Proteins Flashcards
how are proteins denatured?
Heat, pH, chemical solvents (organic and inorganic), mechanical stress
low pH in body to denature proteins
stomach
alcohol as antiseptic
denatures proteins
protein heat stability quantified by
melting curve
increase temperature, less protein folded
Tm by heat
temperature at which 50% of protein is denatured by heat
Tm by chemical
concentration of solvent at which 50% of protein is denatured
can proteins be refolded
Ribonuclease refolding experiment (Anfinson, 1972) showed all information for folding is embedded in protein primary sequence
which protein structures are retained when protein denatured
primary structure (sequence)
timescale for protein folding
proteins fold to lowest-energy conformation in the microsecond to second time scales
Levinthal’s paradox
100aa protein has possible 10^100 conformations, sampling at 10^13/s, need 10^87 years. Implies search for minimum is not random
Two models of protein folding
- Secondary structure first, then loops and tertiary structures
- Hydrophobic amino acids condense to form a molten glubule and then other secondary and tertiary structure features
- Combination of both
amino acids that prefer alpha-helices
alanine, leucine
amino acids that prefer beta-sheets
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globular structure stabilization
side chain interactions between different sections of primary sequence
secondary structure prediction
75% accuracy using two methods:
1) Empirical statistical method
2) Stereochemical method
tertiary structure prediction
major challenge, but use:
1) Homologous modeling (copy other protein fold)
2) Ab initio prediction: Rosetta program
protein refolding after denaturation
May not work because:
1) protein folds as synthesized, not all at once
2) denaturing may insolubilize protein
3) may need chaperone proteins that are no longer available
first class of chaperones
Hsp70/Hsp40
`first class chaperone qualities
prokaryotic homologs: DnaK and DnaJ
induced at elevated temperatures
bind to hydrophobic region of unfolded protein and prevent aggregation
help transport some proteins across membranes in unfolded states
second class of chaperones
chaperonin: GroEL and GroES
GroEL/GroES complex action
GroEL binds, then GroES hydrolizes ATP
protein disulfide isomerase (PDI)
secreted or cell surface proteins
correct disulfide bonds often do not form on their own in proteins with many free cysteines
Protein disulfide isomerase reduces improper disulfide bonds and reform them correctly
Peptide prolyl isomerases (PPI)
move aa (proline) from trans to cis form 6% of proline in mammals, especially frequent in beta turns
cyclophilin
PPI that activates Calcineurin
Calcineurin stimulates Interleukin-2 (IL2) production
IL2 stimulates immune response
Protein mis-folding disease: cystic fibrosis
defects in cystic fibrosis transmembrane conductance regulator (CFTR)
most common mutation is the deletion of F508
F508 deletion causes protein misfolding
Protein mis-folding disease: Prion Disease
CJD (human, genetic mutation in prion disease)
Scrapie (sheep)
Kuru (human cannabalism, ingestion of denatured prion potein)
Mad cow disease (cow)
Chronic wasting (deer, elk
Protein mis-folding disease: Prion disease
Patient brain riddled with holes
Sx: dementia, loss of coordination, fatal
PrP
28kD protein in normal brain, unknown function
diseased form resistant to protease, self-aggregates
infectious! first protein to be identified as such
resists heat, protease, other normal methods of digestion
Protein mis-folding disease: Alzheimer’s Disease
most common neurodegenerative disease, affect 2 million Americans
cognitive impairment: memory, language, perceptual skills, attention, constructive abilities, orientation, problem solving
Protein mis-folding disease: Parkinson’s Disease
primary sx: tremor at rest, muscle rigidity, stooped posture, expressionless face, slow movement
Protein mis-folding disease: AMYLOIDOSIS
amyloid can cause disease in locations other than the brain
Protein purification example: insulin production
originally isolated from dog and cow (preserved across species)
how to separate proteins by size
gel filtration
beads full of holes - large proteins flow quickly past beads, small proteins get stuck in the holes
how to separate proteins by charge
ion exchange chromatography
beads in column carry charges
cation exchange resin is neg charge, binds pos charge proteins (pH dependent b/c zwitterion states)
anion exchange
beads are positively charged, bind neg charge proteins
affinity chromatography
purify protein based on ligand binding properties
- highly specific
- expensive
protein mass determination
mass spectrometry
Edman Degradation
N-terminal sequence of protein reacts with free amine in PITC
pull off each aa in sequence using acid, identify aa
Western Blot
1) Separate protein on polyacrylamide gel
2) transfer protein onto membrane by blotting (literally)
3) incubate membrane with an antibody, leads to binding
4) wash, detect with anigi-Ig coupled to enzyme
Identify HIV infection by WEstern Blot
run known HIV proteins on gel, blot onto membrane, add patients serum. If patient has antibodies, will see reaction and is HIV+
