9.3 Protein Separation, Purification, and Structure Determination

4 steps in protein purification and analysis

1. Extraction: Collecting protein source

2. Solubilization and stabilization: Breaking tissue and suspending protein in supernatant

3. Purification: Fractionation of proteins, collect the fraction of interest

4. Characterization and analysis: Identify and study protein

Crude extract

Supernatant containing desired protein from cell lysis

Cell lysis methods

Mechanical: High pressure homogenizer, ex. french press

Non-mechanical: Physical (Heat related), Chemical (Agents that disrupts cell membrane), Biological (Enzymes, ex. lysozyme, cellulase, zymolase)

Fractionation; Types of fractionation methods

Separate proteins into fractions based on size, charge, or solubility

Types: Solubility (Salting out: Competition for water from salts causes precipitation); Charge (Ion exchange); Size (Size exchange and centrifugation); Hydrophobicity

Ion Exchange Chromatography

Select column based on pI of protein; Uses either cation or anion exchangers; Proteins of same charge elutes first; Use different environments (pH, salt, etc) to change what is eluted/elution rate

Size Exclusion Chromatography

Separates globular proteins based on size; Beads are porous and absorbs smaller proteins, making them slower; Larger beads can’t get stuck and elute through first

Affinity Chromatography

Based on binding affinity; Ex. Histidine tagged proteins binds to nickel, elute everything else out, elute His tagged proteins with competing ligand (Some salts may also work) imidazole

Purification table: Define; Fraction volume, total protein, activity, specific activity; should they go up or down with each step?

Fraction volume: Decreases due to small loss after each step

Total protein: Total mix of proteins, decreases as unwanted proteins are filtered out

Activity: Activity of all proteins, decreases as unwanted proteins are filtered out

Specific activity: Activity of desired protein, increases as unwanted proteins are filtered out

Electrophoresis; proteins migrate due to _______; Visualize using what

Visualize and characterize purified proteins; MW; coomassie blue dye

Proteases; used for what?

Catalyze hydrolytic cleavage of peptide bonds; Used to cleave certain parts of proteins for study

Mass Spectrometry

Gives MW, amino sequence (20-30 AAs), can document entire proteome

Tandem MS (MS/MS)

Two MS filters; Sorts peptide produced by cleavage, then measures m/z ratios of charged fragments to give how much of a AA residue was present in sample

AA sequence can inform

3D structure; function; cellular location

Consensus sequence

Identifies what AA is common at what position to show conserved sequences/functional domains; Informs potential functions

Homologs/homologous proteins

Members of protein families ~25% similarity

Paralogs

Homologs in the same species

Orthologs

Homologs in different species

X-ray crystallography

Pattern of diffracted x-ray collected from protein in crystal form; image is reconstructed using mathematical techniques; Protein must be in crystalized state, the more structurally ordered the crystal is, the better the image

Limitations of x-ray crystallography

Env outside is not representative of physiological env; derived structures only provide static functional conformation (No info on molecular movement); Factors of crystalizing solution may also affect crystals formed

Nuclear magnetic resonance (MNR)

Measures nuclear spin of molecule gives magnetic dipole; excites molecules, when they return to regular energy state, energy is emitted, data is used to determine structure; Gives info on protein conformational changes, folding, and interactions with other molecules; Use higher MW isotope of molecule being measured; limited by size

Cryo-electron microscopy

Sample is vitrified/frozen in noncrystalline ice and kept frozen while being observed in EM; use data from various angle of protein to determine structure