Amino acids, peptides and protein IV LO Flashcards
what is denaturation?
destruction of quaternary, tertiary and secondary structures of protein
what factors denature proteins?
heat, pH, chemicals
how was the ribonuclease refolding experiment conducted?
high concentrations of urea to denature ribonuclease A protein, used dialysis to remove urea, ribonuclease was able to fold itself back together
what were the conclusions from ribonuclease refolding experiment?
- all information required to fold protein is located in the amino acid sequence
- environment provided by the cell is not necessarily required for proper folding
why wasn’t the ribonuclease refolding experiment conclusions completely accurate?
some proteins require chaperone proteins to fold properly
what are the 2 classes of chaperone proteins?
- Hsp70/Hsp40 family
2. chaperonins
what are 3 reasons why some proteins may not refold after denaturization
- protein folds as it is synthesized
- protein may have been irreversibly insolubilized by denaturation process
- requires chaperons
what are the prokaryotic homologs of Hsp70/40 chaperons?
DnaK and DnaJ
how do Hsp70/40 proteins work?
bind to hydrophobic region of unfolded protein and prevent aggregation, sometimes help transport proteins across membranes in unfolded states
when are Hsp70/40 proteins activated?
elevated temperatures
what are chaperonin proteins called in E. coli?
GroEL and GroES
what is the structure of chaperonin proteins in E. coli?
GroEL and GroES form large molecular chambers and GroES is cap
how do chaperonin proteins work?
bind to hydrophobic regions of polypeptide that enter chamber, go through many conformational changes and then release the correctly folded protein
how are chaperonin proteins different in eukaryotes than prokaryotes?
in eukaryotes, subunits that make up the chaperonin protein are not all the same (ex. 7 subunits of GroEL)
when is disulfide isomerase used?
in folding of proteins with many cysteines
what is the function of disulfide isomerase?
reduces incorrect disulfide bonds and reforms them correctly to attain proper protein structure
what environment are proteins with a lot of disulfide bonds usually transported to?
very reduced environment that does not favor the formation of disulfide bonds
why is proline a unique amino acid in regards to conformation?
proline can be cis or trans but all other AA are trans
what is the function of prolyl isomerase?
changes proline between cis and trans conformation depending on the folding pattern needed for the protein
In what diseases is protein mis-folding the major cause of disease?
Prion, cystic fibrosis, alzheimer’s disease, parkinson’s disease, and amyloidosis
what is the most common mutation that causes cystic fibrosis?
F508 deletion
what is the insoluble form of prion?
PrPSc
what misfolded protein causes alzheimers?
Beta-amyloid plaques
what is the histological structure found in brains of parkinson’s disease patients?
Lewy bodies
where does primary amyloidosis occur?
bone marrow produces too many fragments of antibodies which build up in blood stream and then are deposited in tissues
what are the 4 major ways to purify a protein?
- gel filtration chromatography
- ion exchange chromatography
- affinity chromatography
- gel electrophoresis
what is the process of gel filtration chromatography?
- purified based on size
- protein solution poured over porous beads, small proteins get trapped, large proteins make it to the bottom
how are proteins filtrated out in ion exchange chromatography?
- filtered by charge
- beads are negative (cation exchange) or positive (anion exchange)
- proteins of same charge of beads will make it to the bottom
how are proteins filtered out in affinity chromatography?
- based on ligand binding properties
- beads have certain ligands, target protein attaches
- wash target protein off by adding more free ligand
how are proteins filtered out by gel electrophoresis?
- by size
- SDS added to proteins to make them negative
- proteins migrate towards anion end
- small proteins move faster than larger proteins
