . . 150 . . . 160 . . . 170 . . . 180 . . .CEN_Anthirrinum_ :..............................................:SP_Tomato_ 134:ISSAPVSRDQFSSRKFSEENELGSPVAAVFFNCQRETAARRR....:175TFL1_Arabidopsis_136:IFPNIPSRDHFNTRKFAVEYDLGLPVAAVFFNAQRETAARKR....:177FT_Arabidopsis_133:VY.APGWRQNFNTREFAEIYNLGLPVAAVFYNCQRESGCGGRRL..:175Hd3a_Rice_ 135:VY.APGWRQNFNTKDFAELYNLGSPVAAVYFNCQREAGSGGRRVYP:179consensus 141:**-**--*-*****-***-*-**-**************---*----:186

CENAntirrhinum

SP Tomato

TFL1ArabidopsisFTArabidopsis

Hd3a Riceconsensus

CENAntirrhinumSP TomatoTFL1ArabidopsisFTArabidopsis

Hd3a Riceconsensus

CENAntirrhinum

SP TomatoTFL1ArabidopsisFTArabidopsis

Hd3a Riceconsensus

Potential binding pocketDPD-X-P motif GIHR motif

Hd3a shares high homology with other Phosphatidyl Ethanolamine Binding Proteinor Raf Kinase Inhibitor Protein (PEBP/RKIP) in various plant species

Hd3a and FT has 73% homology


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Function of PEBPs (RKIP)

Have diverse roles in animal, yeast and bacteria :

Regulate signaling pathway to control growth anddifferentiation

Inhibitor or signaling components to modulate the flux

through the pathway (Hansawa et al.,2005)

Biological function of Hd3a is unknown

The combination of interacting proteins, resolved crystal structuresand mutant phenotypes will lead to comprehensive understandingof the mechanisms that facilitate the switch from vegetative phase

to reproductive phase


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RKIP /PEBPsfrom structure to function

bPEBP hPEBP

CEN

Hidrogen bond

The arrangement of conserved residues

forming the ligand-binding site

Asp70,His86,Tyr120 and Gly110 (in hPEBP)

(Banfield and Brady, 2000)

Bind phosphoryl group Bind cacodylate ion


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(Ahn et al., 2006)

RKIP /PEBPsfrom structure to function

The two key conserved residues that bind the ligand (His and Asp)

biola(protein)

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