Lecture 2: Biopolymers

• A biopolymer has a common backbone, a defined chemical start and a defined end. • The backbone of a biopolymer does not change with the sequence. • The bonding atom at the junction on the backbone must have at least 3 bonding options. • Carbon can exhibit properties of a metal and a non-metal. • Carbon can catenate – bond to itself and form long chains. • The Carbon-Carbon bond is stronger than the Carbon-Oxygen bond. • Silicon is much more abundant than carbon however the Silicon-Oxygen bond is much stronger than the Silicon-Silicon bond. • Carbon compounds are relatively inert or kinetically stable to hydrolysis and oxidation. • In general, organic reactions tend to be under kinetic control rather than thermodynamic control. • If something is thermodynamically controlled, it means that it is favorable in that direction. • Although many organic reactions are thermodynamically favourable (the product has less energy than the reactant), the rate of reaction is so slow as to be almost non-existent due to high activation energy. • This makes these reactions attractive for control. • The hydrogen has a strong covalent bond and electrons are shared equally. • In an ionic bond, there is an unequal distribution of electrons due to unequal electron affinity between the atoms. • A polar covalent bond has dipole partial charges. • Hydrogen and carbon have similar electronegativity while oxygen has a higher electronegativity. • Major biopolymers: • The four main biopolymers are: fats, , and . • All linear biopolymers have a defined beginning and end. • Biopolymer synthesis is an anabolic process (requires energy input). • All biopolymers are synthesized in one direction only. • Some of the is lost in polymerization, leaving a ‘residue’ incorporated in the growing chain.

• Fats or have the general formula (-CH2-)n. • Lipids are non-soluble due to a long hydrocarbon chain. • or hydrated carbon has the general formula (H-C-OH) n. • Carbohydrates are extremely soluble with significant polarity. 2- • Ionic functional groups: COOH, NH3 and HPO4

• As the functional groups are increasingly oxidized they move from methyl groups to alcohol to aldehyde to ketone to carboxylic acid. • Aldehyde and ketone are carbonyl groups. + • The amine group is ionic, basic, has a pKa and is often positively charged at pH 7 (NH3 ). • The amide group is a derivative of carboxylic acid and is polar but non-ionic (NH2). • Information biopolymers contain a number of different and the order of those monomers when they form a is important and a template is required. The template must be copied faithfully. • are made up of 20 amino acids and differ in their side chains. • The side chains have very different chemical properties, unlike nucleic acid bases. • Proteins can be acidic, basic, polar or hydrophobic. • Two amino acids combine, by condensation polymerization to form a dipeptide. • There is an amide bond and it has the properties of a secondary amide. • This reaction, translation, is extremely thermodynamically unfavourable due to the large amount of water around. • Hydrolysis is always favoured over condensation in an aqueous environment. The process in cells is done via a different reaction pathway.

• The translation reaction occurs on ribosomes and water is excluded from the active site. • RNA catalyzes the reaction but ATP must activate amino acids first. • The has 2 resonance structures, has a polarity so can form H bonds and has a partial double bond character so can’t rotate. • The peptide bond is able to be a proton donor and a protein acceptor.

• Alpha amino acid:

• All amino acids have the above general structure. • The alpha carbon is the carbon next to the functional group. • The R group hangs off the alpha carbon. • The carboxyl group at one end is negative while the amino group at the other is positive. • Hydrophobic amino acids all have a long side chain. • The only bonds in the side chain are C-C and C-H. • Both C and H have similar electronegativities and share the electrons fairly evenly with little or no dipole. • The longer the C-C and C-H chain, the more hydrophobic.

• Aromatic amino acids have an aromatic ring in their side chain.

• Proteins are detected by light absorbance at 280nm and an amino acid with an aromatic ring will absorb light. • Polar non-ionic amino acids have a side chain with a polarity, they will form hydrogen bonds but do not have a pKa i.e. do not become fully charged.

• Carboxylic amino acids are negative at pH 7. • Basic amino acids are positive at pH 7. • Chirality happens if an atom has 4 different groups attached to it. • All amino acids are L isomers. • A D isomer placed into a cell will be a poison.

Lecture 3: Charges on amino acids

• To determine if it is an L or a D isomer, spell C-O-R-N using carbon, oxygen, the R group and nitrogen. • If CORN is spelt in the clockwise digestion it is an L isomer, if CORN is spelt in the anticlockwise direction it is a D isomer. • Charge is related to pH.

• pH is the –log10 [H+] in moles/litre (M). • The lower the pH the higher the hydrogen ion concentration. • At pH 7, [H+] = [OH-]. • Acidic side chains - glutamate and asparate:

• The acid is in equilibrium with its conjugate base. • When the concentration of H+ ions on the right hand side of the equation is increased, the equilibrium will shift to produce more of the acid and the acidic side chain (A-) is neutralised.

• At low pH the acidic side chain becomes neutral, it has been protonated and is in the form COOH above. • Reacting the acid with sodium hydroxide introduces OH- ions which deprotonates the side chain and causes H+ to dissociate from the acid. • The H+ reacts with OH- to form water. • The OH- ions have a stronger affinity for the H + and cause it to dissociate from the AH. • At high pH, the acidic side chain becomes negative.

• At low pHs, the acidic side chain is neutral. • At high pHs, the acidic side chain is negative.

• Basic side chains – arginine, lysine & histidine:

• The basic group is charged when it is protonated. • When the basic group is deprotonated it has no charge. • When H+ ions are added to the right hand side of the equation, they will react with the base to form a positive side chain.

• At low pH the basic side chain becomes positive. • When sodium hydroxide is added, the OH- ions react with H+ ions to form water and the BH+ will denature to form the base (B).

• At high pH the basic side chain become neutral.

• Glycine at pH 1:

• The pKa is the pH at the midpoint of dissociation-association. • Glycine at the pKa of the carboxyl group, pH 2.2:

• The higher the pH, the more the equilibrium will shift towards the right, i.e. the protonated form. • For each 1-pH increase, the number that is in the base form will increase by a factor of 10. • At pH 7, almost all is in the basic form. • At pH 9.6, the pKa of the amino group has been reached:

• Half is protonated; the other half has lost a proton. • At pH 12, the majority is in the base form minus a proton. • The pI is the pH where the charge on the amino acid is exactly zero, it is the average of the highest and the lowest pKa. • The pI for glycine above will be the average of 2.2 and 9.6. • If the amino acid is placed in an electric field at this point it will not move. • If you place the amino acid in a solution at a higher pH it will be negative (due to the COO-). + • If the solution has a lower pH the amino acid will be positive (due to the NH3 ).

• A buffer is a solution that resists a change in pH if you challenge it with an acid or a base. • A solution is a buffer +/- 1 pH unit either side of its pKa.

• A basic side chain will raise the pI, i.e. the pH at which it is neutral is also raised. • The molecule will be increasingly positive below the pI and increasingly negative above the pI.

• An acidic side chain will lower the pI. • Charges on proteins: • The formation of the peptide bond neutralises the carboxyl and amino group charges on the alpha carbon but not the charges on the side chains. • Not all side chains have a charge – only lys, arg, his, glu and asp. • The charge on each side chain is amino acid dependent. • There is always one N terminal and one C terminal in the backbone of a protein. • Different proteins have different native charges and the overall charge on a protein will depend on the sequence and the pH. • Determining the pI of a protein: • It can be predicted from the difference between the sum of the acidic side chains (asp + glu) and the sum of the basic side chains (lys + arg + his). • It is determined experimentally by techniques such as isoelectric focusing. • The protein is placed in a pH gradient and subjected to an electric field. • The protein moves towards its pI. • Those proteins with more acidic residues will have a lower pI. • Those proteins with more basic residues will have a higher pI. • At pH lower than the pI of the protein, the protein will be positive. • At pH higher than the pI of the protein, the protein will be negative. • For example, at pH 7 a protein with a pI of 9 will be positive while a protein with a pI of 5 will be negative.

Lecture 4: Proteins

• Folding of proteins is crucial to cellular function. • Proteins are responsible for , transcription factors, antibodies, receptors and ion channels. • Proteins are only biologically active when they have the right shape or 3D conformation. • The amino acids must be in a specific sequence. • exists between 5 and 50 degrees, with a pH of 7 within all cells. • When an egg is fried, the proteins have denatured but peptide bonds have not been broken. • Proteins can be “hydrolysed” – broken down into their constituent amino acid residues in acidic or basic conditions. • Proteins are easily “unfolded” – lose their unique 3-dimensional shape if they are heated much above 40 ° C. • DNA double helices are easily “melted” – separate and unwind if heated. • Weak forces maintain 3D protein conformation. • These weak forces are one-tenth the strength of the covalent bonds between atoms. • These forces are not rigid and allow the protein to adapt its shape. • Hydrogen bonding: • This results from the small dipole (uneven electron distribution) that exists in certain side chains and in the peptide bond itself. • To have effective H bonding you need an H bond donor and an acceptor. • The amide N of the peptide bond is an H bond donor (partially positive) while the carbonyl O (of another peptide bond) can act as an acceptor (partially negative). These bonds are not sequence dependent. • The polar non-ionic side chains can also participate in H bonding. • Side chains with –OH, -SH and amide N-H all act as H bond donors. • SH forms a weaker, longer form of hydrogen bond called a disulphide bridge. • Side chains with –C=O, OH and SH can act as acceptors. • To be an acceptor it must be delta negative (electronegative). • Side chain interactions are sequence dependent.

• Electrostatic or ionic interactions: • Side chains with a positive charge (His, Arg or Lys) can interact with those side chains with a negative charge (Asp or Glu) if they are close enough. • If side chains with like charges are brought in close proximity they will repel each other and this force also helps maintain 3D conformations. • The strength of the interaction is also dependent on the local environment. The presence of high concentrations of salts (high ionic strength), particularly on the protein surface tends to weaken or dampen down the strength of this force. • This occurs as the Na+ ions in solution shield the O- ions on the side chains. • Van der Waals interactions: • These interactions between or different parts of molecules can result from a charged or polar group coming in close proximity with a non-polar group. • The charged group will induce a small dipole on the non-polar group. Sometimes a transient dipole is induced just from fluctuations in the electron distribution in neighbouring atoms. • While these interactions are quite weak singly, they make a major contribution in a like a protein where many of these interactions occur. • Distant parts of the protein can be brought in close proximity and once there van der Waal’s forces will help stabilize the conformation. • Hydrophobic interactions: • Non-polar side chains will tend to cluster together rather than mix with polar solvents. • This is an entropic effect: polar solvent molecules (usually water) have more options when the non-polar groups are clustered than when they are scattered throughout the polar solvent. • Essentially a non-polar side chain will form a “shell” of ordered water molecules around it. • These water molecules have fewer options for movement than the free flowing molecules contacting only polar groups. • To minimize the effect of this constraint i.e. to minimize the loss of entropy the non-polar groups cluster together and bury themselves in the core of a soluble protein. • This removes them from exposure to the polar environment of the protein surface in contact with water. • In a hydrophobic environment, i.e. membrane bound proteins, the non-polar side chains arrange themselves on the exterior in contact with the membrane lipids. • It is this interaction that drives soluble proteins to fold.

• Disulphide bonds • Disulphide bridges form between cysteine residues (SH). • In a reducing environment such as inside a cell, the cysteine residues exist in a reduced form. • In an oxidising environment such as on the surface of a cell, the cysteine residues exist as disulphide bonds • Shape of the protein: • The shape of a protein is dependent upon the shape of the backbone. • Hierarchy of : • Primary: Amino acid sequence. • Secondary: Alpha helix & Beta sheet. • Tertiary: Overall 3D arrangement of a polypeptide chain. • Quaternary: Organisation of subunits. • Secondary structures: • There are a number of secondary structures a section of polypeptide can form. • The double bond character of the peptide bond (called planarity) places constraints on the number of conformations the backbone can assume. • Because of its restricted rotation the only bonds free to rotate are the bonds between the alpha carbon and the amide N and the alpha carbon and the carbonyl C. • Rotation of the C-N bond is termed phi rotation and rotation of the C-C=O is referred to as psi rotation.

• The other consideration is the polarity of the peptide bond. The amide N can act as an H bond donor and the carbonyl O act as an acceptor. • Two basic structures can form: the alpha helix and the beta sheet. • These are referred to as secondary structures. Sections of a polypeptide chain will form these structures. • Alpha Helix: • All alpha helices found in naturally occurring proteins are right handed due to L-amino acids. • All alpha helices have very similar phi and psi rotation. • Rigid structure with a single polypeptide chain. • Side chains all face outwards. • All peptide C=O H-bond to an amide N. • This secondary structure is characterized by the backbone phi and psi rotation • The side chains all face outwards, thus steric hindrance from large side chains is not a problem. • The helix is maintained by H bonding between peptide N-H and the C=O residues along the chain. • All peptide N-H and C=O are H bonded and are all aligned with the N-H pointing in one direction and the C=O pointing in the other direction. • This gives a dipole along the axis of the helix. • All alpha helices display these characteristics. Because all the peptide dipoles are satisfied by H-bonding the structure is quite rigid. • This is the major secondary structure of globular proteins such as haemoglobin and a key structural element in DNA binding proteins. • Beta Sheet: • The other commonly found secondary structure is the beta sheet. Again H bonding from peptide N-H and C=O maintains this structure. • The strands can form both parallel and antiparallel sheets. • Parallel beta sheets form when the peptide strands align in the same direction (NàC) while antiparallel sheets form when one strand is arranged in the NàC direction and the other in the CàN direction. • The side chains orientate themselves above and below the plane of the sheet, thus again steric hindrance is not a problem. • The tertiary structure:

• The 3D arrangement of the secondary structures within a polypeptide chain is termed the tertiary structure. • A polypeptide chain may have a section of alpha helix, followed by a turn (termed a beta turn) then perhaps a stretch of beta sheet etc. • Each protein will have its own unique fold i.e. beta globin always folds the same way. • Proteins often exist in nature as oligomers i.e. a number of subunits associate together to form the final functional protein. • Each subunit is a single folded polypeptide chain. • The arrangement of the monomers or subunits is termed the quaternary structure.

Lecture 5: Protein structure and introducing thermodynamics

• Molecules can interact with themselves and with other molecules. • The amino acid sequence determines the conformation that a protein will assume. • Most but not all protein folding is spontaneous. • One of the major series of experiments on protein folding was carried out by Anfinsen using the protein ribonuclease. • This protein is a small enzyme (124 residues) containing 4 disulphide bonds, which catalyses the breakdown of RNA. • The enzyme can be treated with urea and reducing agents, which effectively denature the enzyme. In this state it is unfolded, the S-S bonds are reduced and it has no activity. • If this preparation is then slowly dialysed to remove the urea and the reducing agent the activity slowly returns. • After dialysis is complete 100% activity has returned. • The conclusion drawn from these results was that the information necessary to direct the folding of the protein was contained in the primary amino acid sequence.

• Hydrophobic interactions drive proteins to fold. • The second law states that everything moves to disorder. • More scientifically reactions proceed in the direction that will give molecules more options. • Proteins spontaneously fold in a reproducible manner to produce functional proteins. • While the protein may become more ordered i.e. entropy lowered, the entropy of the whole system, solvent molecules included will increase. • This is because non-polar side chains will become buried in the core of the protein and the water molecules that were surrounding the side chains will be freer to assume more options. • Chaperones protect the peptide during the folding and prevent inappropriate association with other proteins. • Chaperones assist in the folding of proteins that do not fold spontaneously. • DNA binding proteins: • There are two classes of interaction, non-base sequence specific e.g. DNA packaging proteins and DNA polymerases, and base sequence specific e.g. restriction enzymes. • DNA and RNA are composed of ; protein is made of amino acids and cannot base pair to the DNA. • The DNA sequence is also buried in the core of the double helix. • Base sequence specific: • Amino acid residues in the protein need to make contact with the -phosphate backbone and the bases. • Most DNA binding proteins are one of three classes: helix-turn-helix (H-T-H), zinc finger and leucine zipper. • The alpha helix fits into the major groove of the DNA, allowing access to the information. • The alpha helix is a protein. • The alpha helix is a polypeptide chain, made of amino acids and hydrogen bonded to itself in a rigid fashion with parts of the peptide bond groups. • Enzymes: • Enzymes increase the rates of reactions and are highly specific for their preferred substrate. • Enzymes can be regulated, localised in certain organelles and organised into pathways. • Enzymes are usually proteins although some RNA molecules have now been found to behave as enzymes. • Usually have molecular weights between 10000 and 1000000 D. • Some enzymes require cofactors and some are multi-enzyme complexes with regulatory functions. • Holoenzymes have regulatory factors as well as catalytic factors. • Living Systems: • Living systems, including cells, obey the laws of thermodynamics and are an open system. • They exchange both matter and energy with their surroundings. • The change in free energy in a reaction is described by the term ΔG. • The change in free energy (ΔG) is the amount of energy available to do useful work. • The change in free energy (ΔG) is a combination of enthalpy (ΔH) and entropy (ΔS): ΔG = ΔH - TΔS.

• Living systems only achieve equilibrium upon death. • Equilibrium occurs when there is no net flow of energy in either direction of a chemical reaction. • Equilibrium occurs when the ΔG = 0 (no change in free energy).

• The concentration of reactants and products at equilibrium is described by the equilibrium constant Keq. • The higher the temperature, the higher the movement of molecules and the higher the entropy. • As a reaction approaches equilibrium the amount of energy to do useful work decreases. • The cell needs to operate far from equilibrium. • You don’t want to lose the capacity to do useful work to an increase in entropy. • Because are an open system matter and energy can continually flow in and out of the cell the cell can exist in a steady state that is NOT at equilibrium.