Carbohydrates Protein Stability and Protein Folding and Dynamics

Protein Stability & Protein Folding and Dynamics How Can Proteins Fold So Fast? • Proteins fold to the lowest-energy state in the µsec– sec time scales. How can they find the right fold so fast? • It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest-energy one is found (Levinthal’s paradox). • Search for the minimum is therefore not random; there must be a PATHWAY toward the native structure, which is thermodynamically most favorable. Protein Stability & Protein Folding and Dynamics Kinetics How? Protein folding Protein Folding Follows a Distinct Path <10 ms 2° Structures form 3° Structural Domains form <5-1000 ms Molten Globule 3° Structure forms/4° structure forms Protein Stability & Protein Folding and Dynamics Computer Simulation of Protein Folding: A 40 residue protein (video is on Web site under announcements) Simulating protein folding on the millisecond timescale has been a major challenge for many years. Recently, Folding@home researchers Vincent Voelz, Greg Bowman, Kyle Beauchamp, and Vijay Pande have broken this barrier. This is a movie of one of the trajectories that folded (i.e. started unfolded and ended up in the folded state). See Voelz et al. (2010) J. Am. Chem. Soc., 132:1526 for more details. Protein Stability & Protein Folding and Dynamics Protein folding help While the primary structure dictates what the fold will be, IN THE CELL, proteins often need help. Kinetic help 1. Protein disulfide isomerase (PDI) 2. Peptide Prolyl Isomerase (PPI) *Energetic help (prevent aggregation; maintain equilibria) *Thermodynamics 1. Molecular chaperones – heat-shock proteins (Hsp) • Hsp70, Hsp90, etc. 2. Molecular chaperonin • GroEL-GroES Protein Stability & Protein Folding and Dynamics Kinetic help Protein Disulfide Isomerase (PDI) Catalyzes Disulfide Interchange Incorrect disulfides Correct disulfides Protein Stability & Protein Folding and Dynamics Kinetic help Peptidyl Prolyl Isomerase (PPI) Catalyzes trans-to-cis isomerization Without catalysis this takes 8-10 min! Activation energy reduced 20 kcal/mole by PPI à 1015-fold rate increase Pro trans-proline cis-proline Protein Stability & Protein Folding and Dynamics Thermodynamic help Chaperones Prevent Misfolding and Aggregation Heat-shock proteins (HSP) Protein Stability & Protein Folding and Dynamics Thermodynamic help Chaperones Prevent Misfolding and Aggregation Heat-shock proteins (HSP) Nucleotide-exchange factor Protein Stability & Protein Folding and Dynamics Thermodynamic help Facilitated folding: Chaperonin Protein Stability & Folding Disease Misfolding and Disease Amyloid Plaques Protein Stability & Protein Folding and Dynamics Protein Prediction If the 1° structure is known, but only the 3° structure of a related homologous protein is known, a prediction of your protein can be done by “homology modeling” (threading). BUT, WHAT IF NO STRUCTURE? o Given all the known 3D structures, predictions of propensities to find residues and/or sequences of residues in certain structures have been effective. • e.g., already discussed propensities of residues to be in a-helices, bsheets, and b-turns. o Computer programs can now predict to about 90% certainty where these 2° structures will be in a given 1° sequence. And, the overall-fold prediction is pretty good (>80%). See Alpha-Fold: Protein Stability & Protein Folding and Dynamics Protein Stability Key Concepts • Protein stability depends primarily on hydrophobic effects and secondarily on electrostatic interactions. • A protein that has been denatured may undergo renaturation. • Protein structures are flexible and dynamic; may include unfolded regions. Protein Folding Key Concepts • Primary sequence dictates tertiary fold. • A folding protein follows a pathway from high energy and high entropy to low energy and low entropy. • Protein disulfide isomerase (PDI) catalyzes disulfide bond formation/exchange and Peptidyl Prolyl Isomerase (PPI) catalyzes cisproline peptide bond formation. • A variety of molecular chaperones assist protein folding via an ATPdependent bind-and-release mechanism, including the Chaperonin “Anfinsen Chamber.” • Amyloid diseases result from protein misfolding, with many misfolded proteins forming fibrils with extensive β structure. Carbohydrates Carbohydrates Carbohydrates Definition • Carbo-Hydrate: have formula Cn(H2O)n (for n ≥ 3) • The precursor-macromolecule relationship is: – Monosaccharide–polysaccharide (or oligosaccharide) • Carbohydrates are everywhere (ubiquitous) and versatile in function; fulfill a variety of functions. – Can be covalently linked with proteins and lipids; are intimately involved in nucleic acids ROLES Monosaccharide Polysaccharide 1. Energy source/storage glucose, fructose, etc. 2. Structure glucose, glycerol Cellulose, chitin, lipids & membranes 3. Information 4. Recognition Nucleic acids Glycolipids & glycoproteins ribose (nucleotides) many Starch, glycogen Carbohydrates The 4 S’s Size Shape Solubility Stability Range from as small as glyceraldehyde (Mw = 90 g/mol) to as large as amylopectin (Mw > 200,000,000 g/mol) Depends on size and glycosidic bond Very polar, very soluble, until large polymers Stable due to glycosidic bond Carbohydrates Monosaccharides C3(H2O)3 • Basic nomenclature: triose, tetrose, pentose, hexose – Use the suffix “-ose” – Aldehyde & ketone functions solve problem of a “carbohydrate” H–C O = yde h n 1a alde H–C–OH H2O–CH Ma H–C–OH H2O–CH Make C H 2a ket H–C–OH H2O–C one ke C OK, we know enough chemistry to drawwith a What is wrong this structure? carbon-hydrate! H–C–OH H–C–OH H H H–C–OH C=O H–C–OH H = aldoses aldotriose (triose), aldotetrose(tetrose), aldopentose, aldohexose = ketose Ketotriose (triulose), ketotetrose (tetrulose)… (pentulose, hexulose). Carbohydrates Monosaccharides • • An aldose is a carbohydrate with aldehyde functionality. A ketose is a carbohydrate with ketone functionality. Are there any chiral carbons? Carbohydrates Monosaccharides Glyceraldehyde L-Glyceraldehyde • Chemical Features: D-Glyceraldehyde – Chirality • One or more asymmetric carbons • Linear and ring forms – Derivatives: the chemistry of carbohydrates – Polymerization • The Glycosidic Bond • Non-covalent bonds in macro-molecular structure Carbohydrates Monosaccharides Fischer projections • Vertical bonds are between carbons, with highest oxidation state at the top, AND project away from you. • Horizontal bonds are pointing toward you. • If hydroxyl is on the left; its L • If hydroxyl is on the right; its D • Here D=R & L=S It turns out that the L form of glyceraldehyde is called L because it is “levorotary,” meaning it will rotate plane-polarized light to the left, or counter-clockwise. D rotates “dextrorotary.” Carbohydrates Nearly all sugars in biology are “D” GREEN =functional group RED=highest numbered chiral carbon Right (sort of like most amino acids are “L”). As you go to tetrose, pentose, etc., you are adding more chiral carbons. Rib A D- or L-sugar is defined by the chirality of the highest numbered carbon. Right Right Right Another complete set of 15 L-aldoses exist, and are enantiomers of their D-aldose relatives shown here. Glc Gal Man Right Left Right Right Carbohydrates D-ketoses The first chiral sugar is a tetrulose. Rul There is also another complete set of 7 L-ketoses exist, and are enantiomers of their D-ketose relatives shown here. Fru Fructose is the ketose form of glucose Right Ribulose is the ketose form of ribose Right Left Right Carbohydrates Monosaccharides: Stereoisomer Nomenclature • Enantiomers – stereoisomers that are nonsuperimposable complete mirror images – Example: D-sugars & L-sugars • Diastereomers – stereoisomers that are not complete mirror images – Diastereomers have different physical properties (e.g., water solubility) – Example: Carbohydrates Monosaccharides: Stereoisomer Nomenclature • Epimers – Epimers are stereoisomers that differ at only one chiral center – Epimers are diastereomers; diastereomers have different physical properties (i.e., water solubility, melting temp) – Example: – Glc & Man (2 epimer); Glc & Gal (4 epimer); Rib & Ara; Rib & Xyl • Anomers – Anomers have different chirality at carbon involved in ring formation Carbohydrates Monosaccharides: Stereoisomer Nomenclature • D-Mannose • D-Mannose and D-galactose are both epimers of D-glucose. and D-galactose vary at more than one chiral center and are diastereomers, but not epimers. Left Right Right Left Left Left Right Right Left Right Right Right Carbohydrates Monosaccharides: The most important sugars • Glyceraldehyde and dihydroxyacetone are the simplest (3 carbon) aldose and ketose, respectively. • Ribose (Rib) is the standard five-carbon sugar. • Glucose (Glc) is the standard six-carbon sugar. • Galactose (Gal) is an C4-epimer of glucose. • Mannose (Man) is an C2-epimer of glucose. • Fructose (Fru) is the ketose form of glucose. • Ribulose (Rul) is the ketose form of ribose. Need to know, recognize, draw Fisher Projection, name, abbreviate Carbohydrates • Enantiomers Monosaccharides: Stereoisomer Nomenclature – stereoisomers that are nonsuperimposable complete mirror images – Example: D-sugars & L-sugars • Diastereomers – stereoisomers that are not complete mirror images – Diastereomers have different physical properties (e.g., water solubility) – Example: ribose & lyxose • Epimers – Epimers are stereoisomers that differ at only one chiral center – Epimers are diastereomers; diastereomers have different physical properties (i.e., water solubility, melting temp) – Example: – Glc & Man (2 epimer); Glc & Gal (4 epimer); Rib & Ara; Rib & Xyl • Anomers – Anomers have different chirality at carbon involved in ring formation Carbohydrates Monosaccharides: What are these “ring” forms? • SUGARS WITH ≥5 CARBONS RAPIDLY AND STABLY FORM RINGS THROUGH HEMIACETAL (ALDOSES) AND HEMIKETAL (KETOSES) BONDS. Hemiacetals – : Not chiral Chiral • If the aldehyde (R1) and the alcohol (R2) are on the same molecule, you have a RING! • Due to the oxygen, the ring is heterocyclic Carbohydrates Monosaccharides: What are these “ring” forms? The interconversion of α and β anomers is called mutarotation. Remember “b-up” Attack sp2 carbon from top Attack sp2 carbon from bottom a The carbon (with 2 bonds to oxygen) is now chiral; its called the “anomeric” carbon. b •Anomers –Anomers are different chirality at carbon involved in ring formation –These are designated a (OH down) or b (OH up) *for D-sugars Carbohydrates Monosaccharides: What are these “ring” forms? Hemiketals a (OH down) or b (OH up) Besides the a & b, what is this pyran and furan? Its all about which alcohols are used in these reactions. Carbohydrates Monosaccharides: What are these “ring” forms? • Six-membered oxygencontaining rings are called pyranoses after the pyran ring structure. • Five-membered oxygencontaining rings are called furanoses after the furan ring structure. a (OH down) b (OH up) The way we are drawing these sugars is called a Haworth projection….. Carbohydrates Haworth projections right • Highest carbon with hemiacetal/hemiketal alcohol in back; oxygen to right. • If chiral (as for D-sugars), next highest carbon up if D, or down if L. • The anomeric carbon is usually drawn on the right side. • Other chiral carbons are down if right in Fisher projection and up if left in Fisher • b-D-Glc pyranose goes up, down, up down, up as you go from C5 to C1 • b-D-Rib furanose goes up, down, down, up as you go from C4 to C1 left right right down left up D-Glucose right down b-D-Glucopyranose right right right right Carbohydrates • Pentoses and hexoses readily undergo intramolecular cyclization. • The former carbonyl carbon becomes a new chiral center, called the anomeric carbon. • ALL forms are in equilibrium in solution Process is called “mutarotation” – When the former carbonyl oxygen becomes a hydroxyl group, the position of this group determines if the anomer is a or b. – If the hydroxyl group is on the opposite side (trans) of the ring as the CH2OH moiety, the configuration is a. – If the hydroxyl group is on the same side (cis) of the ring as the CH2OH moiety, the configuration is b. Just remember, for D-sugars, a (OH down) or b (OH up) Carbohydrates Why is the b-Glc more stable than a-Glc, and visa versa for Man? to answer this, we need to look at ACTUAL structures. These Haworth Projections are not the actual conformation…. What is? ~56% Carbohydrates Actual Conformations of Cyclized Monosaccharides • Cyclohexane rings have “chair” or “boat” conformations. • • Pyranose rings favor “chair” conformations. Multiple “chair” conformations are possible but require energy for interconversion (~46 kJ/mole). Furanose rings are envelopes • OH H H Also termed “pucker” a OH We can understand why there is a 2:1 ratio of b:a glucose….. steric hindrance AND why a-mannose is favored (2:1 ratio of a:b) Carbohydrates Monosaccharides: Chemistry • Chemical Features: – Chirality • One or more asymmetric carbons • Linear and ring forms – Derivatives: the chemistry of carbohydrates ① • Oxidation – C1 – C6 ② ③ ④ • Reduction – C1/C2 – Other carbons • Ester formation • Amino sugars – Polymerization • The Glycosidic Bond • Non-covalent bonds in macro-molecular structure Carbohydrates ① Monosaccharides: Derivatives Oxidation: These make “sugar acids” or “acid sugars” • Oxidation of aldehyde/ketone to acid (2e– loss); reaction from the C1 of aldoses – named as “onic” acids (“onate” for conjugate base) – these sugars can reduce Cu2+ to Cu+ (Fehling’s/Benedict’s test) or Ag+ to Ag0 (Tollens’ test) – Example: gluconic acid Blue This reaction is called Benedict’s test H2O Red (ppt) The copper is reduced and the sugar is oxidized. So, the sugar is called a Reducing Sugar. Reducing sugars have a free anomeric carbon. These acids can participate in internal condensation to form lactones Carbohydrates ① Monosaccharides: Derivatives Oxidation: These make “sugar acids” or “acid sugars” Colorimetric Glucose Analysis d-D-Gluconolactone b-D-Glucose CH2OH CH2OH O OH OH HO OH O Glucose oxidase OH HO O2 O OH H2O2 NH NH2 OCH3 OCH3 2 H2 O Peroxidase OCH3 OCH3 NH NH2 Oxidized o-dianisidine (bright orange) Reduced o-dianisidine (faint orange) • Enzymatic methods are used to quantify reducing sugars such as glucose. – The enzyme glucose oxidase catalyzes the conversion of glucose to glucono-d-lactone and hydrogen peroxide. – Hydrogen peroxide oxidizes organic molecules into highly colored compounds. – Concentrations of such compounds is measured colorimetrically. • Electrochemical detection is used in portable glucose sensors. Carbohydrates ① Monosaccharides: Derivatives Oxidation: These make “sugar acids” or “acid sugars” Oxidation of alcohol to acid (4e– loss) – Reaction for C6 groups; like C1 oxidation, normally on aldoses – Named as uronic acids (“uronate” for conjugate base) – Many L-sugars are Uronic acids – Examples: D-glucuronic acid, L-iduronic acid CHO CH2OH D-Idose L-Idose L-Iduronic acid Carbohydrates ② Monosaccharides: Derivatives Reduction: These make “sugar sugar alcohols alcohols” or “deoxysugars” deoxysugars Reduction of aldehyde/ketone to alcohol (2e– gain) – Only carbon not already an alcohol is the anomeric carbon (C1/C2) – Named as sugar “ol” or “itol” – Examples: glycerol, mannitol, glucitol (sorbitol) Reduction of alcohol carbon to methyl/methylene (2e– gain) – Can react at any except the anomeric carbon – Named as “x-deoxy” sugar with x being the reduced carbon – Many are L-sugars, and have specific trivial names – Examples: 2-deoxyribose, L-Fucose (Fuc) and L-Rhamnose (Rha) Fuc is also 6-deoxy L-Gal Rha is also 6-deoxy L-Man OH OH b-D-Gal Notice that for L-sugars the b-anomer is down and a-anomer is up b-D-Man Carbohydrates Monosaccharides: Chemistry • Chemical Features: – Chirality • One or more asymmetric carbons • Linear and ring forms – Derivatives: the chemistry of carbohydrates ① • Oxidation -onic -uronic – C1 – C6 ② • Reduction -tol – C1/C2 – Other carbons deoxy- ③ ④ • Ester formation • Amino sugars – Polymerization • The Glycosidic Bond • Non-covalent bonds in macro-molecular structure Carbohydrates ③ Monosaccharides: Derivatives Esters: condensation of alcohol (sugar) and an acid • Most important sugar esters use a phosphoric acid • These are called phospho-sugars or sugar phosphates • Examples: nucleotides, Glc 6-P, Fru 1,6-P2, glycerol 3-phosphate D- a-D-Glucose 6phosphate b-D-Fructose 1,6-bisphosphate