What are three ways to classify proteins?
Globular- soluble
Fibrous- regular repeating elements
Membrane- very hydrophobic
Which side chains move the most in a protein?
Lys, Arg because they are long and floppy
What are two reducing agents that can unfold a protein?
BME, DTT- break disulfide bonds
Is protein folding reversible?
yes
What does urea do to a protein?
Denatures the protein, alters the original shape
What is the order of protein folding pathways?
Hydrophobic interactions
Secondary structures
Supersecondary structure
molten globule
What is the purpose of a chaperone?
To prevent a protein to enter low energy, but incorrect folded states
What are examples of chaperones?
PDI- enzyme that mediate disulfide bridge formation
Heat shock proteins- molecular chaperon
GroES/GroEL chaperonin- hydrophobic patches bind unfolded structures
What is the Sanger method of protein sequencing?
reduce disulfide bonds with BME
use proteases to cut polypeptides into smaller peptides
use Edman’s reagent/TFA to cut out a amino acid
can reconstruct the sequence of each polypeptide
What is a source of getting protein?
E. Coli
What are the two types of centrifugation?
Differential centrifugation separates with g force,
Density gradient centrifugation separates based on density
What are the four types of chromatography?
affinity, gel filtration, ion exchange, and hydrophobic
How does affinity chromatography work?
attach ligand/substrate/antibody to matrix
requires affinity tag to bind ligand to matrix
How does gel filtration work?
separation based on mostly size and shape
How does ion exchange chromatography work?
separation based on charge (pK, pI, solvent pH)
uses a cation or anion exchange
How does hydrophobic chromatography work?
hydrophobic groups will bind to a high salt buffer solution
What is SDS-Page and what is it for?
based on the idea of electrophoresis
protein samples move based on mass and purity
What is Western Blotting for?
run electrophoresis to separate proteins and put it on a membrane surface
What is absorbance spectroscopy?
Use Beer’s Law, through absorbance
What is circular dichroism
used to see if a protein has certain secondary structures
What is X-ray crystallography?
expose protein crystals to see a snapshot of structure
Correlation of resolution and numerical number
Higher resolution: lower numerical value, sharper image
What is NMR?
NMR gives distances between nuclei
What is cyro-electron microscopy?
get a direct image in a 3D way
