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Unformatted text preview: Subject for Today: Water and Proteins Subject Questions? Problems? Chapters 1-4 are Intro and Review Chapters 8 and 9 are Methods Unique Properties of Water Unique Water is the most abundant molecule in cells. Water The properties of water are particularly important for your understanding of its effects on the structure of proteins and membranes structure Liquid at room temperature Is an electrolyte: Can dissociate to H+ and OH High dielectric constant (water, 78; vacuum, 1; High air, 1). air, Good solvent for ions and polar molecules High melting point, heat capacity, boiling point, High and heat of vaporization show that there are strong forces between water molecules strong Structure of Water Structure What is the concentration of water in cells? Hydrogen Bonds in Water Hydrogen Hydrogen Bond Strength 5 Kcal/Mole Covalent bond strength is ~ 100 Kcal/Mole Tetrahedral orientation Water H bonds with 4 other water molecules Greatest strength in preferred orientation Formation is sensitive to temperature Cooperativity Cooperativity Dramatic Changes in Structure and Fluidity -9 Dynamic: Half life approx. 1 x 10 -9 sec Dynamic: Current work suggests that liquid water exists in clusters of 21 Current water molecules in a geodesic dome like structure T. S. Zwier. Science 304, 1119 (2004) Water as a Solvent: Free and Bound Water Water Ordinary Water Bulk Water Bulk Solvent Water Solvent Free Water Free Osmotically Active Osmotically Water Water Bound Water Structured Water Water of Hydration Much of the Water in Much Cells is Bound Water. Cells Bound Water is Very Bound Important Water Affects the Thermodynamics of Cellular Reactions Cellular ∆Γ = ∆ H - T ∆ S Free Energy, Free Enthalpy, Entropy Enthalpy, Reaction proceeds if Reaction u∆ G is negative ° ∆ G is related to the is equilibrium constant equilibrium ∆ G = - RT ln K Example: Tubulin Example: assembles to form microtubules. microtubules. Which has the more Which entropy, the free tubulin or the microtubule? microtubule? Proteins Proteins Chain of Amino Acids Connected by Peptide Bonds 19 of the 20 natural amino acids are alpha amino acids Review amino acid side chains Post the structures and the single letter and 3 letter code in Post your notes your Large Variation in Length and Sequence of aa’s Sequence of aa side chains distinguishes proteins Native form folds spontaneously Minimum free energy state Most stable state Most probable state (But, IT IS NOT MADE OF CONCRETE). Other proteins may aid, but do not instruct folding Molecular chaperones Protein disulfide isomerase Forces That Direct Protein Folding Forces WEAK FORCES Hydrogen bonds Hydrophobic Interactions Electrostatic Interactions Dipole Interactions Strong Forces Covalent Bonds Protein disulfide isomerase Protein (PDI) can rearrange to optimize folding optimize Hydrogen Bonds, Electrostatic Bonds, and van der Waals Forces Hydrophobic Interactions Covalent Bonds in Proteins Since covalent bonds are stronger than non-covalent bonds, they stabilize protein structures. Protein Structure Protein Levels of Protein Folding Primary- the amino acid sequence Secondary- local folding Alpha-helix primarily dependent on hydrogen bonds Beta-Sheet primarily dependent on hydrogen bonds Tertiery Structure- domains Quaternary Structure Assembly of multiple peptides to form the native structure e. g. homodimer; heterotrimer, etc. Conformational Change Induced by physical factors (pH, temperature) Induced by non-covalent interaction: binding another Induced molecule molecule Ligand substrate Induced by covalent modification (e.g. phosphorylation) Binding Site Alpha Helix Beta Sheet Binding Site
Can you recognize the amino acid side chains? Can you recognize the ligand that is bound to the protein? Binding of ligand can cause a conformational change. Energy Drives Protein Conformational Change See also 3-64 and 3-71, Alberts 5th Ed. RAS: guanine nucleotide regulates activity RAS:
Guanine Nucleotide is bound GDP vs GTP conformations Regulated by: GAP: GTPase Activating Protein GNRF: Guanine Nucleotide Exchange Factor Many other G proteins: ran, rab, arf, rac heterotrimeric G proteins Structure of a Cyclin Dependent Protein Kinase Structure Protein phosphorylation
serine, threonine, tyrosine Induces conformational change
depends on site Can increase or decrease activity Kinases regulated by various events Humans have > 500 Protein Kinases Ways to Study Proteins
(see chapters 8 and 9) (see Chemical Electron Methods- Amino Acid Analyzer, Amino Acid Sequencer, or Mass Spectrometer Microscopy Centrifuge- sedimentation coefficient. Sed. Coeff.
depends on M.W. and shape, and chemical composition. Chromatography- Separation based on differential partition between a mobile phase and a stationary phase. Electrophoresis- size and charge. function. X-ray Activity- Enzymatic, regulatory and or structural diffraction- Localize atoms in protein. Binding Reactions hormone (insulin) binds to its receptor on cell surface RNA polymerase binds to promoter site on DNA Non-Covalent Interactions: Binding Reactions Scatchard Plot Scatchard Measures affinity and stoichiometry of Reaction Measures affinity stoichiometry Scatchard Equation V/[L] = nK - vK (I.e. Y = mx + b; y = V/[L]; x = V) K = Equilibrium Association Constant [L] = Concentration of Ligand free at Equilibrium N = number of binding sites on protein V = moles ligand bound per mole protein Plot v/[L] vs v Plot Slope = -K Slope X-intercept = n Practical Application Scatchard Ideal Example Scatchard
V/[L] = nK - vK If v/[L] = 0, then n = v If [L] = Kd = 1/Ka, then v = n/2 This is the ligand concentration at This which the sites are half saturated. which Steeper Slope Larger Ka Lower Kd higher affinity higher X-intercept Normally an interger (# of sites) Scatchard Real Example Scatchard MLCK = myosin light chain MLCK kinase kinase CDR = calmodulin Stoichiometry n=1 Affinity Ka = 2 x 10 7 Ka Kd = 5 x 10-8 ...
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This note was uploaded on 04/03/2011 for the course CBIO 3400 taught by Professor Shen,kipreos during the Spring '08 term at UGA.
- Spring '08