Lecture 6: Amino Acids, Peptides, and Proteins

Naming Peptides

Full amino acid names (serylglycyltyrosylalanylleucine)

Three letter abbreviations: Ser-Gly-Tyr-Ala-Leu

One letter code abbreviations: SGYAL

Peptide Size

Biologically active peptides and polypeptides occur in a vast range of sizes and compositions

Length of naturally occurring peptides is from 2 to many thousands of amino acid residues

Multisubunit Protein

2 of more polypeptides associated noncovalently

Oligomeric Protein

At least 2 identical subunits

Identical units are called protomers

Conjugated proteins

Contain permanently associated chemical components and the non protein group is called the prosthetic group

Protein Separated

Protein can be separated by size, charge, binding properties, protein solubility

First Step for Purifying Proteins

Break open tissue or microbial cells

This is the crude extract and will release proteins in solution

Second Step for Purifying Proteins

Fractionation: separate proteins into fractions based on size or charge

Salting out (a from fractionation): lower solubility of proteins in salt to selectively precipitate proteins

Third Step for Purifying Proteins

Dialysis use semipermeable membrane to separate proteins from small solutes

Methods for Purifying Proteins

Column Chromatography

Ion-Exchange Chromatography

Size-Exclusion Chromatography

Affinity Chromatography

Column Chromatography

First: Buffered solution (mobile phase) migrates through porous solid material (solid phase)

Second: Buffered solution containing protein migrates through solid phase

Protein properties effect migration rate

Ion exchange, size exchange, and affinity are all forms of column chromatography

Ion Exchange Chromatography

Separates based on sign and magnitude of the net electric charge

pH and concentration of free salt ions affect protein affinity

Use bound charge groups (if cation exchanger, stationary phase is full of negative charge and negative charge eludes first)

Size Exclusion Chromatography

Also called gel filtration chromatography

Separates based on size

Large proteins emerge from the column before small proteins do because the small proteins get stuck in small pores that are like tunnels and large proteins do not

Affinity Chromatography

Separates based on binding affinity

Eluded by high concentration of salt or ligand

FLAG-tagged purification is one common example of affinity purification

After elusion protein is subject to denaturation