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Deroo's Lectures (Topics 1 - 5).docx

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Western University
Biochemistry 2280A
Chris Brandl

Deroo’s Lectures Topic 1: The Central Dogma of Biology  Biochemistry is the study of the “molecules of life”, what makes the organs work at the cellular level  Dogma doesn’t necessarily mean it’s true, but in science there’s a factual basis  Central dogma refers to the way genetic information is stored and retrieved in living cells, how nucleic acids code for protein o DNA (nucleic acids)  RNA  proteins o DNA functions as information storage molecule and read out into RNA molecules and carry out information to produce proteins  Three ways central dogma has been expanded: 1) Epigenetics: modifications on the DNA such as acetylation, affect ability of transcription to occur o Ex: chromatin is extremely important in controlling transcription 2) MicroRNA made in antisense format and can bind to pieces of the genome and block transcription 3) In post-translation modification, proteins can be altered in function as well (phosphate group / acetyl / methyl groups added to them)  Genotype: actual genome, sequence of nucleotides in the DNA – sum of inheritable potential  Phenotype: result from genotype, what you can see – sum of observable characteristics o Ex of genotype VS phenotype: anyone can have a 6-pack if you lower your body fat and work out… but an 8-pack is genetic  Defects in DNA can lead to defects in proteins  Everybody’s genotype is different, response to medicine may be different  Proteomics studies the full set of proteins encoded by a genome  Pharmacogenomics analyzes how genetic makeup affects an individual's response to drugs o Sample of DNA is run through sequencer to find out of their DNA, based on their individual genotypes you can cut out trial & error and give proper treatment to patient (save $ and pain)  Cost of sequencing your entire genome has gone down due to advances in technology, feasible to do this patient-to-patient o Ex: BRCA-1 – 10% of developing breast cancer, if this is found in your genome then you can do preventative measures  Complicated by the fact that a lot of diseases aren’t just based one gene  Proteins are the most abundant molecules in living systems, made up of amino acids strung together in long polymer chains  These chains fold and coil in three dimensions to achieve a structure with a biological function  Most diverse class of macromolecules, all synthesized from combinations of the 20 amino acids (linear polymers)  Amino acids encode for various proteins through the genetic code  Structure of amino acid has amino group, carboxylic acid group, and a side chain (which differs each amino acid from each other) o When 4 different groups are attached to a C, stereoisomers are possible designated as D– and L– acids o Only active part in the body is the L-form  The 20 amino acids can be divided in 4 classes  Broadly divided into polar and non-polar amino acids, polar are more hydrophilic than the non-polar (hydrophobic)  Side chains tells you a lot about polarity and solubility, classified by these properties 1) Polar (- charge): R-side chain is an acid 2) Polar (+ charge): basic 3) Polar (uncharged) 4) Non-polar: hydrophobic side chain, can’t carry a charge at all  Properties of the side chain determine a protein’s character o Hydrophobic / hydrophilic (charged or uncharged) o Physical size o Ability to form hydrogen bonds  Non-polar side chains are hydrophobic and largest group o Protonated R-groups depend on the pH o Proline is the only amino acid that rejoins itself – imino acid (least flexible) and can be hydroxylated (take on a hydroxy group)  Polar charged side chains are hydrophilic  Polar uncharged side chains are also hydrophilic o In post-translational modifications, by having a phosphate group added to them from the OH (serine, thereonine, tyrosine) o Phosphorylation of these 3 amino acids can have a dramatic effect on protein function o Acidic amino acids mimic phosphorylation o Replace “phosphorylated” amino acid with alanine (no charge), what happens to the function of the protein? o Replace amino acid with an acidic amino acid (aspartic acid = -1), you should see no change  For 8 amino acids, know: properties, 3 letter code, 1 letter code, and structure Name Structure 3- 1- Polarity Side Letter Letter Chain 1) Alanine -CH3 Ala A Non Polar Non Polar 2) Glycine -H Gly G Non Polar Non Polar 3) Proline -CH 2CH 2CH 2 Pro P Non Polar Non Polar 4) Phenylal -CH 2enzene Ring Phe F Non Polar Non Polar anine 5) Cysteine -CH SH Cys C Non Polar Non Polar 2 6) Serine -CH 2H Ser S Polar Uncharge d 7) Lysine -CH 2H C2 CH2NH 2 3+ Lys K Polar Positive + 8) Aspartic -CH COO Asp D Polar Negative - 2 Acid  What bond links amino acids together? Peptide bond forms a protein  Formation of a peptide bond – reaction in which two amino acids (amino terminus on the left and carboxyl on the right), loss of water = peptide bond o Resulting peptide, you still have the N-terminus of the left and C- terminus on the right  Amino Acid “Residue”: bit leftover when it’s in a peptide, not really an amino acid anymore (missing a few atoms) – amino acid in a protein / peptide o Due to water being lost in the course of creating peptide bond  Limited rotation around the peptide bond (CONH) due to resonance character  Polypeptides (dipeptide, tripeptide) – refer in signals and codes (three letter / one letter code)  Peptide bonds are always added to the carboxy end  Polypeptides are elongated by the addition of amino acids to the C- terminal end of the growing chain  When writing sequence of amino acids, amino side always comes first Topic 2: Amino Acids  To predict the properties of a protein in solution, we need to calculate the protein’s net charge  Net charge is derived from the ionization of weakly acidic or basic groups  On a group, net charge changes as the hydrogen ion concentration (pH) changes because of the association of hydrogen ions with the groups  Start by calculating the net charge of a single amino acid in solution  Three parts of an amino acid can carry a charge in solution (added up for the net charge): o Amino group o Carboxylic acid group o Side chain (some amino acids)  Amino acids in solution are surrounded by lots of water molecules which plays an important role in characteristics  Protons move continuously in aqueous solutions, moving from one molecule to the other  To calculate net charge of an amino acid, you need pKa (tendency of the groups to attract a proton) and pH (number of protons available in solution o pKa – how much does that carboxylic acid want to take on a proton? o pH scale (logarithmic) = -log[H+]  Acidic and basic amino acids ionize (become charged) when placed in water, chemical reaction known as dissociation  When the reaction is in equilibrium, Ka = dissociation constant  Dissociation Constant: quantitative measure of the strength of an acid / personal scale of +cidi-y o HA  H + A o Ka = ( [H ] [A ] ) / [HA]  Equilibrium is characterized by a constant Ka for each individual group + o Lowering pH (increasing H )-will drive equilibrium to the left, resulting in decreased A and increased HA – fraction of the molecules that are ionized will decrease, thus net charge on group will decrease o For basic groups, the effect is opposite – fraction of molecules that are ionized increases with decreasing pH  Adding charges of each group in an amino acid gives you amino acid net charge  Amino group loses its proton at a higher pH than the carboxyl group  At pH = pKa, 50% of the carboxyl or amino group will be ionized Topic 3: Protein Structure  Proteins are linear polymers of amino acids (size: 50 to >30,000 amino acids)  Condensation reaction – water molecule is formed  Covalent bond (shares electrons)  Peptide Bond: resonance gives partial double bond character, no rotation around the bond, atoms involved are coplanar, restricts possible folding patterns of the chain o Written as single bond, but has characteristics of double bond due to resonance between the C-O and C-N bonds o The six atoms are involved are coplanar, there is not free rotation around the C-N axis constraining the flexibility of the chain and preventing some folding patterns  Backbone: repeating part of polypeptide –N-C-C-N- along the length of the molecule o Side chains of the individual residues project outwards from this backbone  Side Chains: project from backbone  Peptides: small numbers of joined residues  Residues: remaining part of amino acids (not water)  Proteins fold into many different shapes based on general rules that guide how they’ll be form  Certain structural elements are found in many proteins (primary, secondary, tertiary, quaternary)  Primary Structure: sequence of residues o Involves only the covalent bonds linking residues together o Small protein would consist of a sequence of 50 or so residues  Secondary Structure: local folding pattern of the backbone o Stabilized by hydrogen bonds between the backbone N-H and C=O groups o and β-strand make up over 50% of proteins, other uncommon types  Hydrogen bonds are strongest when the three atoms are in a straight line  α-helix: helical arrangement of single polypeptide chain (coiled spring) o Precise dimensions: 3.6 residues per turn, 0.54 nm per turn o C=O and N-H groups are oriented parallel to the axis o Each carbonyl is linked by a hydrogen bond to the N-H of a residue located 4 residues further on in the sequence within the same chain o Side chains project outward and contact any solvent, no interior space  Aromatase contains a lot of α-helices, converts testosterone to estrogen  β-sheet: polypeptide chain folds back on itself so that polypeptide strands lie side by side held together by hydrogen bonds forming very rigid structure o H-bonds occur between backbone of two or more polypeptide chains arranged adjacently and in parallel o Side chains project alternately up and down o Bonds are formed between neighbouring polypeptide (β) strands o Folds back on itself in either a parallel or antiparallel arrangement o β-sheets can be formed from two or more separate chains or folding back on itself  Silk fibroin consists mostly of β-sheets  Most proteins have both α-helices and β-sheets, plus turns and other less regular structure  Primary sequence determines type of structure formed  One chain may have different regions that take on different secondary structures  Major determinant of whether a particular part of a sequence will fold into one or the other of these structures is the interactions between side chains of the residues in the polypeptide o Steric hindrance between nearby large side chains, charge repulsion between nearby similarly-charged side chains, presence of proline, presence of other chemical groups o Proline contains a ring that constrains bond angles so that it will not fit exactly into an α-helix or β-sheet (hydrogen cannot bond due to lack of H)  Tertiary Structure: concerns the 3D arrangement of the polypeptide chain stabilized by bonds between side chains (or side chain to backbone) o Describes how regions of secondary structure fold together o Interactions between residues distant in sequence o Gives overall shape of protein o All four “weak” forces contribute to tertiary sutrcture  Hydrophobic interactions are most important, tendency of hydrocarbons to form intermolecular aggregates in an aqueous medium o Hydrophobic side chains are hidden from water by clustering together and excluding water from interior while charged side chains are mainly exterior  Tertiary structure is stabilized by multiple weak bonds and a very important covalent bond o Weak forces other than hydrophobic – H-bonds, Van der Waals forces, ionic bonds (far weaker than covalent bonds) o Disulfide Bond (covalent bond) occur between cysteine residues in distant parts of the protein only formed in oxidizing environments (hold protein in tight shape)  Hydrogen bonds can form in many different places: backbone to backbone, backbone to side chain, side chain to side chain  Domains: critical concept for larger proteins, distinct region of a protein o Often can fold independently, provide structure / function o Separated by loosely folded region and may create clefts between them  Structural Domain: independently-folded part of a protein that folds into a stable structure  Many proteins made up of connected domains, related domains often found in different proteins (evolution)  Quaternary Structure: more than one polypeptide, known as a protein’s subunit (number and arrangement of the individual polypeptide chains) o Within each of these subunits, there can be domains  Forces / bonds involved are the same as for 3° (hydrophobic, H-bonds, van der Waals, ionic, disulfide)  Specialized arrangement – “Coiled Coil”: requires two polypeptides to wrap around each other  Structure is stabilized by a hydrophobic surface on each α-helix that is created by a heptameric repeat pattern of hydrophilic / hydrophobic residues o Strip along each polypeptide (hydrophobic residues) causes the two strands to wrap around each other o Each α-helix has a hydrophobic surface that matches the other that get buried together with the coiled coil forms o Becomes coiled coil when you take two of these to put together  How to indicate protein structure: o Wire – side chains & proximity, predict amino acids involved in function o Ribbon – visualize secondary structure o Space-Filling – gives feel for shape, shows which amino acids are on the surface, predicts interactions with water / proteins  Post-translational modifications o Lipoproteins (binds lipids), metalloproteins (metal ions), hemoproteins (attached heme group) o Adds to diversity of proteins and protein function  Protein folding pathways are specified by sequence  Folded biologically-active protein is considered to be in its native state  Cooperativity: thousands of weak interactions  Random coil (newly-translated polypeptide) becomes its native state (3D structure)  Molecular Chaperones: proteins that help with folding (outside of primary sequence) o Proteins that must cross membranes must stay unfolded until their destination  Doesn’t fold to the lowest energy form…most likely a stepwise process starting with hydrophobic interactions  2° structure  3°  Conformational Changes: various types of motion possible for proteins depending on the structure o Proteins are not completely rigid, some are flexible o Often domains can move, connected by flexible linkers  Organic solve
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