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Lecture 6 - Post translational modifications and protein structure
Post translational modifications- regulate protein activity and protein interactions
Phosphorylation-Ser, Thr, Tyr-Control protein activty and structure, as well as protein-protein and protein/nucleic acid interactions-Kinases phosphorylate, phosphatases dephosphorylate-Kinases are major drug targets
OP
O
O OOH
Glycosylation-Ser, Thr, Asn-regulated by glycosyl transferases-Control protein structure,stability, and trafficking. Regulate protein activity.
O
OH
XHNAc
HOHO
Carboxylation-most common is -carboxy-glutamate-Vitamin K, CO2, O2 dependent.ex. Prothrombrin
CO2HHO2C Ca2+ localizedat membrane thrombrin blood
clotting
Hydroxylation-Pro, Lys-Proline hydroxylation is important in transcriptional control and protein structure.-Hydroxylation and subsequent crosslinking of lysine residues in collagen cause conformational restriction and stabilize the coil-coil structure.
N
OH
O NH
NH2HO
O
SH SOH
Thiol oxidation-caused by reactive oxygen species-unclear whether this has natural regulatory activity
Acetylation-N-terminus, Lysine side chains-Affects chromatin structure and gene expressionMethylation-Glutamate, arginine, lysine.
NH
CO2H
ONH
CO2Me
O
NH3 NH2Me NHMe2 NMe3
-methylation affects chromatin structure and gene expression-methylation is mediated by S-adenosyl-methionine (SAM)
NNN
NNH2
O
OH OH
SMe
MetVitamin K dependent process; Warfarin inhibits turnover of Vitamin K by epoxide reductase and prevents clotting
ADP ribosylation (NAD+)-Arg, Glu, Asp, dipthamide-G-proteins, EF-2
O
OHOH
OHPO
O OPO
OHO
O
OHOH
ProteinA
Iodination-Tyr-Thyroid peroxidase
OHII O
I
IOH
Proteins: Primary Structure
HNHN
O
ORH
RH
N
OHN O
H
Above =0, =0
(0, 0)
HNO
O
HN
RH
+180o
H
R
N
O
H
O
NH
~ -120o
- angles from -40 to -160o are populated - angles from 0 to +90o and +90 to +180o are populated
Ramachandran Plots:We can analyze amino acids in terms of a hard sphere model and come up with a map of favored anglesA Ramachandran plot is a plot of and angles for various residues.
Gly -branched Ile, Val
Pro (-80o, +150o)
O HN
O HN
ON
Would you want to build proteins with lots of glycines?-There's a huge entropic penalty to pay going from unfolded proteins where glycine can be all over the ramachandran plot, to a folded protein where it is contained in one region.
I
I
Below =0, =+180
Newman Projection:
rotate -120o around
Note: clockwise rotation is +
disfavored -relieves unfavorable amide-amide steric interaction in (0,0) structure
folded unfolded
Gly conformationally restricted Gly conformationally free
disfavored
-On the other hand a Val, Ile or Pro with allowed conformation in the folded structure is stabilizing due to its lower conformational entropy in the unfolded state.
Proteins: Secondary StructureThree main structures: - helices, -strands, loops
-helix-right handed favored due to L-structure of amino-acids-3.6 residues/turn of helix-rise of 1.5Å per residue-5.4Å rise per turn
-Hydrogen bonding between amino acids i and i+4 .-Solvent exposed R groups-3.6 residues required to form first backbone hydrogen bond
NH
O0.42
0.2
-Average helix length ~10 residues-Left handed helices are poorly favored for L amino acids due to the close approach of side chains with C=O, only observe short regions of 3-5 amino acids
helix dipole
NH3
disfavorable dipole-chargeinteraction
N
O
Hacetylate to removepoint charge
Allowed -helix(-57,-47)
Helical Wheels
L1
K2A3
F4S5
N6
A7
L K A F S N A
H2N COOH
i i+4
Lys Asp/Glu
generates a salt bridge
-In many cases helices are amphipathic with one side containing hydrophilic residues and the other hydrophobic residues
-Strand2 types: antiparallel and parallel -sheets
OHN
NHO
HN
O
R
R
RO
HN
NHO
HN
O
R
R
RO
NH
HNO
NH
O
R
R
R
N-terminus
C-terminus C-terminus
N-terminusC-terminus
N-terminus
-Hydrogen bonds between adjacent strands-R groups alternate between up and down faces-3.5Å per residue, extended structure
Antiparallel Parallel
OHN
NHO
HN
O
R
R
RO
HN
NHO
HN
O
R
R
R
N-terminus
C-terminus C-terminus
N-terminus
(-139,+135)
(-119,+113)
(-57,-47)
-view helix as a helical wheel
-Proline is disfavored for helix formation except in the first turn (loss of H-bond donor)-Side chains have an -helix preference:
Good: Ala, Glu, Leu, MetBad: Pro, Gly, Tyr, Ser
i i+4
NH3
Loops-Proteins are typicaly built up of -helices and -strands connected by loops typically at the surface with main chain C=O and NH groups exposed to solvent in order to satisfy Hydrogen bonding.-Loops are typically the location of deletions and insertions in phylogenetic species comparison. Introns occur at loops. -Phylogenetic species comparison shows that core elements are relatively insensitive to loop structure. -Loops act as connectors but also play a role in binding and active sites. -Loops which connect antiparallel -strands are hairpin loops. The two most commonly occuring loops are type I' and type II'
O
HN
NH
NH
O
O
HN
O
R
R
RR
-most common loops have 2 residues in the turn and 2-5 residues in the loop- i+1, i+2 residues are often Gly, Ser, Thr, Asn.
Motifs-Connecting together secondary structural elements in specific geometric arrangements produce motifs-there are four common motifs seen in proteins
1) Loop-helix-loopDNA binding and Ca2+ binding
2) Hairpin motiffFrequently occuring, present in most antiparallel -sheets
3) Greek Key4 adjacent antiparallel strands
4)2 parallel -strands connected by an -helix
3 2 1 4
The sum of secondary structures motiffsThe sum of motiffs domains
Proteins can consist of one or several domains:e.g. TIM barrel is built up of repeating motiffs
N- ...
i
i+1i+2
i+3
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