Life cycle of a protein
-10 steps
- Synthesis
- Folding
- Processing
- Covalent modification
- Translocation
- Activation
- Catalysis
- “Aging”
- Ubiquitination
- Degradation
Highly purified proteins are essential for….
Examination of its physical and functional properties
Selective precipitation
Exploits differences in relative solubility of individual proteins as a function of:
- pH
- Polarity
- Salt concentration
Column Chromatography
- Stationary phase: consists of small beads loaded into a cylindrical container.
- Mobile phase: liquid.
Liquid from the mobile phase is collected into fractions.
High pressure liquid chromatography
Pumps a sample mixture or analyte in a solvent (mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
Gel filtration (size exclusion) chromatography
- Based on their size.
- Funnel, column (gel beads, consists of a hydrated polymer).
- Small proteins: fit into the gel beads, they will emerge last at the bottom.
- Large proteins: do not fit inside the gel beads, emerge first at the bottom.
ONLY USE this method if there is a large size difference between protein.
Ion- exchange chromatography
- Based on their net charge.
- Special gel beads, can have positive or negative charge (carbohydrate polymer).
- Proteins interact with the stationary phase.
- Proteins with a net positive charge will adhere to beads with negatively charged functional groups and vice- versa
- Bound proteins are then displaced by raising the ionic strength of the mobile phase, thereby weakening charge–charge interactions.
Hydrophobic interaction chromatography
- Based on the reversible absorption of bio molecules according to their hydrophobicity.
- 4 main stages - equilibration, sample application and wash, elution, regeneration.
- Polarity of the mobile phase is decreased by gradually lowering the salt concentration of the flowing mobile phase.
Affinity Chromatography
- Based on a protein’s affinity to bind to specific molecules.
- Gel beads are modified, specific group immobilized ligands are attached to it.
- Only proteins that interact with the immobilized ligand adhere.
Gel Electrophoresis (SDS-PAGE)
- In the presence of sodium docecyl sulfate (SDS).
- Based on the rates at which they migrate in an applied electrical field.
- SDS binds at a ratio of ONE MOLECULE OF SDS PER TWO PEPTIDE BONDS.
- SDS breaks the non- covalent interactions between the proteins denaturing it.
- SDS has a charge of -1, after many the protein net charge will be -1.
- Top: larger proteins band.
- Bottom: smaller proteins band.
Isoelectric focusing
- Based on isoelectric point.
- pH gradient is created and connected to a voltage source.
- Left side: low pH, acidic.
- Right side: high pH, basic.
- Proteins move until they reach a pH at which the net charge is 0.
Who was the first to determine the sequence of a polypeptide?
Frederick Sanger
- Reduced the disulfide bonds.
- Cleaved each chain into smaller peptides.
- Resulting peptides were isolated.
- Each peptide was reacted with Sanger’s reagent.
- Then the amino acid was identified.
Edman’s reagent and Sanger’s reagent
Edman’s reagent: to label the amino-terminal residue of a peptide.
Sanger’s reagent: used to generate a new amino- terminal residue.
Edman reaction
- React the protein with a special molecule called phenyl isothiocyanate.
- Molecule will attach the proteins uncharged alpha amino terminal.
- First peptide bond will cleave without breaking any other peptide bonds.
- PTH + amino acid and peptide with one less amino acid.
Name of the method that replaced Edman technique
Mass spectrometry
-ionizes chemical species and sorts the ions based on their mass-to-charge ratio.
Basic components of a simple mass spectrometer
- Mixture of molecules is vaporized in the chamber.
- Molecules are then accelerated down the tube.
- An electric field is applied to accelerate the ions toward the detector at the end of the flight tube.
Tandem Mass Spectrometry
- Complex peptide mixtures can now be analyzed, without prior purification.
- The first mass spectrometer separates individual peptides based upon their differences in mass.
- By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined.
Proteome
The set of all the proteins expressed by an individual cell at a particular time.
Genomics
The analysis of the entire oligonucleotide sequence of an organism’s complete genetic material.
Physicochemical properties of proteins
-Water solubility depends on…
the structure of the protein
Proteins can be denaturated by.. (3)
- Heat
- Strong pH changes.
- Organic solvents
Protein denaturation
A loss of 3-dimensional structure sufficient to cause loss of function
Protein renaturation
Certain globular proteins that were denatured regain their native structure and their biological activity if returned to conditions in which the native conformation is stable.
Protein purification techniques
Are based on….
Solubility
Ionic charge
Size
Ability to bind ligands
