PROTEIN FOLDING

PROTEIN FOLDING

1. proteins = polypeptides folded into 3D structure due to...

   • alpha-helix
   • beta-pleated sheet
   • ionic interactions
   • hydrophobic interactions
   • van der Vaal forces
   • disulphide bridges

2. FUNCTIONAL STRUCTURES

   • number of functional structures ~ 1 (possible few variations retain protein function)
   • may be > 100,000 different proteins in an organism
   • each requiring specific folding for appropriate function
   • functional strucure = soluble, non-functional structure = insoluble/aggregate

3. MECHANISMS OF FOLDING

   • normal in vitro protein folding occurs in a crowded complex environment
   • possible correct folding in vitro under appropriate conditions
   • amino acid sequence alone could 'direct' protein folding
   • reduced, unfolded ribonuclease A -- spontaneous --> native (active) state ribonulease A

4. native folded state = free energy minimum (lowest entropy)

   • therefore, thermodynamic interaction of protein (polypeptide) with it's aqueous surrounding 'drives' folding

5. FOLDING OF SMALL PROTEINS

   • small = < 150 amino acids
   • small proteins denatured by 8M urea or 6M guanidine HCl
     • refolding - spontaneous or takes a few secs
   • not random or would take a long time
   • proteins with tertiary structure reliant on 2nd and 3ary interactions between close amino acids fold faster than those reliant on interactions between distant aa sequences
   • implies a requirement of energy input to bring together distant sequences
     • A DECREASE IN ENTROPY

6. FOLDING OF LARGE PROTEINS

   • in vitro conditions
     • v. low protein concentration
     • long incubation time
     • v. low temperatures
   • in vivo conditions
     • high protein concentration
     • short time available
     • ambient (37 degrees) temperatures
   • in vivo conditions require additional help
     • MOLECULAR CHAPERONES!!

7. CHAPERONES

   • recognise non-native proteins (exposed hydrophobic residues)
   • guide the folding procedure
   • prevent aggregation and misfolding
   • prevent interaction with other cellular components
   • activity may be modulated by binding and hydrolysis of ATP
   • act as catalysts (not present in folded product)
   • few very specific to proteins they assist in folding
   • examples...
     • heat shock proteins
     • protein disulphide isomerase
     • peptidyl prolyl isomerase

8. AMYLOIDOSIS

   • whole or fragments of proteins (normally soluble) deposit extracellularly as insoluble fibrils
     • fibrils accumulate
       • physically disrupt structure and function in tissues/organs
         • disease
         • death
         • apoptosis
   • FEATURES
     • ultrastructural morphology - common beta sheet 4nary structure
     • histochemical properties - stains with Congo red
     • deposits rich in glycosamingoglycans non-covalently bound
     • all contain serum p component (SAP)
   • CAUSES
     • sustained abnormally high levels of serum amyloid A protein (SAA) - an acute phase protein and produced during chronic infections and inflammatory disease
     • increased concentration of b2microglobulin - occurs as a consequence of haemodialysis
     • normal concentration of amyloidogenic protein over a prolonged period of time
     • inherited variant of protein with an abnormal structure or acquired variant of a protein with an abnormal structure
   • SITE OF DEPOSITION
     • focal - small amounts
     • localised - organ specific
     • systemic - present in ground tissue of the viscera and all tissues except brain
   • DIAGNOSIS
     • histological - congo red staining (+ve), requires biopsy
     • immunochemical - determines amyloid type, Abs to known amyloid protein available
     • SAP scintigraphy - serum amyloid protein radiolabeled when given localises to amyloid deposits proportionally to quantity of amyloid present - can be used to monitor levels
     • ECG - cardiac deposits not show readily with SAP scinitigraphy but deposition shown on ECG and by echocardiography
   • TREATMENT
     • reduce supply of amylloid precursor e.g. immunosuppressants for RA, renal transplant for haemodialysis, liver transplant for FAP
     • research going into developing a drug to inhibit action of SAP