Proteins

Proteins

1. Amino Acids

   • Non-polar side chains
   • Polar side chains
   • Electrically charged side chains
   • Polypeptide chains
     • Primary Structure
       • Linked by peptide bonds
     • Secondary Structure
       • Linked by loops and B-turns
       • Arranged into tertiary 3D structures
       • Some proteins have quaternary structure e.g Immunoglobulin G
       • A single polypeptide chain folds into 2 or more compact and spatially distinct units called domains
         • A single polypeptide chain can form several domains but only ONE subunit
         • Many domains appear in more than one protein, like building blocks that nature shuffled to build different proteins.
         • Domains can move with respect to each other
       • Only 20% of eukaryotic proteins are single domain proteins.

2. Electrophoresis

   • Gel acts like a 3D sieve slowing down larger and more globular proteins compared to small and elongated ones.
   • Velocity of charged protein moving in a gel depends on: protein charge density, protein size and protein shape
   • Native PAGE (Polyacrylamide Gel Electrophoresis)
     • Carried out under conditions that preserve proteins in their native state. Useful for separation of enzymes.
     • Proteins are separated according to the combined effect of charge density, size and conformation.
     • conformational changes self-association or aggregation binding of other proteins or compounds.
   • SDS PAGE (PAGE with addition of sodium dodecyl sulfate)
     • SDS is included in the protein sample, gel and running buffer
     • Binds to proteins at a ratio of 1 molecule SDS per 2 amino acid residues which give proteins high negative charge masking protein's own charge causing unfolding of proteins
     • Charge density same for all proteins
     • Also included in the protein sample is mercaptoethanol that breaks disulfide bonds.
     • Protein separation by size
     • Coomassie Blue staining
     • SDS PAGE can be used to determine the size of a protein
     • Western blot
       • SDS PAGE of a cellular lysate from cell culture of human origin
         • Transfer to membrane
           • Incubate with primary antibody (e.g. raised in mouse against human protein of interest)
             • Incubate with secondary antibody (anti-mouset IgG raised for example in rabbit)
               • Detect emited light
               • Secondary antibody linked to enzyme that catalizes a reaction producing light (horseradish peroxidase).
   • Isoelectric focusing (IEF)
     • Electrophoretic separation of proteins according to their isoelectric points.
     • Polyacrylamide gel with a pH gradient generated with carrier ampholytes (a mixture of aliphatic amines and either carboxylic or sulfonic acid).

3. Fibrous and Globular Proteins

   • Globular Proteins
     • Globe like shape
     • Highly soluble in water
     • Functions as enzymes, transporters, antibodies, signalling proteins
   • Fibrous Proteins
     • Rod-like shape
     • Low solubility in water
     • Structural proteins
     • Collagen
       • Primary Structure
         • Localiztion: Cornea, skin and connective tissue, bones and teeth
         • Amino acid composition: glycine, proline, hydroxyproline, hydroxylysine
         • Regions with several hundreds of repeated tripeptides: Gly-X-Y (X is proline and Y is hydroxyproline) and Glycine appears at every third position.
         • Peptide bonds between prolines and hydroxyprolines are in cis conformation.
       • Secondary structure
         • Peptide bonds between prolines and hydroxyprolines are in trans conformation.
         • 3 residues per turn. No H-bonds within the helix
       • All collagen molecules are trimers
     • Keratin
       • All keratin molecules are dimers
       • Two α-helices intertwined to form a double helix called coiled-coil.
       • The two helices are cross-linked by weak interactions (van der Waals and ionic) and strong covalent bonds (disulfide bridges between numerous cysteines).
       • 3.5 residues per turn of the α-helices in keratin, so exactly 7 residues per 2 turns of the helix.
       • Tandem repetitions of 7 residue segments (heptad), in which the 1st and 4th residue are hydrophobic.
       • The more cysteines, the more disulfide bonds in keratin. Hair cysteine content is 10-14%; Nail cysteine content is 20-24%
       • A reducing agent breaks the disulfide bridges between α-helices of keratin.
       • Hair is arranged into the desired shape and an oxidizing agent causes reformation of the disulfide bonds.