week%209-9 - Amino acid and protein structures Outline •...

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Unformatted text preview: Amino acid and protein structures Outline • amino acid structures • protein structure, conformation, and functions • protein analysis techniques Reading assignments: Molecular Biology of the Gene (Watson, 6th ed) chapter 5, chapter 21 p764-771 Amino Acids are the building blocks of proteins Amino Acids Amino acids can be grouped based on their side chains: • neutral-nonpolar • neutral-polar • acidic 3 • basic Neutral Amino Acid Side Chains: I. non-polar Neutral Amino Acid Side Chains: I. non-polar Neutral Amino Acid Side Chains: II. polar Neutral Amino Acid Side Chains: II. polar Charged Amino Acid Side Chains: I. acidic Compare to: Charged Amino Acid Side Chains: II. basic Formation of a Peptide Bond The peptide bond is a planar amide linkage. N C The polypeptide (protein) • Proteins are linear polymers typically from ~100-1000 amino acid residues in length, but may be a few times larger or smaller. • The average amino acid has a molecular weight (MW) of 110 daltons (Da), so most proteins have a mass in the range of ~10-100 kDa. • Adjacent amino acids are covalently bonded by peptide bonds. Protein Conformation • Native conformation-the normal folding structure of a protein. • Denaturation-the partial or complete disruption or unfolding of the native protein conformation. Treatment with heat, detergent, or strong salts(ions) cause denaturation. • Renaturation-restoration of the native conformation. May be accomplished by heating to completely denature and then slowly cooling to allow proper refolding. Cells have proteins called chaperones that assist in protein folding. The side chains (R Groups) determine the folding of the polypeptide • Polar amino acids tend to be on the protein surface. • Non-polar (hydrophobic) amino acids tend to be internal. • Two cysteines can form covalent disulfide bonds. • Hydrogen bonding between the C=O group of one peptide bond with the NH group of a different peptide bond. Hierarchies of Protein Structure • Primary structure: The linear sequence of amino acids from the N- to C-terminus. • Secondary structure: Arrangement of segments of the polypeptide chain into regular structures. • Tertiary structure: The overall three-dimensional conformation (shape) of the polypeptide resulting from folding of secondary structures and unstructured regions. • Quarternary structure: The association of two or more polypeptides (called subunits) to form a functional protein (complex). Structures may be: di-mers, tri-mers, etc., homomers or hetero-mers, Example: homodimer Four levels of protein structure Hydrogen Bonding Stabilizes Regular Secondary Structures -helix -sheets • H-bonding between the C=O group of one peptide bond and the N-H group of another occurs in helices and -sheets. The -helix • H-bonding between the C=O group of peptide bond n and the N-H group of peptide bond n+3 • 3.6 amino acids and 5.4 Å per turn and diameter of 2.3 Å (the DNA helix is about 20 Å in diameter). Another View of an -Helix • Proline residues disrupt -helices. -Sheets • The R groups project upward and downward, in alternation. Two types of -Sheets parallel sheets the strands are arranged in the same orientation with respect to their Nand C-termini antiparallel sheets adjacent strands alternate in orientation with respect to their Nand C-termini. Turns Join Stretches of Secondary Structure • Glycine and proline are frequently found in turns, because glycine has no side chain and proline has a natural bend in its carbon backbone. Locally connected 2° structures can form motifs Zinc-Finger Coiled-Coils Almost All Proteins Contain -Helices and/or -Sheets • Regions of secondary structure may be joined by regions of undefined structure Many proteins are composed of modular domains • Domains are contiguous segments of proteins that independently maintain their own structure and/or function. • Different domain “modules” may be combined to create a protein with two or more discrete functions. Determination of Protein Structure • Secondary structures and motifs can sometimes be predicted from the primary sequence. • X-ray crystallography can provide a high-resolution (~ 2 Å) structure of proteins, but is technically challenging, time-consuming, and many proteins resist analysis. NMR is also useful but limited to small proteins (< 20 MDa). • Comparison of the primary sequence to known sequences of proteins for which the threedimensional has been previously determined can reveal the likely structure of the unknown protein. Protein Functions • Catalysis (metabolism, macromolecule synthesis, modification of protein structure/function). • DNA binding/regulation (DNA compaction, replication, transcription, recombination, etc.). • Structural (cell structure, scaffolds for enzymes, etc.) • Signaling (hormones, intracellular signal transduction). • Recognition (cell-cell interactions, immune response). • Others DNA Binding Proteins • May interact with specific DNA sequences, or nonspecifically. • The most common structure of a protein that interacts with DNA is the -helix. • Interactions with the negatively charged phosphate backbone of DNA mediate non-specific interactions. Regulation of protein structure and function I. • Allostery is a change of conformation of one site of a protein (e.g.: enzyme’s active site) through binding of a ligand to or covalent modification of a second (allosteric) site on the protein. • Cooperativity is the facilitated binding of one molecule to a ligand through interaction with another molecule. For example, a DNA-binding protein may bind another protein, thus recruiting the second protein to DNA. Allosteric regulation of protein function activation repression Allosteric regulation of protein function --- the Lac repressor Negative Feedback Regulation by an Allosteric Regulator • In metabolic pathways, a final reaction product sometimes will inhibit an enzyme responsible for its synthesis. This is negative feedback inhibition. Regulation of Protein Structure and Function II. • Covalent modification of amino acid side chains can act as a “switch” (on, off, alter binding properties, target for degradation, etc.). This may result in an allosteric change in the protein. Alternatively, the modification may directly affect ligand binding or cooperative interactions. The chemical reactivity of R groups in formation of covalent bonds • The hydrophobic groups are essentially non-reactive. • The sulfhydryl group (S-H) group of cysteine often forms a disulfide bond with another cysteine • Hydroxyl groups (serine, threonine, tyrosine) react to form O-linkages, including phosphorylation (this adds a negative charge) and glycosylation (addition of a carbohydrate group). • The amide group of asparagine may be glycosylated (Nlinked). • The amino group of lysine may be methylated or acetylated (this neutralizes the charge), or ubiquitinated. • ………. Phosphorylation Is a reversible modification that alters the chemistry of proteins • Protein kinases catalyze phosphate addition. • Protein phosphatases catalyze phosphate removal. Allosteric Regulation by Protein Binding and Phosphorylation • The binding of two proteins often induces a conformational change in one or both molecules. • Phosphorylation adds negative charge and can induce a conformational change. Regulation of protein structure and function III. • Proteolysis eliminates proteins by degradation. • Ubiquitin is a small protein (7 kDa) that may be covalently linked to lysine. Isolation of proteins with chromatography • Ion exchange chromatography exploits differences in electrostatic characteristics. • Gel filtration chromatography separates proteins based on their sizes. Assays are required for protein isolation • To track the purification of a protein and further analyze its biochemical characteristics, some type of biochemical assay is required. • Examples: antibody binding, enzymatic assay, ligand-binding, mass, others. SDS-PAGE electrophoresis of proteins---separate proteins by size • Proteins are coated with sodium dodecyl sulfate (SDS), which is highly negatively charged. • Polyacrylamide gel • With an equal mass:charge, migration through the gel will be inversely proportional to their mass. • Proteins are visualized with a stain or other method. Isoelectric focusing of proteins--separating proteins by charge • Proteins vary greatly in their charges due to different amino acid compositions. • The isoelectric point (pI) is the pH at which a particular molecule or surface carries no net electrical charge • A protein molecule will stop migrating at its isoelectric point. Two-Dimensional Gel Electrophoresis • First, isoelectric focusing. • Then standard SDS-PAGE. Immunoblotting (Western blotting) for specific protein detection • The antibody that recognizes the protein of interest (the primary antibody, 1o) is incubated with the blot. • Next, the blot is incubated with an antibody (secondary, 2o) that recognizes the constant fragment (Fc) of the 1o antibody. • The 2o antibody is attached to an enzyme that produces a chromogenic or chemiluminescent reaction in the presence of the appropriate substrate. The quaternary structure of Immunoglobulin G IgG specificity is determined by its variable regions Fig. 4-10 The variation occurs through DNA rearrangements in antibody producing immune cells (B cells). Antibodies are also important research tools • specifically identify a protein in a very complex mixture (e.g.: Western blotting). • localize proteins in a cell (e.g.: Immunofluorescence) • protein purification (e.g.: chromatography). • Can be used diagnostically (e.g.: HIV). • Can be used as inhibitors of protein activity (in research and therapeutically). Direct sequencing of a protein--Edman degradation ...
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This note was uploaded on 10/20/2009 for the course BISC 320L taught by Professor Baker,aparicio during the Spring '07 term at USC.

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