9 Protein Folding

1. preproinsulin (inside the cell → in a reduced environment)

• 2. proinsulin (oxidized in ER)
• - upon entering the ER lumen, it goes from a reduced to oxidized environment
• - disulfide bonds get oxidized

• • final: Insulin (secreted & oxidized)
• - releases the pro bit upon secretion

• AA involved in disulfide bonding = Cysteine

• cytoplasm: reducing

• extracellular (eg. plasma) + ER lumen: oxidizing

• • sulfides on cysteine tend to form disulfide bonds with each other (→ cystine) in an OXidizing environment
• - this imparts high stability to proteins

• • major protein of the human body
• • procollagen contains ~1500 AA residues; mature collagen contains ~1000
• • mature has 3 polypeptide chains called alpha chains
• • it’s a fibrous protein that doesn’t dissolve in water
• • they give structural integrity to tendons, cartilage, skin

• • found in Bone (bONE), Skin, Tendon
• • they have similar, not identical, AA sequences & are encoded by 1, 2 or 3 differernt genes

found in Reticulin (supporting mesh in soft tissues, also skin)

1. collagen triple helix

2. modified amino acids (hydroxy-proline & -lysine)

Fibroblasts

• collagen fibers (comprising fibrils) give structural rigidity to the skin

• problem though: don’t want fiber assembly INSIDE the fibroblast cell

• 1. Synthesis of polypeptide chain
• 2. Hydroxylation of proline & lysine
• 3. Glycosylation
• 4. Disulfide bond formation at C-terminus
• 5. Triple-helix formation
• 6. Secretion
• 7. Hydrolysis of propeptides (result = tropocollagen)
• 8. Assembly of fibril (result = collagen fibril)
• 9. Fiber assembly & formation of cross (X) links

• result = mature collagen fiber!

• Shit Head Gary Doesn't Talk Smack, He Asks for X •

• hydroxylate proline & lysine in step #2 of collagen formation to make hydroxyproline & hydroxylysine

• require the cofactor is ascorbic acid (a form of Vitamin C)

• example of postranslational modification

lack of ascorbic acid (Vitamin C) which leads to a decrease in the hydroxylation of lysines & prolines in collagen which makes collagen unstable

damage & fragility of many tissues & organs such as the gums, as well as skin lesions & blood vessel fragility

• • hydroxylysines are attachment sites for simple sugars
• • at this point, the 3 pro alpha chains have been translated & modified

• unlike simple proteins (which fold from N → C), procollagen begins to fold by disulfide bond formation in the C TERMINUS (zips up toward the N term)

• disulfide bond formation should be a clue that protein has moved from reducing location (cytoplasm) → oxidizing (ER lumen)

• collagen adopts a 2ndary structure formed by H-bonding among the 3 chains, which involves the NH groups of glycine on one chain & the COOH group on an adjacent chain

• a glycine at every 3rd residue allows close packing of each helical segment (it's small size allows close approach of the chains in the triple helix)

• A MUTATION WOULD DISRUPT ASSEMBLY!

the steric repulsion between the prolines & hydroxyprolines

• • conversion of pro → tropo collagen is catalyzed by specialized propeptidases in the ECM
• • tropocollagen is still 3 collagen polypeptide chains complexed together
• • N & C-terminal propeptides are cleaved to make collagen alpha chains

• Tropocollagen spontaneously assembles into a fibril with gaps between each tropocollagen molecule

• these are important for bONE formation (collagen forms the matrix on which calcium phosphate is deposited during osteogenesis)

Osteogenesis Imperfecta

• • a severe forms of the mutation replaces collagen's glycine with a cysteine → interfering with the collagen triple helix
• • for a multi-subunit protein, all the chains have to be functional in order to get an overall functional protein
• • if you’re heterozygous for OI, you’ll still have bad collagen

bone deformities, fragile blood vessels, brittle bones & teeth

collagen fibrils assemble into fibers, which are strengthened by cross-links between lysine or hydroxylysine

lysyl oxidase oxidizes lysine to allysine

this forms a cross-link with unmodified lysines

• lysyl hydroxylase (step #2): catalyzes hydroxylation of lysine to hydroxylysine [requires cofactor Vit C]

• lysyl oxidases (step #9): oxidizes NH3+ group at the end of the lysyl side-chain resulting in formation of modified amino acid residue → allysine

• allysine condenses with the side-chain of an unmodified lysine forming a covalent cross-links in mature collagen

• steps 1-5, up to triple helix formation occur inside the cell

• after the triple helix is formed, a procollagen molecule is secreted from the cell to undergo C & N-terminus hydrolysis (severing), spontaneous assembly into a fibril, then cross-linking & further final fiber formation

• 1. Improper processing of collagen caused by lack of enzyme (eg. lysyl oxidase deficiency)
• - heterozygotes are usually okay because having 1 copy of a functional enzyme is usually sufficient; most defects occur with homozygous “recessive” mutations

• 2. genetic defects in the collagen genes
• - like what’s described in OI; heterozygosity is equally destructive as homozygosity

hyperelastic joints, skin hyper-extendibility

• • Mad Cow disease
• • Scrapie (sheep)
• • Creutzfeldt-Jakob disease
• • Kuru

1. Inherit a missense mutation (eg. Creutzfeldt-Jacob disease)

2. Come into contact with "bad" form of the protein

• prion protein can exist in either a soluble or aggregated form; aggregated forms are disease causing

• classical C-J disease presents later in life, whereas new-variant disease presents at an average age of 29

FALSE: different types of collagen are encoded by separate genes

hydroxyPROLINE & hydroxyLYSINE

hydroxyproline content

it's typically found at every third residue

Tropocollagen; these molecules spontaneously assemble to form a collagen fibril

disulfide bonds & non-covalent interactions