Native Conformation (Protein)
The folded shape of the protein that is both stable and functional
Refolding insulin
Is 25% but increases to 75% if renatured if chemically cross linked that mimics C chain
Hydrophobic core as a determinant of protein folding
non-polar amino acids want to remain in the hydrophobic core of protein.
Helices and sheets as a determinant of protein folding
Fill space and are hierarchically organized ( folds in a particular order)
Surrounding amino acid sequences as a determinant of protein folding
Insertion of amino acids that should produce a specific 2D structure will not as the other 2D structures effect what that part of the sequence should be
X-ray Crystallography
-need 97% purity
-Optimize conditions
-Can't do flexible proteins
NMR
- Limit of 35KDa
-Can do flexible proteins
-need concentrated proteins
-2D can analyze proximity of specific amino acids
Cyro-Electron Microscopy
-View in native enviroment
- Can't do small over 80KDa
-liquid N temp
Anfinsen's Experiment
unfolded proteins can return the their native states
Hydrophobic collapse
Biggest driving force of protein folding
Levinthal Paradox
That proteins fold completely randomly through possible confirmations
Stopped Flow Instrument
Mixes the denatured proteins with the buffer rapidly to kickstart refolding
NMR- Pulsed H/D exchange
Follow as H in protein are exchanged for D in the solvent. Allows for the formation of secondary structure to be viewed
1/3 in protein folding pathway
Hydrophobic collapse
-rapid but side chains are still in disarray
2/3 in protein folding pathway
Tertiary structure formation
- subdomains folded but not docked together yet, side chains still mobile.
3/3 in protein folding pathway
Final folding
-side chain forms final conformation, remaining water is expelled
Landscape Theory
polypeptides fold through various conformational adjustments that reduce free energy state until reaching their native state. (Funnel)
Paper Chromatography
stationary is cellulose based paper and the mobile phase is a solvent more or less polar, Molecules seperated based on polarity
Thin Layered
silica gel on glass or plastic plate with a mobile phase of a more or less polar solvent.
Hydrophobic Interactions
Stationary phase is hydrophillic lightly substitued with hydrophobic groups which during the mobile phase will attract the protein. Will be eluted by salt gradient
Reverse Phase
The stationary phase is a non polar liquid subbed with n-alkyl chains the mobile phase is a polar solvent. Seperation is by hydrophobic forces by solute and ligands
Dialysis
Rebuff of the protein. With a semipermable memberane allow for the excess solvent and protein to reach equilimbrium
Ultrafiltration
Used to rebuff protein sample or to concentrate the sample.
Loose leaf tea basket cup but spin it and toss the tea (buffer)
Ultracentrigution
seperate heavy particles by spinnn
Zonal Centrigfution
Sep according to sedimentry coeffecient and fraction out the results
Equilimbrium density centrigfution
Separation by the density of the particles from a uniform solution, form bands in their isopycnic positions
Western Blotting
- Gel electrophoresis and blot onto nitrocellulose
- Block unoccupied spots
- Incubate in antibody for protein of interest
- Wash and incubate with a labeling antibody
- Assay for coluorimetri