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