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“There are many more things in heaven and earth, Horatio, than are dreamt of in your philosophy.” Hamlet, Act 1, Scene 5 Biochemistry Medicine The Great Unknown E. coli “to scale” artwork by David Goodsell Red Blood Cell and Blood Serum Biomolecular Scale 1 Angstrom = 0.1 nm atoms and bonds: 1-3 Angstrom typical single domain globular protein: 35-50 Angstroms in diameter Classes of Biomolecules Fats, Oils, Lipids Co-Factors (Metal ions, vitamin derivatives) Carbohydrates Metabolites Nucleic Acids Proteins Minimal Ingredients for Life on Earth? •liquid water •physical scaffold •glue •organization •semi-permeable barriers •source of energy •capacity to harvest, store, and transduce energy •information storage •information copying, and retrieval •capacity for sensing environment •logic for information use, integration, and response •capacity for regulation (switches and throttles) •catalysts •systems for quality control: error management and exploitation •capacity for reproduction Minimal Ingredients for Life on Earth? Proteins play major, usually dominant, roles in… •liquid water •physical scaffold •glue •organization •semi-permeable barriers •source of energy •capacity to harvest, store, and transduce energy •information storage •information copying, and retrieval •capacity for sensing environment •logic for information use, integration, and response •capacity for regulation (switches and throttles) •catalysts •systems for quality control: error management and exploitation •capacity for reproduction Proteins are linear polymers of amino acids • 20 different amino acids: many combinations • Proteins are made in the RIBOSOME amino acids ribosome protein (short proteins are called petides or polypeptides) How Significant are the Roles of Proteins in Human Disease? Consider “simple” inherited diseases… 5000 known simple disorders. Mutations involving about 2000 different genes have been documented. 53,000 mutations now logged What are the consequences of these known mutations? Heritable Genetic Lesions Logged Heritable Mutations Linked to “Simple” Diseases 7000 Human Gene Mutation Database (Cardiff) www.hgmd.org 6000 5000 4000 3000 2000 1000 1986 1988 1990 1992 1994 1996 Year 1998 2000 2002 Heritable Mutations Linked to “Simple” Diseases 53,000 Total Chromosomal DNA Mutations: 10%: involve gross DNA changes 10%: affect RNA splicing 80%: fall in protein open reading frames of these, 30,000 are missense mutations that code for a single amino acid change in a protein (missense mutation = single DNA base change) Mutations can cause protein: loss of function altered protein function “gain of function” Diabetes insipidus: a disease caused by a protein’s loss of function. Notes on protein nomenclature: Human Vasopressin V2 Receptor •beginning of chain is “N-terminus” •end of chain is “C-terminus” •amino acids (“residues”) are numbered from N-terminus to C-terminus HN 2 P E L Q S S N P L T T S A M L M 1 S 20 P R D R C G A F A E P 200 300 A E P W P 192 W E V N G L G R C D D R R D G E A 112 D R W Q D L T S G P R A A C 180 G T R V A 100 F T A A L A L 289 W V Y L I K P K Q V A F F A W Y R L L T V V F L 309 L L Q W Q 39 A E 209 P Q F I L P L L A 120 M S L V P A L A L M M V 171 G L L L L W F L M V 92 Q L A L L F C S S Y A S V V F L I V A S Y 319 A W A N F V A S P 279 V A S V V L T 50 A M T V L V L V D I A I V 160 I A N G V L 131 A L L P P W A L M R S A 220 81 L C T I N T N C H L M Y W L R Q A G H G D V V V L S I L A I R 269 T F A A 60 K H F F V L R A S G H R 230 V S A A 140 A S I E 330 R G I A S P A I R H 150 W V H C H A G R 260 R S H R Y P E D Q E G A G 70 G M L R P P V L A E R S S S P P C G T R T G P G T 240 R G 360 G R R R 250 T cytosol S P L S 10 A S H G P V 30 F S P S Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 8-arginine vasopressin (the anti-diuretic hormone): binds to the vasopressin V2 receptor, turning on its signaling state ectoplasm V S L E S S G C L L 340 C A R S G R L A S R S 350 S P P T K A S L D T S 370 S - COO Diabetes Insipidus and the Vasopressin V2 receptor aquaporin Diabetes Insipidus 90% of cases caused by X-linked mutations that lead to a loss of function in the vasopressin V2 receptor. Mostly affects males (1/250,000). Symptoms start soon after birth: chronic severe thirstiness, dilute urine, high blood sodium, fever, constipation, etc. Disease is manageable. Lots of water, low sodium diet, and certain diuretic drugs. (diuretic = promotes urine formation and loss of sodium) Protein in Complex Multi-Factorial Diseases Proteins play central roles in 11/15 of the leading causes of death in the U.S. How many human proteins are there? How Many Protein-Encoding Genes Are There? M. genitalium: 500 E. coli: 4000 M. tuberculosis: 4000 Yeast: 6000 C. elegans: 18,000 Drosophilia melanogaster: 13,000 Rice: 60,000 Humans: 22,000 (best current estimate) Protein Sequence Defines Identity The average human gene contains 9 introns. 40-60% of all human genes are expressed in multiple splice variant forms. There are many more proteins than the ca. 22,000 genes that encode them. From: Alberts parent gene RNA splicing, then translation post-translation modifications splice variants Nature often generates diversity based on combinatorial principles. Proteins Can Be Classified According to Molecular Function The vast majority of drugs target proteins. Known drugs appear to target <150 proteins. There are at least 600 potential targets. Targets of marketed small-molecule drugs by biochemical class. (G protein-coupled receptors) 99% are proteins This Concludes The “Biomedical Significance of Proteins” Section Useful Observations About Protein Biochemistry… • Water water everywhere… • Temperature range of life is limited. • Think velcro. • Functional redundancy is a recurrent concept. • Just because you take it away and nothing happens doesn’t mean it’s not important. • Reductionism is great, but it does have limits. • Pleiotropy is a recurrent concept. • Thermodynamics and kinetics matter, but you don’t have to be J. Willard Gibbs. • Patterns and paradigms go a long way. The Energetic Governance of Biomolecular Interactions Total Energy = Kinetic Energy + Potential Energy Energy Units Quantities of Matter Used in PE Joules moles (biochemicals) Calories coulombs (electricity) 1 Calorie = 4.184 Joules partial pressure (gas) Chemical Potential Energy Units: kJ/mole or kCal/mole Electrical Potential Energy Units: volts: (Joules/Coulomb) Faraday’s Constant: 96,500 coulombs/mole Kinetic energy in biology = random thermal energy =“kT energy” Boltzmann distribution. Brownian motion and the random walk. From: Krell Institute Aquaporin Membrane Bilayer Shape isn’t everything. Experimental Electron Density Maps for the 20 Amino Acid Side Chains What does it mean when something is at equilibrium? R+L RL At equilibrium: There is no net flux between states. Free and bound receptor and ligand populations are constant. forward rate = reverse rate kforward.[R]..[L] = kreverse . [RL] Indeed: Keq = kforward/kreverse The Equilibrium Constant kforward.[R].[L] = kreverse . [RL] [RL] kforward/kreverse= = Keq [R] . [L] R +L RL Keq Position of an equilibrium is described by its equilibrium constant, Positions determined by the free energy difference. reactants Energy DGo products By definition: DGo = -RTlnKeq DGo is known as the “standard Gibbs free energy”. Negative DGo: products favored. Note the logarithmic relationship between energy and Keq. Note also that being at equilibrium does not mean static. Coupled Equilibria R+L RL + L RLL R+L RL Keq,1 RL + L RLL Keq,2 Overall equilibrium constant: R + 2L RLL Keq,overall Keq,1 x Keq,2 = Keq : DGo1 + DGo2 = DGooverall Important Classes of Equilibria in Biological Systems Ligand binding to a “receptor” carrier-ligand enzyme-substrate antibody-antigen protein-nucleic acid protein-protein Membrane partitioning (ligand in solution vs. ligand in membrane) Keq = [Membrane-Bound Ligand]/[Ligand in Solution] Biochemical Reactions (reactants vs. products) [C] . [D] For: A+B C+D Keq = [A] . [B] Protein Folding (unfolded form versus folded form) Keq = [Folded]/[Unfolded] Acid/Base Buffering (the “ligand” is the proton) Ectoplasm N Equilibria Involved in Signaling By G Protein-Coupled Receptors (such as the Vasopressin V2 Receptor) Cytoplasm C hormone (i.e., vasopressin) K basal << 1 “off” signaling state K activation >>> 1 “on” signaling state “on” signaling state In the absence of hormone, only the first equilibrium is operative, and so the receptor is mostly in its off signaling state. When the hormone becomes abundant, the overall equilbrium shifts towards the “on” signaling state because Kactivation is >> K basal. In other words, under normal physiological conditions, the receptor significantly populates the signaling-active state only when the hormone is present. In “loss of function” disorders such as diabetes insipidus, some of the causitive mutations can lower the free energy of the “off” state to the exent that it becomes the predominate state of the receptor, even under conditions where the hormone would normally drive the equilibrium completely towards the “on” state. In other words, the basal equilibrium constant is reduced to be even smaller than usual. Alternately, mutations can induce “loss of function” by mutating the hormone binding site so that the receptor does not significantly bind the hormone at normal activating concentrations. In other words, some mutations lower the activation equilibrium constant dramatically, making the receptor unresponsive to hormone at normally-stimulating concentrations. Equilibria Involved in Signaling By G Protein-Coupled Receptors How mutations can can cause constitutive “gain of function”. hormone (i.e., vasopressin) K basal<< 1 “off” signaling state K activation >>> 1 “on” signaling state “on” signaling state Above: Normal Conditions for Signaling For the vasopressin V2 receptor (and a number of other related receptors), there is another class of mutations that can change the difference in free energyConstitutive between the basalActivation “on” and “off” states, such that the “on” state structure becomes Below: Mutation-Induced the low energy state (meaning becomes the energetically favored state even in the absence of hormone). This is illustrated below. K basal > 1 “off” signaling state K activation >>> 1 “on” signaling state “on” signaling state In this case, the signaling active form of the receptor predominates even in the absence of its cognate hormone. This is inappropriate. The unstimulated signaling from such “constitutively active” mutant receptors can cause disease. Human Vasopressin V2 Receptor and NSIAD HN 2 P E L D R D P L L 39 A E L I 50 A L L V V S L V A cytosol L S S N S P L S P L T T S A M L M 1 S 20 P R R C G F A E P 300 E A P A 200 W P 192 W E V N G L G C D D R R D G E A 112 R W D Q L T S G R A A C 180 T R V G 100 A F T A A A L 289 V W Y K I P A K F V Q F A W R L Y L L T V V L F Q W L Q 309 P 209 Q F I L P L L A M 120 S L V P A A L L M M V 171 G L L L F W Q M V 92 L A L S L F C S Y A V S V F L A S Y 319 A W A N F V A S P V 279 A S V V L T M V T L V L D I A 131 I A V 160 I G N V L A L W P A L M R P C A 220 81 T I T N N L H C L M R Y Q A G H W G D V V L S L I A I R 269 T F A 60 K H F F V R A S G H R 230 V S A A A 140 S I E 330 R G I A P A S I R W H 150 V H C G R A H 260 R S H A R P Y E E D Q G 70 G R G M L P A P V L E R S S S P P C G T R P T G G T 240 R G 360 G R R R 250 30 T Q H G P 10 S V A F P ectoplasm V S L E S S G C L L 340 C A R S G R L R S 350 P P T K A L S D T S 370 S Change of arginine-137 to A S S either cysteine or leucine causes NSIAD: Neprhogenic Syndrome of Inappropriate Antidiuresis. It has been shown that these mutations result in constitutive activation of the receptor, such that signaling occurs at all times. Nephrogenic Syndrome of Inappropriate Anti-Diuresis (NSIAD) A recently discovered disorder (May 5 2005 NEJM, 2 baby boys). Very similar to the Syndrome of Inappropriate Anti-Diuretic Hormone Secretion (SIADH). This disorder is caused by higher-than-usual levels of vasopressin, the hormone that triggers vasopressin V2 receptor signaling. Phenotype is opposite of diabetes insipidus: inappropriate retention of water, resulting in hypoosmolar blood (specifically, low blood sodium) and overly concentrated urine. One patient suffered seizures. Patients are babies, so can’t be treated by restricting liquid intake. Instead, urea was administered orally– an agent that increases osmolarity and thereby triggers the formation of higher volumes of urine and fluid reduction. Babies now OK (so far). Vasopressin levels normal in these babies. Cause of NSIAD is mutations of Arg137 of the vasopressin V2 receptor that make it constitutively active. Recall that turning on the V2R turns on water absorption into the body, leading to concentrated urine. When are systems not at equilibrium? For: R + L RL Keq If [RL] ≠ Keq [R] . [L] Then, the system is not at equilibrium. Potential (“driving force”) energy towards equilibrium: Examples of non-equilibrium conditions: Ligand binding after mixing. Metabolic pathways. Nascent proteins. Energetically-trapped protein structures. Enzyme reaction following mixing. ΔG (0 at eq.) What Are the Molecular Determinants of DGo? DGo = DHo - TDSo H: enthalpy. Negative is favorable. T: temperature S: entropy: degree of disorder. Positive is favorable. Organizing anything involves a loss of entropy: Creation of a concentration gradient Connecting molecules together (whether covalent or not). Folding a protein. Binding of a flexible drug molecule to a rigid protein site. Solvation. The Hydrophobic Effect. free alkyl chains in water aggregation genesis of oil droplet Interactions That Are Primarily Electrostatic Van der Waals interactions All atoms undergo VDW interactions. What counts is intimate molecular packing. Repulsive VDW forces are basis for “steric hindrance” Ionic Interactions + d q1 x q2 Energy Between Two Ions in Solution = kxexd q: charges of the ions e: dielectric constant of solution between the ions 78 for water 2 for hydrocarbons k: constant (get from a physics book) Bond Dipoles Helix Dipole partial negative charge partial negative charge C-terminus C=O H-O H-N N-terminus partial positive charge partial negative charge C-O partial positive charge H net dipole (dipole moment) of water H partial positive charge when salt dissolves, salt bridges beween Na+ and Cl- are replaced by energetically favorable ion-water electrostatic interactions Hydrogen Bonding H-Bond Donors: H-N H-O H-S not C-H : H-Bond Acceptors: N: O: Some Terminology Hydrophobic (“hates water”) = Nonpolar = Apolar Hydrophilic (“loves water”) = Polar •Unfavorable for polar molecule to be in a hydrophobic environment •Hydrophobic molecules prefer to be in hydrophobic environment •Water is a polar solvent •Membrane bilayers have hydrophobic interiors How much energy are these various interactions usually worth in aqueous solution? H-Bond and salt bridges: -1 to -5 kCal/mol Loss of 1 degree of conformational entropy when rotation around a single bond is frozen out: +1 kCal/mol Hydrophobic Effect: transfer of 1 hydrocarbon –CH2- or –CH3 from water to hydrocarbon phase: -0.9 to -2.1 kCal/mol How Different Contributions to Molecular Energetics Can Create Order: Formation of Lipid Bilayers, the Critical Structural Unit of All Biological Membranes polar head group apolar tail (acyl chains) A bilayered lipid vesicle-- also called a liposome. By forming sealed vesicles, the remaining hydrocarbon tails are shielded from water.