Chapter 3- Protein Analyses and Structure Predictions

Peptide Functions

Hormone and Pheromones

Neuropeptides

Antibodies

Protections (toxin)

Proteins

Larger peptides

May Include

• Cofactors-

  • functional non amino acid component

  • metal ions or organic molecules

• Coenzymes

  • Organic cofactors

  • NAD+ in lactate dehydrogenase

• Prosthetic Groups

  • Covalently bonded cofactors

Column Chromatography

Column chromotography allows seperation of a mixture of proteins over a solid phase using a liquid phase to mobilize the proteins

Proteins with lower affinity for the solid phase wil wash off first then proteins with higher affinity will retain on the column longer and wash off later

Ion-Exchange

Charges on the protein stick to the charges on the resin causing them to not flow through You can get your protein off after by changing the pH to change the charge of the protein or adding a salt which will take the place of the protein and allow it to flow out

Size- Exclusion

Small proteins get stuck in beads

Big proteins go around and come out first

Affinity Chromotography

Solution of ligand is added to column

Protien mixture is added to column containing a polymer-bound ligand specific for protien of interest

Hydrophobic Interaction Chromotography

Hydrophobic proteins bind resin in low salt concentrations

When more salt is added the hydrophobic proteins are released

Electrophoresis

The elctric field puls proteins according to their charge

the gel matrix hinders mobility of proteins according to their size and shape

The gel is commonly polyacrylamide so seperation of protiensvia electrophoresis is often called polyacrylamide gel electrophoresis (PAGE)

SDS PAGE

SDS Makes the protien negativly charged

BME removed disulfide bonds

Glycerol adds density so the protien doesnt float out of the well

SDS micelles bind to proteins and facilitate unfolding

• SDA gives all proteins a uniformly negative charge

• The negative shape of proteins does not matter

• The rate of movement will only depend on size: small protiens will move faster

Calculating Molecular Weight (Electophoresis)

Can estimate based on the molecular ladder

Can graph the distance from the well for the molecular ladder and mathmatically figure out or estimate the wieght

Determining pI

A protien sample may be applied to one end of a gel strip with an immobilized pH gradient

After strining proteins are shown to be distributed along pH gradient accoring to their pI values

The largest staining spot is the pH where the most protien loses its charge

2D Electrophoresis

Useful for looking at many proteins- Example: Many protiens from one organism

Steps:

• Seperate protiens in first dimention on gel strip with isoelectric focusing

• Seperate protiens in second dimention using SDS-PAGE for size-based separation.

Western Blotting

After doing a gel electrophoresis the protein is transfered to a membrane

a processes of adding a primary antibody to bind to the protein then a colorful secondary antibody that binds to the first is done to the protien bands can be seen

Light Absorption Protiens

Proteins and peptides with known extinction coeficients, concentration can be determined by UV-visible spectrophotometry using the Beer-Lambert Law

Beer-Lambert Law

A = Ecl

A = Absorbance

E = Extinction Coeficent

C = Concentration

L= constant

Protien Purity

The ratio of activity to [Total Protien]

“Specific Activity”

Edman’s Degradation

Successive rounds of N-terminal modification, cleavage, and identification can be used to identify protein with known sequence

Mass Spectrometry

can precisley identify the mass of a peptide and thus the amino acid sequence

Can be used to determine posttranslational modifications

Proteins are normally very big so they are cleaved into parts and then mass spec is done on the parts

Evoluntionary Relationships

Sequence of proteins with identical functions from a wide range of species can be aligned and analyzed for differences

Differences indicate evolutionary divergences

Analysis of multiple protien families can indicate evolutionare relationships between organisms and ultimatley can show the history of life on earth