PROTEIN struktur dan fungsi
Laboratorium Biokimia Fakultas Biologi UGM
KUIS
Apa dasarnya anda mengambil matakuliah Biokimia Lanjut Dengan mengikuti kuliah Biokimia Lanjut, harapan anda mendakitkan
Central dogma
Translasi proses pembacaan kodon menjadi asam amino yang saling bergabung melalui ikatan peptida Komponen dalam translasi
1. mRNA tersusun dari kode genetik
2. Ribosom 3. tRNA pembawa a.a 4. enzim
Translation process consists of 3 main stages
Initiation
Elongation
Termination
Initiation Activation of amino acids for incorporation into proteins.
Activation of amino acids for incorporation into proteins.
Kode genetik Tiga nukleotida - kodon kode untuk asam amino pada protein
Tidak semua kodon digunakan dengan frekuensi yang sama. Terdapat variasi kodon yang digunakan antar spesies yang berbeda.
Wobble Hypothesis
Relationships of DNA to mRNA to polypeptide chain.
Transfer RNA (tRNA)
Terdiri dari asam nukleat dan asam amino spesifik berfungsi untuk menghubungkan sekuen asam nukleatprovide the link between the nucleic acid mRNA dengan asam amino yang dikodenya Antikodon sekuen yang terdiri dari 3 nukeotida pada tRNA yang komplemen dengan kodon
Struktur tRNA
Only tRNAfMet is accepted to form Twothe initiation initiation factors complex. (IF1 &IF3) bind to a 70S All ribosome. further charged tRNAs require promote fully assembled the dissociation (i.e., 70S) of 70S ribosomes ribosomes into free 30S and 50S subunits. The Shine-Dalgarno sequence help ribosomes mRNA and IF2, which and carries mRNA aligns correctly for the - GTP start of translation. - the charged tRNA Ribosome consists of - A bind site to aminoacyl a free 30S subunit. - P site After these peptidyl have all - bound, E site exit the 30S initiation complex is complete.
Peptide bond formation catalyzed by an enzyme complex called peptidyltransferase Peptidyltransferase consists of some ribosomal proteins and the ribosomal RNA acts as a ribozyme.
The process is repeated until a termination signal is reached.
Termination of translation occurs when
one of the stop codons (UAA, UAG, or UGA) appears in the A site of the ribosome. No tRNAs correspond to those sequences, so no tRNA is bound during termination. Proteins called release factors participate in termination
Posttranslational Processing of Proteins
Folding Amino acid modification (some proteins) Proteolytic cleavage
Before a newly translated polypeptide can be active, it must be folded into the proper 3-D structure and it may have to associate with other subunits.
FOLDING
Enzymes/protein involve in folding process
1. Cis-trans isomerase for proline
Proline is the only amino acid in proteins forms peptide bonds in which the trans isomer is only slightly favored (4 to 1 versus 1000 to 1 for other residues).
Thus, during folding, there is a significant chance that the wrong proline isomer will form first. Cells have enzymes to catalyze the cis-trans isomerization necessary to speed correct folding. 2. disulfide bond making enzymes 3. Chaperonins (molecular chaperones)
a protein to help keep it properly folded and non-aggregated.
Insulin is synthesized single polypeptide preproinsulin has leader sequence
(help it be transported through the cell membrane)
Specific protease cleaves leader sequence proinsulin. Proinsulin folds into specific 3D structure and disulfide bonds form Another protease cuts molecule insulin 2 polypeptide chains
Chaperones a. Some proteins capable to Function to its keep a newly fold into proper 3-D synthesized protein structure by itselffrom without either folding or any improperly help of other molecules aggregating b. Some proteins need chaperones to fold After synthesized, protein (example in human hsp 70) needs to fold in order to have its function c. Some proteins need bigger protein chaperonins to The folding pattern is dictated be able to fold correctly. in the amino acid sequence of the protein. Chaperonins a polysubunit protein form a cage like shape give micro environment to protein
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-fide examples are known all 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 14-33s. The best current model is that 14-3-3 can variously either mask or expose nuclear import and/or export signals on target proteins [47 and 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 (grey green). Interaction with 14-3-3 masks -COP binding sites, preventing entry into the retrograde transport system [49 and 50].
Protein Targeting
Nascent proteins contain signal sequence determine their ultimate destination.
Bacteria newly synthesized protein can: stay in the cytosol, send to the plasma membran, outer membrane, periplasmic, extracellular. Eukaryotes can direct proteins to internal sites lysosomes, mitochondria etc.
Nascent polypeptide E.R and glycosylated golgi complex and modified sorted for delivery to lysosomes, secretory vesicle and plasma membrane.
Translokasi
Protein ditranslokasi( disebut proprotein)) 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.
Signal recognition particle (SRP) detects signal sequence and brings ribosome to the ER membrane
Banyak protein mitokondrial disintesis di sitosol dan diimport ke organel
Bagaimana mempelajari fungsi protein?
Molecular Model of the Hd3a Protein
Hd3a protein contains a large central b -sheet (yellow ribbon) which is flanked on one side by a smaller b-sheet and on the other by an a-helix (red ribbon).
This molecular model of the Hd3a protein was built using the Swiss-Prot automated comparative protein modeling server, based on its sequence homology to two members of the RKIP protein family whose structures have been determined by x-ray crystallographic methods : Arabidopsis thaliana FT and TFL1 (Protein databank accession numbers 1WKP and 1WKO, respectively).
. . . .10 . . . .20 . . . .30 . . . .40 . . . .50 . . . .60 . . . .70 CEN_Anthirrinum_ 1: ...MAAKVSSDPLVIGRVIGDVVDHFTSTVKMSVIYNSNNSIKHVYNGHELFPSAVTSTPRVEVHGGDMR: 67 CEN Antirrhinum SP_Tomato_ 1: ...[Link]...KHVYNGHEFFPSSVTSKPRVEVHGGDLR: 63 SP Tomato TFL1_Arabidopsis_ 1: [Link]....QVSNGHELFPSSVSSKPRVEIHGGDLR: 65 TFL1 Arabidopsis FT_Arabidopsis_ 1: ..MSIN..IRDPLIVSRVVGDVLDPFNRSITLKVTYGQR....EVTNGLDLRPSQVQNKPRVEIGGEDLR: 62 FT Arabidopsis Hd3a_Rice_ 1: ..MAGSGRDRDPLVVGRVVGDVLDAFVRSTNLKVTYGSK....TVSNGCELKPSMVTHQPRVEVGGNDMR: 64 Hd3a Rice consensus consensus 1: ---*****--*!!***!!*!*!*!-!--****-!*!*-*-----!*!!****!!-!***!!!!**!*!*!: 70 GIHR motif Potential binding pocket DPD-X-P motif . . . .80 . . . .90 . . . 100 . . . 110 . . . 120 . . . 130 . . . 140 CEN Antirrhinum CEN_Anthirrinum_ 68: SFFTLIMTDPDVPGPSDPYLREHLHWIVTDIPGTTDSSFGKEVVSYEMP.....................:116 SP Tomato SP_Tomato_ 64: SFFTLIMIDPDVPGPSDPYLREHLHWIVTDIPGTTDCSFGREVVGYEMPRPNIGIHRFVFLLFKQKKRQT:133 TFL1 Arabidopsis 66: TFL1_Arabidopsis_ SFFTLVMIDPDVPGPSDPFLKEHLHWIVTNIPGTTDATFGKEVVSYELPRPSIGIHRFVFVLFRQKQRRV:135 FT Arabidopsis FT_Arabidopsis_ 63: NFYTLVMVDPDVPSPSNPHLREYLHWLVTDIPATTGTTFGNEIVCYENPSPTAGIHRVVFILFRQLGRQT:132 Hd3a Rice Hd3a_Rice_ 65: TFYTLVMVDPDAPSPSDPNLREYLHWLVTDIPGTTAASFGQEVMCYESPRPTMGIHRLVFVLFQQLGRQT:134 consensus consensus 71: *!*!!*!*!!!*!*!!*!*!*!*!!!*!!*!!*!!*-*!!*!**-!!*!********-*******--***:140 . . . 150 . . . 160 . . . 170 . . . 180 . . . CEN Antirrhinum CEN_Anthirrinum_ : ..............................................: SP Tomato SP_Tomato_ 134: ISSAPVSRDQFSSRKFSEENELGSPVAAVFFNCQRETAARRR....:175 TFL1 Arabidopsis 136: TFL1_Arabidopsis_ IFPNIPSRDHFNTRKFAVEYDLGLPVAAVFFNAQRETAARKR....:177 FT Arabidopsis FT_Arabidopsis_ 133: [Link]..:175 Hd3a Rice Hd3a_Rice_ 135: [Link] consensus consensus 141: **-**--*-*****-***-*-**-**************---*----:186
Hd3a shares high homology with other Phosphatidyl Ethanolamine Binding Protein or Raf Kinase Inhibitor Protein (PEBP/RKIP) in various plant species Hd3a and FT has 73% homology
Function of PEBPs (RKIP)
Have diverse roles in animal, yeast and bacteria : Regulate signaling pathway to control growth and differentiation 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 structures and mutant phenotypes will lead to comprehensive understanding of the mechanisms that facilitate the switch from vegetative phase to reproductive phase
from structure to function
RKIP /PEBPs
Hidrogen bond
Bind phosphoryl group Bind cacodylate ion
bPEBP
hPEBP The arrangement of conserved residues forming the ligand-binding site Asp70,His86,Tyr120 and Gly110 (in hPEBP)
CEN
(Banfield and Brady, 2000)
from structure to function
RKIP /PEBPs
The two key conserved residues that bind the ligand (His and Asp)
(Ahn et al., 2006)
