o Nonpolar molecules tend to cluster in water due to the increase in entropy when water Chapter 1 Relationship between G and the spontaneity of a reaction o G 0 spontaneous process o G 0 non spontaneous process o G 0 system is at equilibrium Gibbs free energy equation o G H T S o H S ALWAYS spontaneous o H S spontaneous lower T o H S spontaneous higher T o H S NEVER spontaneous Equation relating G and Keq o G RT ln Keq Equation relating Keq and temperature Chapter 2 Hydrophobic effect molecules are liberated pH of solutions of weak acids o Henderson Hasselbalch equation o pKa calculation pH log H Be able to predict charge of a weak acid given pKa and pH Neutral pH pOH 7 Acidic H OH 7 Basic H OH 7 Chapter 3 Be able to draw the Watson Crick base pairs o o Structures of purines and pyrimidines o o DNA o RNA Structural differences between RNA and DNA Deoxyribose Adenine guanine cytosine and thymine Ribose Adenine guanine cytosine and uracil o General features of DNA double helix o Antiparallel strands o Right handed o Deoxyribose phosphate chain on outside o Bases stacked in middle like steps in a spiral staircase o 5 3 Determining handedness o DNA sequencing o Sanger Method Uses DNA polymerase enzyme Extends ssDNA in the 5 to 3 direction based on template Can only add nucleotides to existing DNA DNA synthesis depends on template DNA primer DNA and nucleoside triphosphates NTPs Uses dideoxynucleotides which are added to randomly stop extension A set of nested fragments is produced o Agarose gel nucleic acid is detected using the fluorescent compound ethidium bromide Chapter 4 Be able to draw the structures of the 20 common amino acids and their 3 and 1 letter abbreviations o Be familiar with their classification polar charged nonpolar and approximate pKas o pKa s Backbone amino group 9 Backbone carboxyl group 2 Acidic side chains 4 Basic side chains 11 Histidine 6 Properties of amino acids o Zwitterions contain charged groups of opposite signs o Functional groups consist of amino carboxyl and side chain o 20 different side chains o Amino acids are polyprotic can donate more than one hydrogen Amino acid stereochemistry o Natural amino acids are L o All amino acids have the same stereochemistry o This allows for handedness and specificity o Energetically efficient to have just one stereoisomer Isoelectric point calculation o pH at which of negative charges equals of positive charges o pI pKa 1 pKa 2 2 Amino Acids o o Chapter 5 SDS PAGE o Gives protein uniform mass to charge ratio o Linear relationship between mobility and log of molecular weight o Can determine mass of protein by running standards o Protein visualized using Coomassie stain which stains proteins bright blue Methods used in protein purification o Solubility pH dependence on solubility proteins are least soluble at their isoelectric points Salt dependence on solubility Salting In low ionic strength solubility increases as salt concentration Salting Out high ionic strength solubility decreases as salt concentration increases increases o Charge Polyacrylamide gel electrophoresis separate proteins based on charge SDS Page Ion exchange chromatography Anion Exchange media is charged Cation Exchange media is charged Below pI protein is charged Above pI protein is charged Isoelectric focusing Used to separate proteins based primarily on their pI After voltage is applied protein migrates to region of matrix where pH pI Affinity chromatography column media functionalized with protein binding ligand Size exclusion chromatography sorts proteins by size and shape o Polar character o Size o Specific binding characteristics Protein sequencing o Step 1 Denature Separate oligomers of same protein 8M urea or 6M guanidine o Step 2 Cleave disulfide bonds if any Oxidation with performic acid converting S S to two SO3 groups o Step 3 Fragment into peptides Enzymatic hydrolysis trypsin chymotrypsin etc o Step 4 Determine sequence of peptides Edman degradation o Step 5 Piece together overall sequence from overlapping fragments Chapter 6 Four levels of organization of protein structure 1 2 3 4 o Primary Structure amino acid sequence o Secondary Structure local arrangement of peptide backbone into common motifs o Tertiary Structure three dimensional structure of entire protein o Quaternary Structure spatial arrangement of multiple subunits Phi and psi angles o It takes three amino acids to measure phi and psi o Measured on either side of alpha carbon o N Ca is phi o Ca C is psi o Limited in range of angle they can adopt o Plot of phi vs psi angle for a peptide bond is called a Ramachandran plot Significance of Ramachandran plot o Ordered secondary structures have repeats of phi and psi angles along the chain o Looks the same for most amino acids except proline and glycine o Gly because side chain is H most free o Pro because side chain loops back to N Properties of alpha helices beta sheets and beta turns o Alpha Helices Right handed H bonds are maximized H bonds are parallel to chain Side chains point out from helical axis Amide planes are nearly parallel to helix o Beta Sheets Parallel and antiparallel Antiparallel H bonds are perpendicular to the chain direction Parallel H bonds are at an angle relative to the chain direction Side chains alternate up and down H bonds interstrand with Beta pleated sheets in comparison to alpha helix o Beta Turns Often connect secondary structural elements Type I and II make 180 degree turn in 4 residues Residue 1 H bonds with residue 4 Differ in conformation of bonds between residues 2 and 3 Residue 2 usually Pro Residue 3 usually Gly Thermodynamics of protein folding Mb O2 MbO2 MbO2 Mb O2 Kd o Proteins fold in less than a few seconds o Helices sheets and turns form first o Next is hydrophobic collapse o Then slow formation of long rage interactions o Overall energy landscape forms folding funnel o For a protein to fold Delta G must be negative o Protein folding is entropically driven can also be characterized by fractional saturation YO2 can combine with previous equation o Monomer binds O2 independently o Binds heme between helix E and F which carries O2 YO2 Kd O2 o Fe2 binds O2 Chapter 7 Binding of oxygen to myoglobin Mb MbO2 MbO2 O2 pO2 Kd pO2 YO2 pO2 is partial pressure o o Hyperbolic o Kd is dissociation constant smaller Kd means tighter binding pO2 pO2 pO2 0 5 Binding of oxygen to hemoglobin o When kd pO2 YO2 MbO2 Mb O2 o Tetramer binds 4 O2 o Binds heme which carries O2 Mb O2 Kd o Fe2 binds O2 MbO2 o