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Protein Structure and Function

Lecture 6: Post-translational modification

Handouts

Chemistry happens

• Proteins are molecules.• Everything you learned (and forgot) about

chemistry can be applied to proteins. • Proteins undergo chemical reactions.

Hydrolysis, ester formation, amide formation, oxidation-reduction, nucleophilic attack, etc.

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Common post-translational modifications

• Proteolytic cleavage • Glycosylation• Disulfide bond formation• Hydroxylation• Phosphorylation• Amidation• Acetylation• Methylation• Ubiquitination• + many more

Proteolytic cleavage

• Some proteins/polypeptides are activated by snipping off part of the chain (proproteins)

Proinsulin to insulinProenzymes to enzymes, e.g., chymotrypsinogen

S

S

S

S

S S

H2N

COOH

S

S

S

S

S S

H2N

COOHH2N

COOHCleave

Cleave

Proinsulin Insulin

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Cysteine protease zymogens

Cathepsin L, K, S, …

Cathepsin B

Papain

Cathepsin C

Cathepsin X

Cathepsin F

205 aa

250 aa

105 aa

95 aa

215 aa

235 aa

215 aa

62 aa 242 aa

215 aa

38aa 242 aa

Procathepsin L

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Proteolytic cleavage (preproteins)

• Preproteins, contains peptide fragment used for targeting

• Signal peptidesLeader sequences to direct transport of the protein to proper location in the cellThe sequence is often clipped off when it reaches its destination

Translocation across bacterial plasma membrane

Stryer, Biochemistry

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Glycosylation• Most common form of postranslational modification

But much less common in bacteriaTakes place in Golgi, ER and extracellular surfaces

• O-glycosylationSer, Thr, hydroxy-lysine

• N-glycosylationAsn, occurs at -Asn-X-Ser- or –Asn-X-Thr- motif, X ≠ Pro, Asp

• Degree of glycosylation varies greatlyCollagen has 0.4% sugarCartilage has 95% sugar

• Cell surface full of glycoproteins and proteoglycansLong thought to be only on extracellular side

Now known to be also present in the nucleus and cytosolIntercellular recognition

• Common monomer unitsGalactose, mannose, glucose, fucose, N-acetylglucosamine, N-acetylgalactosamine, sialic acids, xylose

Glycosylation, cont’d

N-acetylgalactosamine

O

CH2OH

OH

OH

NH

NH

O

Asn

OCH3

N-acetylglucosamine

O

CH2OHOH

OH

NH

O Ser

OCH3

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Glycosylation, cont’d

OSer

NH

O

Asn

First sugar usually N-acetylglucosamine or N-acetylgalactosamine

Glycosylation• Most common form of postranslational modification

But much less common in bacteriaTakes place in Golgi, ER and extracellular surfaces

• O-glycosylationSer, Thr, hydroxy-lysine

• N-glycosylationAsn, occurs at -Asn-X-Ser- or –Asn-X-Thr- motif, X ≠ Pro, Asp

• Degree of glycosylation varies greatlyCollagen has 0.4% sugarCartilage has 95% sugar

• Cell surface full of glycoproteins and proteoglycansAlways on extracellular sideIntercellular recognition

• Common monomer unitsGalactose, mannose, glucose, fucose, N-acetylglucosamine, N-acetylgalactosamine, sialic acids, xylose

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Hydroxylation• Hydroxyproline, hydroxylysine

• CollagenStructural protein: bone, tendons, skin, ligaments, blood vessels(Gly-X-Y)n, one-third of X and Y are ProMany of the Pro and Lys in Y position are hydroxylated

hydroxyPro stabilizes triple helical structure of collagenHydroxylysine may be glycosylatedVitamin C deficiency inactivates prolyl hydroxylase

Results in skurvy

N

OX

Y

OH

N

OH3NOH

HX

Y

+

Disulfide bond formation

• Cys – Cys bond, intra-chain or interchain

• Cys in a disulfide is called cystine vs cysteine.• S-S can be converted back to S-H with DTT (dithiothreitol).• S-S bond formation rigidifies the overall structure• e.g., α-keratin cross-links in hair, fingernails

N

S

O

Y

H

X

N

S

O

Y'

H

X'

S

S

S

S

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Cystine knot

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Oxytocin

Amidation, Acylation

• Edman degradation fails for acylated proteins• Fatty acid acylation alters physical and functional properties significantly

Effects include increasing affinity for membranes, stabilization of protein-protein interactions, inhibition of enzymatic activityGenerally reversibleRole in signal transduction and metabolic regulation

H3NO

ProteinR

+ N

R

O

ProteinO

R'H

ProteinN

O

O

H

R– Protein

NO

NH2

H

R

Acylation

Amidation

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Deamidation

• Asn to Asp or isoAsp• Less common for Gln to Glu• Amino acid must be solvent-accessible and in a

flexible region of the protein.• Biological function of non-enzymatic deamidation

is not clear.Postulated to be a signal for protein degradation thus regulating intracellular levelsAt physiological (near neutral) pH, occurs by intramolecular reorganization

Deamidation of Asn in proteins

N

ONH

R

O

NH

O

NH

ONH

ONH2

R

O

NH

N

ONH

R

O

NH

O

– NH3

NH

ONH

OO

R

O

NH

ONH

ONH

R

O

NH

O

Asn Succinimide intermediate

isoAsp

Asp

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Phosphorylation

• Ser, Thr, Tyr, His

• Phosphorylation/dephosphorylation regulates protein activity

Kinases phosphorylate, phosphatasesdephosphorylateATP, source of phosphate

O PO

OH

NH

O

O

Y

X–O P

O

OH NH

O

O

X

Y–

Methylation

• Occurs at α-amino or Lys, Arg, His side chains

• Example: Reversible methylation of receptor proteins in bacterial chemotaxis

Motile response toward or away from a diffusible chemicalMethylation/demethylation turns motor on/offTied to local concentration of chemical

H3NO

ProteinR

+ N

R

O

ProteinCH3

H

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Cyclization

NN

NN

R1

O R2

O R3

O R4

O R5H

H

H

H

OHN

NH

N

O R

OHO

etcN

O R

OH

NH2 etc

OH

O

Ubiquitination

• Ubiquitin, discovered in 1975, present in all eukaryotes

• Ubiquitination pathway elucidated in the 1980s by Ciechanover, Hershko and Rose

Nobel prize in 2004• Ubiquitin is a tag• MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKE

GIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRG

• Modification sites: Lys48, Lys63

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Roles of ubiquitination• Antigen processing • Apoptosis • Biogenesis of organelles • Cell cycle and division • DNA transcription and repair • Differentiation and development • Immune response and inflammation • Neural and muscular degeneration • Morphogenesis of neural networks • Modulation of cell surface receptors, ion channels and the secretory

pathway • Response to stress and extracellular modulators • Ribosome biogenesis • Viral infection

Ubiquitin conjugation

Nat Rev Cancer 6:776-788 (2006)

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Different types of modifications

Nat Rev Cancer 6:776-788 (2006)

Different modifications of p53 and mdm2

Nat Rev Cancer 6:776-788 (2006)