BCHM461 EXAM 2 REVIEW GUIDE 3 2 Peptides and Proteins Peptides are chains of amino acids two amino acid molecules can be covalently joined through a peptide bond to yield a dipeptide through removal of water dehydration synthesis from one alpha carbon group of one a a and one alpha amino group of another when a few amino acids are joined the structure is called an oligopeptide when many amino acids are joined the structure is called a polypeptide N terminal on left and C terminal on right R group of amino acids can ionize and contribute to the acid base properties of the molecule multisubunit proteins have two or more polypeptides associated noncovalently the individual polypeptide chains in a multisubunit protein may be identical oligomeric or different the identical units consisting of one or more polypeptide chains are referred to as protomers i e hemoglobin 3 3 Working with Proteins proteins can be separated and purified extracted proteins are subjected to treatments that separate the proteins into different fractions based on a property such as size or charge a process called fractionation Salting out solubility of proteins is lowered in the presence of some salts addition of certain salts in the right amount can selectively precipitate some proteins i e ammonium sulfate 1 Column Chromatography most powerful method for fractioning proteins Takes advantage of differences in protein charge size binding affinity and other properties Individual proteins migrate faster or more slowly through column depending on properties 2 Ion exchange Chromatography Exploits differences in the sign and magnitude of the net electric charge of proteins at given pH Column matrix is a synthetic polymer resin containing bound charged groups o Those with bound anionic groups cation exchangers o Those with bound cationic groups anion exchangers Affinity of each protein for the charged groups on column is affected by pH and the concentration of competing free salt ions in surrounding solution 3 Cation exchange Chromatography Solid matrix has negatively charged groups In the mobile phase proteins with a net positive charge migrate slowly through matrix while those with a net negative charge migrate faster 4 Size exclusion Chromatography gel filtration Separates proteins according to size Large proteins emerge from column sooner than small ones Large proteins have to take a shorter path through column small ones take a longer path 5 Affinity Chromatography Based on binding affinity Beads in the column have a covalently attached chemical group called a ligand which binds to proteins When a protein mixture is added to column proteins with affinity for this liganbs binds to it and its migration is slow retarded 6 Electrophoresis Separation of proteins based on migration of charged particles Can help determine size isoelectric point and number of proteins in the mixture Larger proteins move slowly while smaller proteins move fast through gel SDS sodium dodecyl sulfate a detergent that unfolds proteins and gives them an overall negative charge which means they are separated mainly by size 7 Isoelectric focusing Procedure used to determine the isoelectric point pI of a protein A pH gradient is established by allowing a mixture of low molecular weight organic acids and bases to distribute themselves in an electric field generated across the gel When a protein mixture is applied each protein migrates until it reaches the pH that matches its pI proteins with different pIs are distributed differently throughout gel 8 Two dimensional electrophoresis Combining isoelectric focusing and SDS electrophoresis Permits the resolution of complex mixtures of proteins separates proteins of identical molecular weight that differ in pI or proteins with similar pI values but different molecular weights 3 4 The Structure of Proteins Primary Structure Primary structure a description of all covalent bonds mainly peptide bonds and disulfide bonds linking amino acid residues in a polypeptide chain sequence of amino acid residues determines 3D folding Secondary structure refers to stable arrangements of amino acid residues giving rise to recurring structural patterns Tertiary structure describes all aspects of the 3D folding of a polypeptide Quaternary structure when a protein has two or more polypeptide subunits The Edman degradation procedure labels and removes only the amino terminal residues from a peptide Proteases catalyze the hydrolytic cleavage of peptide bonds Trypsin cleavage catalyzes the hydrolysis of only those peptide binds in which the carbonyl group is contributed by either a Lys or Arg residue regardless of length or sequence of chain Mass Spectrometry offers an alternative method to determine amino acid sequencing 4 1 Overview of Protein Structure spatial arrangement of atoms in a protein or any part of a protein is called its confirmation a proteins conformation is stabilized largely by weak interactions which are important in the folding of polypeptide chains into their secondary and tertiary structures hydrophobic interactions are important in stabilizing conformation interior of a structured protein is a densely packed core of hydrophobic amino acid side chains the interaction of oppositely charged groups that form an ion pair or salt bridge can have either a stabilizing or destabilizing effect on protein structure Van der Waals interactions are dipole dipole interactions involving the permanent electric dipoles in groups such as carbonyls weak bonds and contribute little to protein stability hydrophobic residues are largely buried in protein interior number of hydrogen bonds and ionic interactions within the protein is maximized The peptide bond is rigid and planar Linus Pauling and Robert Corey C a C N C a The six atoms of the peptide group lie in a single plane with the oxygen atom of the carbonyl group trans to the hydrogen atom of the amide group From these findings Pauling and Corey concluded that the peptide C N bonds because of their partial double bond character cannot rotate freely Rotation is permitted about the N C a and the C a C bonds Thus backbone will look like a rigid plane limiting the range of conformations possible for a polypeptide chain Peptide conformation is defined by three dihedral angles phi psi omega planes are defined by bond vectors in peptide backbone 2 successive bond vectors describes a plane three successive vectors describe two planes In principle phi and psi can have any