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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 properties2. 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 groupso Those with bound anionic groups= cation exchangerso 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 solution3. 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 faster4. 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 path5. 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 size7. 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 gel8. Two-dimensional electrophoresis- Combining isoelectric focusing and SDS electrophoresis - Permits the resolution of complex mixtures of proteins; separates proteins of identical molecular weight that differin pI, or proteins with similar pI values but different molecular weights3.4 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 sequencing4.1 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


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UMD BCHM 461 - EXAM 2 REVIEW GUIDE

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