Amino Acids, Peptides, and Proteins Chapter 3 Amino Acids, Peptides, and Proteins

Amino Acids

Amino Acids 20 Residues Numbering of carbons 1,2,3... from C of COO- a , b, g… from C bonded to NH3+ and COO- Absolute configuration of amino acids L amino acids in biological system 1 2 a

Classification of Amino Acids Nonpolar UV absorption at 280 nm

Classification of Amino Acids Nonpolar Structural role

Classification of Amino Acids

Uncommon Amino Acids Collagen Myosin Cell wall Collagen A few proteins Incorporation during translation Prothrombin Ca2+ binding proteins Elastin

Amino Acids as Acids and Bases Zwitterion Acts as either an acid or a base Ampholytes Amphoteric substances Substances with dual nature Amino acid is a diprotic acid

Titration of Amino Acids Two pKa and two buffering regions pI: ioselectric point or isoelectric pH The point with zero electric charge Above pI : negative charge Below pI : positive charge pI = (pK1 + pK2)/2 = 5.92

Effect of Chemical Environment on pKa

Amino Acids with Ionizable R Group pI = (pK1 + pKR)/2 = 3.22 pI = (pKR+ pK2)/2 = 7.59

Peptides and Proteins

Peptides and Proteins Peptide bond Dehydration reaction Polypeptide vs. protein Polypeptide: Mr<10,000 Amino-terminal (N-terminal) Carboxyl-terminal (C-terminal)

Ionization of Peptide Ionization of peptide One free a-amino group One free a-carboxyl group Inonizable R groups pKa of R groups in peptide Different from pKa of free amino acid

Biologically Active Peptides and Polypeptides Size Small peptide Vertebrate hormones Oxytocin (9), thyrotropin-releasing factor (3), insulin (30 + 21) Antibiotics Large proteins Titin: vertebrate muscle protein (27,000 a.a.) Most of the proteins < 2,000 a.a. Oligomeric status Single polypeptide chain Multisubunit proteins Oligomeric : at least two subunits are identical Protomers : identical units Calculation of the number of amino acid residues Mr / 110 Average Mr of 20 a.a. : 138 Average Mr of protein a.a : 128 Removal of water during peptide bond formation : 128 -18 =110

Conjugated Proteins Conjugated proteins Proteins with permanently associated chemical components Prosthetic group Non-amino acid part of a conjugated protein

Working with Proteins

Protein Purification Cell lysis Crude extract Fractionation Use differences in protein solubility Depending on pH, temperature, salt concentration etc. Salting out Addition of ammonium sulfate ((NH4)2SO4) for differential precipitation of proteins Dialysis Exchange of salts and buffer using semipermeable membrane Column chromatography Separation of proteins based on charge, size, binding affinity etc. Stationary phase and mobile phase containing protein

Ion-exchange chromatography Cation-exchange chromatography Solid matrix : negatively charged group Positive charged proteins migrate slowly Anion-exchange chromatography Solid matrix : positively charged group

Size-Exclusion Chromatography Solid matrix : Beads with engineered pores of cavities Small proteins enter the pores Slow migration

Affinity Chromatography Beads with covalently attached chemical group Binding of proteins with affinity for the chemical group

Protein Purification HPLC (high-performance liquid chromatography) Use high pressure pump that speed the movement of the protein molecules Limited diffusion  High resolution Determining the methods for protein purification Mostly empirical

Separation of Protein by Electrophoresis Separation of charged proteins in an electric field Electrophoretic mobility of proteins Depending on size and shape of proteins SDS gel electrophoresis (SDS PAGE) SDS (sodium dodecyl sulfate) binds to proteins roughly proportional to the molecular weight of the protein Binding of 1 SDS/ 2 amino acids Similar chare to mass ratio for all the proteins Similar shape for all the proteins Separation of proteins depending on the mass Useful to determine molecular weight Visualization of the bands by staining (e.g. Goomassie blue)

Determining Molecular Weight of a Protein SDS PAGE (polyacrylamide gel electrophoresis)

Procedure to determine the pI of a protein Isoelectric Focusing Procedure to determine the pI of a protein Establishment of pH gradient Gel containing a mixture of low molecular weight organic acids and bases (ampholytes) Application of electric field Each protein migrates until it reaches the pH corresponding to its pI

Two-Dimensional Electrophoresis 1o : Isoelectric focusing 2o : SDS-PAGE

Quantification of Proteins During the Purification Enzyme assay 1 unit of enzyme activity The amount of enzyme causing transformation of 1.0 mmol of substrate/min at 25oC under optimal conditions Activity Total units of enzyme in solution Decrease during purification Specific activity The number of enzyme units/ mg of total proteins Measure of enzyme purity Increase during purification Other assays Biological activities DNA binding, alteration of cell growth Detection of the presence of the target protein Western blotting