Lesson 5: Protein Folding and Methods in Protein Biochemistry Flashcards
Heat
tends to be irreversible (think fired egg)
pH
tends to be reversible (but not advisable) too high/too low pH can damage amide backbone (i.e. hydorlysis)
chaeotropic salts
reversible precipitation and/or unfolding
urea
interacts with amide backbone (and/or solvation shell of protein); denature proteins
ammonium sulfate (Na4)2SO4
changes the activity of water in molar excess; precipitates folded proteins
Tm
melting temperature - temperature where 50% of protein is denatured
ex: if you “remove” one s-s bond, then less heat is required to reach Tm
% protein folded in native state vs. [urea]
urea interacts with the peptide backbone - as [urea] increases, there is a linear decrease in the % of proteins in native state
% protein folded in native state vs. [ammonium sulfate]
added in molar excess - changes the activity of water. Non-linear because all proteins are differential
1st (on top) - more hydrophobic, less solvation, ppt first
2nd (on bottom) - more hydrophilic, more solvation, ppt first
how to determine % folded correctly if there are 8 -HS
1/7 X 1/5 X 1/3 X 1/1 = 1/105 = 0.95%
*** take away 2 each time
result of native molecule –> reduce (BME) –> denature (urea) –> denatured molecule –> remove urea –> oxidize
nearly 100% of protein folded correctly
- with urea removed first, the only thing then that is missing is to “turn on” disulfides
result of native molecule –> reduce (BME) –> denature (urea) –> denatured molecule –> oxidize –> remove urea
leads to completely random disulfide formation b/c there no tertiary structures yet –>
results of Anfinsen experiment
- nearly 100% original enzyme activity and structure was restored (when Urea was removed first)
- protein folding is not random
- primary sequence dictates higher order structures (as long as primary structure is not disturbed)
characteristics of protein
- every protein has a pI where the net charge = 0
- solubility of protein tends to be minimized when pH approaches pI
- acidic proteins have pI < 7 and basic proteins have pI > 7
at lower valus of pH, proteins will carry a more () charge
positive
at higher values of pH, proteins will carry a more () charge
negative
sonification
- a way to prepare a protein extract from prokaryotic and eukaryotic cells *
- high frequency sound
enzymatic and physical
- a way to prepare a protein extract from prokaryotic and eukaryotic cells *
- lysozyme and freeze/thaw
column chromatography types
- gel filtration
- ion exchange
- affinity
gel filtration
- size exclusion
- larger molecules elute first
ion exchange
- charged matrix
- exploits charge difference between proteins
- uses 2 matrices: DEAE (positively charged) –> anion exchange, CMC (negatively charged) –> cation exchange
** remove bound proteins w/ elution buffer
** ions that have the same charge (either positive or negative) as the matrix elute first
affinity
- highly specific, binds to ligand then elutes from column
– to deptrotonate from bound matrix, add free ligand (DNA) or something that mimics DNA or urea
visualizing proteins
proteins are charged, therefore they will migrate in an electric field
» acidic and basic proteins do not migrate in the same manner
SDS-PAGE
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- confers the same shape and charge so proteins can be separated by their size
how does SDS work
- SDS binds to proteins w/ a specific stoichiometry (one SDS molecule for every 2 amino acids)
- binds to peptide backbone and disrupts all of non-covalent forces that hold the peptide together
SDS breaks up
noncovalent bonds
BME breaks up
disulfide bonds
gel electrophoresis
- largest molecules have least electrophoretic mobility (more friction, slower movement)
- add BME before running gel to ensure protein is completely linear (also boil it), and has no disulfide bonds
order of flow for electrophoresis
(-) cathode to (+) anode
