Lecture 7: Protein Purification, Characterization, and Levels of Protein Structure

Electrophoresis

Separation and characterization technique for proteins

Can be used to estimate the number of different proteins in a mixture, degree of purity, isoelectric point, and approximate molecular weight

Electrophoresis for Protein Analysis

Uses cross-linked polymer polyacrylamide gells

Proteins migrate based on charge-to-mass ratio

Visually, blue color binds to proteins

Calculation of Migration of Proteins during Electrophoresis

u = V/E = Z/f

u is electrophoretic mobility

V is velocity

E is electrical potential

Z is net charge

f = frictional coefficient

The migration of protein in a gel during electrophoresis is proportional to the function of size and shape

Sodium Dodecyl Sulfate (SDS)

A detergent

Binds and partially unfolds proteins

Gives all proteins a similar charge to mass ratio

Electrophoresis in the presence of SDS separates proteins by molecular weight (smaller proteins migrate faster, s is for small)

Estimating Molecular Weight of a Protein

Plot log Mr of marker proteins vs relative migration during electrophoresis

This should be linear

Heterologous Expression of Proteins

Is the expression of a gene in a host that naturally don’t have that gene

This process is facilitated by the use of recombinant DNA technology

Endogenous Expressions of Proteins

Refers to expression of a gene in an organism where the gene is naturally found

Protein is expressed physiologically

Homologous Expression of Proteins

Refers to over expression of a gene in an organism where the gene is naturally found

Levels of Protein Structure

Primary, Secondary, Tertiary, and Quaternary

Function is Dependent on Amino Acid Sequence

Amino acid sequence confers 3D structure

3D structures confers function

Most human proteins are polymorphic meaning they have amino acid sequence variants

Edman Degradation

Classic method of sequencing amino acids

How to Study Protein Structure

Protein Structure is studied using methods that exploit protein chemistry

Traditional protein sequencing techniques are labeling proteins and breaking proteins into parts

Studying Protein Structure through Bond Breaking

Oxidation with performic acid or reduction by dithiothreitol breaks disulfide bons and denatures protein

Relationship Between Structure and Function

Proteins can assume an uncountable number of special arrangements or conformations

Chemical or structural function relate to unique three dimensional structure (native structure, functional and folded)

Protein Conformations

Limited number of conformations predominate under biological conditions

Conformation is the thermodynamically most stable and has the lowest free energy

Stability

Tendency of protein to maintain a native conformation

How are proteins stabilized

By weak interactions

Unfolding of have high conformational entropy

Chemical interactions stabilize native conformations

What chemical interactions stabilize native conformations

Weak (noncovalent) interactions and forces are numerous

Hydrogen bonds, hydrophobic effect, ionic interaction

Strong disulfide bonds are uncommon

Protein Secondary Structure

Describes the spatial arrangement of the main chain atoms in a segment of a polypeptide chain

Common types are alpha helix, beta sheets, and random coils

Alpha Helix

Simplest arrangement, maximum number of hydrogen bonds

Backbone wound around an imaginary longitudinal axis

R group protrude out from backbone

Each helical turn is 3.6 residues

Axis is parallel to hydrogen bond

Handedness of an alpha helix

Right handed: R groups protruding away from the helical backbone (most common)

Extended left hand: Theoretically less stable, not observed in proteins

The helix goes with way thumb points when palm faces towards you

Intrahelical Hydrogen Bonds

Between hydrogen atoms attached to the electronegative nitrogen atom of residue n and the electronegative carbonyl oxygen atom of residue n+4

H bonding confers significant stability

Amino Acid effects on alpha helix

Amino acid residues have an intrinsic propensity to form an alpha helix

Interactions between R chains spaced 3-4 residues apart can stabilize or destabilize an alpha helix

Charge, size and shape of R chains can destabilize

Formation of ion pairs and hydrophobic effect can stabilize

Proline and Gylcine

Occur infrequently in an alpha helix

Proline introduces destabilizing kink in helix due to nitrogen atom being part of a rigid ring making rotation around the N-C bond not possible. It also has double bond like character

Glycine is highly flexible and is mostly take up coiled structures or turns

End Amino Acids

Amino acid residues near the end of the alpha helix segment affect stability

Small electric dipoles in each peptide bond align through hydrogen bonds

Negatively Charged amino acids often found near the NH3+ terminus

Positively charged amino acids often found near the COO- terminus