BCMB2002 Weeks 1-4 Basics PDF

BCMB2002 → What are proteins? Proteins are versatile macromolecules in every living organism and have endless diversity of functions in biological processes. Proteins: Main Agents of Biological Function Catalysis: Catalysis is the increase in rate of a chemical reaction due to an added substance...

BCMB2002 → What are proteins? Proteins are versatile macromolecules in every living organism and have endless diversity of functions in biological processes. Proteins: Main Agents of Biological Function Catalysis: Catalysis is the increase in rate of a chemical reaction due to an added substance known as a catalyst - enolase (in the glycolytic pathway) - DNA polymerase (in DNA replication) Transport: - Haemoglobin (transports O2 in the blood) - Lactose permease (transports lactose across the cell membrane) Structure: Collagen (connective tissue) Keratin (nails, hair, feathers, horns) Motion: - Myosin (muscle tissue) - Actin (muscle tissue, cell mobility) Protein structure Primary → amino acid residues (linear sequence of amino acids) Secondary → alpha helix and beta sheet and random coils Tertiary → polypeptide chain Quaternary structure→ assembled subunits 20 abundant amino acids MEMORISE STRUCTURE AND NAME OF 20 ABUNDANT AMINO ACIDS LEARNNNN Acid base properties Charge properties ect. Amino Acids: Building Blocks of Protein Proteins are linear heteropolymers of a-amino acids Amino acids have properties that are well-suited to carry out a variety of biological functions – Capacity to polymerize – Useful acid-base properties – Varied physical properties – Varied chemical functionality When combined in various sequences, the functional groups contribute to the function Most a- amino acids are chiral The alpha carbon always has four substituents and is tetrahedral All except proline have - An acidic carboxyl group - A basic amino group - An alpha hydrogen connected to the alpha carbon The fourth substituent (R) is unique In glycine the fourth substituent is Hydrogen. RULE OF CORN: L amino acid the one that we use Changing R will change amino acid The CORN rule is a convention for determining the chirality of an amino acid as either L or D Clockwise is L Anti clockwise is D Amino Acids: Atom Naming Organic nomenclature: start from one end Biochemical designation: start from a-carbon and go down the R-group Common amino acids can be placed in five basic groups depending on their R substituents: Nonpolar, aliphatic (7) Glycine Gly G Alanine Ala A Proline Pro P Valine Val V Leucine Leu L Isoleucine Ile I Methionine Met M Aromatic (3) Phenylalanine (Phe F) Tyrosine (Try Y) Tryptophan Trp (W) Polar, uncharged (5) Serine Ser S Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q Positively charged (3) Lysine Lys K Histidine His H Arginine Arg R Negatively charged (2) Aspartate Asp D Glutamate Glu E Need to known name, 3 letters and one letter code Methionine STARTS PROTEIN SYNTHESIS (MET) 7 nonpolar → Order GAPVLIM (increasing length of chain or structure 1. Glycine single H 2. Alanine CH3 only 3. Proline (cyclic makes a box) 4. Valine (short upside down v) 5. Leucine makes an upside down y 6. Isoleucine longer chain 7. Methionine has S group + longest chain on R substituent Aromatic 3 → PTT ABSORB UV LIGHT at 270-280 nm Phenylalanine (single aromatic ring) 1 benzene group Tyrosine (single aromatic ring + OH group) 1 benzene group + OH Tryptophan (benzene with extra C=CH and NH POLAR UNCHARGED R GROUPS (5) Side chains form amino acids + cysteine forms disulfide bonds Serine (CH2OH) Threonine Cysteine (CH2 + SH) important for disulfide bonds → reversible covalent linkage Asparagine CH2- C-H2N C=O Glutamine long chain with H2N + C=O Lysine → long chain of CH2 + NH3 + (1 nitrogen) Arginine → long chain of CH2 + (3 nitrogens) c = NH2+ Histidine → 2 CH double bonds 2 nitrogen (pentagon structure) 2- Negatively Charged R groups → ATE Aspartate → Ch2 COO- Glutamate → CH2 CH2 COO- Uncommon Amino Acids in Proteins Not incorporated by ribosomes - except for Selenocysteine and Pyrrolysine Arise by post-translational modifications of proteins Reversible modifications, especially phosphorylation, are important in regulation and signaling All chains have amino acid start and carboxyl terminal end Serine Glycine Tyrosine Alanine Leucine -SER-GLY-TYR-ALA-LEU Or serylglycltyrosylananylleucine Or for longer do single letter codes SGYAL Peptides: A Variety of Functions Peptides are anything less than 100 amino acids. More than 100 amino acids → proteins Hormones and pheromones Insulin → think sugar Oxytocin → childbirth Sex peptides → fruit fly mating Neuropeptides Substance p ( pain mediator) Antibiotics - Polymyxin (gram negative bacteria) - Bacitracin (gram positive bacteria) Protection eg toxin Amantin (mushrooms) Conotoxin (cone snails) Chlorotoxin (scorpions) Quesitons: FORMATION OF A PEPTIDE BOND The formation of a peptide bond occurs during protein synthesis when two amino acids are joined through a dehydration synthesis (condensation) reaction. In this process, the amino group (–NH₂) of one amino acid reacts with the carboxyl group (–COOH) of another. This reaction releases a molecule of water (H₂O) as a byproduct, with the remaining atoms forming a covalent bond between the carbon of the carboxyl group of one amino acid and the nitrogen of the amino group of the next. The resulting bond, called a peptide bond or amide bond, has the structure –CO–NH–. This bond links amino acids into a linear chain, forming a dipeptide initially, and, through successive peptide bond formations, leads to longer polypeptides and ultimately functional proteins. The peptide bond is strong and planar, with a partial double bond character due to resonance, which restricts rotation around the bond and helps stabilize protein structure. DISULFIDE BRIDGE disulfide bridge: There’s no mention of cysteine in the sequence, so no disulfide bridge can form. Two amino acids of the standard 20 contain sulfur atoms. They are: c) methionine and cysteine Explanation: Out of the 20 standard amino acids, cysteine and methionine are the only two that contain sulfur atoms. Cysteine has a thiol (-SH) group in its side chain, which can form disulfide bonds with other cysteine residues. Methionine has a thioether (-S-) group in its side chain. The other amino acids listed do not contain sulfur atoms, so options a), b), d), and e) are incorrect. Thus, the correct answer is c) methionine and cysteine. Lecture 2: Protein structures Learning objectives Know the non covalent interactions that help in the formation of secondary, tertiary and quaternary structures Understand the nature of secondary structures and how are these formed Explain the proteins interactions at quaternary level of protein structures Know the conformation of the peptide backbone - Adopt a 3D conformation → gives rise to native fold structure - 3D conformation gives rise to a specific biological function - Native fold has a large number of favourable interactions within the protein (structure where it has the lowest possible delta G) Peptide bond achieves most things within the protein - There is cost in conformational entropy of folding the protein into one specific native fold 4 Favourable interactions: TWO MAIN RULES THAT DICTATE THE FOLD (hydrophobic and hydrogen bonds) 1. Hydrophobic → release of water molecules from structured solvation layer around molecule as protein folds increases the net entropy If you bury the hydrophobic gain lots of London dispersion and hydrophobic contractions inside the protein which will stabilise them. Water molecules are going to be forming massive shields surrounding them. 2. Hydrogen bonds → interaction of N-H and C=O of the peptide bond leads to local regular structures such as a-helices and B-sheets H-BONDS DICTATE SECONDARY STRUCTURE OF PROTEIN 3. London dispersion → The medium-range weak attraction between all atoms contributes to the stability in the interior protein 4. Electrostatic interactions → One is negative one is positive → salt bridges in a hydrophobic environment stabilise the protein We have 3 positive and 2 negative amino acids so when they are nearby they can react and form electrostatic interactions + salt bridges to stabilise protein. PROTEIN IS NOT IN ITS ACTIVE FORM UNTIL IN ITS NATIVE FOLD!! - Misfolding causes mutations Native state is in tertiary structure Structure of the peptide bond: - The Peptide bond is the bond formed with the carboxylic group + the amino group - this is the planar region → bc the N has 2 electrons (free and not paired) - means nitrogen can donate and form a double bond → becoming positively charged → small electric dipole: VIA the carbonyl O2 has a partial negative charge and the amide nitrogen has partial positive - All peptide bonds in proteins occur in this trans configuration - Structure of protein = properties of peptide bond - Peptide bond is a resonance hybrid of two structures - Resonance = → bonds that are less reactive (esters = more reactive) → rigid and nearly planar → large dipole moment in favoured trans configuration ONLY POINT OF ROTATION IN PEPTIDE CHAIN IS CARBON ALPHA Decrease shaking and moving around The polypeptide is made up of a series of planes linked at a-carbons: - The carbon-alpha is the only point of rotation - 2 planes can rotate - The phi angle and phsi angle …? - Retrain certain angles - Side chains restrict movement also The rigid peptide plane and partially free rotations: Rotation around the peptide bond is not permitted Rotation around bonds connected to the alpha carbon is permitted f (phi): angle around the a-carbon—amide nitrogen bond y (psi): angle around the a-carbon—carbonyl carbon bond In a fully extended polypeptide, both y and f are 180° Ideal angles Alpha helices → PHi -57 and PSI -47 → angles are always negative bc twists in one direction Beta conformation → Antiparallel: - 139 or + 135 → one is negative and one is positive - allows zig zag structure to form Some f and y combinations are very unfavourable because of steric crowding of backbone atoms with other atoms in the backbone or side chains - Some f and y combinations are more favourable because of a chance to form favourable H-bonding interactions along the backbone - A Ramachandran plot shows the distribution of f and y dihedral angles that are found in a protein → shows the common secondary structure elements → reveals regions with an unusual backbone structure Secondary structure: - Secondary structure refers to a local spatial arrangement of the polypeptide backbone - Two regular arrangements are common: The a helix – stabilised by hydrogen bonds between nearby residues The b sheet – stabilized by hydrogen bonds between adjacent segments that may not be nearby - The irregular arrangement of the polypeptide chain is called the random coil ALPHA HELIX: - The backbone held by hydrogen bonds between the backbone amindes of an n and n+4 amino acids - Right-handed helic with 3.6 residues (5.4A) per turn - Peptide bonds are aligned parallel with the helical axis - Side chains point out and perpendicular wiht helical axis TOP VIEW: - Inner diameter (no side chains) is 4-5 A - Outer diameter (with side chains) is 10-12 A  - Fit well into the major groove of dsDNA - Residue 1 and 8 align nicely on top of each other → what kind of sequences give a helix with one hydrophobic face? NOTHING FITS INSIDE GROOVE TOO SMALL The A helix - Side chains always point out Sequence affects helix stability: - Not all polypeptide sequences adopt a-helical structure - Small hydrophobic residues such as Ala and Leu are strong helix formers - Pro acts as a helix breaker because the rotation around the N-Ca bond is impossible → IT FORMS A CYCLE BACK ON ITSELF - cant form helix - Gly acts as a helix breaker because the tiny R-group supports other conformations (more rotation too flexible)→ GlY terminates the alpha helics - Attractive or repulsive interactions between side chains 3–4 amino acids apart will affect the formation The helix dipole: - Peptide bond = strong dipole moment → carbonyl O negative → amide H positive - All peptide bonds in the helix have similar orientation - The a helix has a large macroscopic dipole moment - Negatively charged residues occur near postive end of helix dipole BETA SHEETS:  - The planarity of the peptide bond and tetrahedral geometry of the a-carbon create a pleated sheet-like structure - Sheet-like arrangement of backbone is held together by hydrogen bonds between the backbone amides in different strands (Up to 15 residues long) - Side chains protrude from the sheet alternating in up and down direction - 2 to 22 strands in globular proteins (average is six), right handed twist, usually form the central core Parallel and Antiparallel b Sheets: Form hydrogen bond network → at least 2 hydrogen bond strands to form beta sheet. - Parallel or antiparallel orientation of two chains within a sheet are possible - In parallel b sheets the H- Bonded strands run in the same direction → Resulting in bent H-bonds (weaker) (> 5 residues) - In antiparallel b sheets the H-bonded strands run in opposite directions - Resulting in linear H-bonds (stronger) ANTIPARALLEL = MORE STABLE BC ITS MORE EXTENDED - HYDROGEN BOND IS LINED UP DIRECTLY WITH AMINO GROUP !! - IF THEY ARE ALIGNED THEN ELECTRONS FLOW MUCH BETTER - Antiparallel happens continuously and linear - Interspaced by 4 amino acids that will do the turn and return and continue that B turns: b turns occur frequently whenever strands in b sheets change the direction → in position 2 -Position 2 proline and make a break and stabilise proline makes a break and puts amino acid to the other side - The 180 ° turn is accomplished over four amino acids - The turn is stabilised by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence - Proline in position 2 (type 1) or glycine in position 3 (type )are common in b turns How to measure secondary structure: - CIRCULAR DICHROISM (CD) - CD measures the molar absorption difference of left and right circularly polarised light - Chromophores in the chiral environment produce characteristic signals - CD signs from peptide bonds depend on the chain conformation - Alpha helix → has phi and psi negatively charged = has double peak - Beta - goes positive then negative - - Random coil = flat Determine if a mutation will cause disruption of beta sheet or alpha strand as this will determine the content. Or if the mutation causes no harm → then function is the same. Protein Tertiary Structure: - Tertiary structure refers to the overall spatial arrangement of atoms in a protein - Stabilised by numerous weak interactions between amino acid side chains → Largely hydrophobic and polar interactions → Can be stabilised by disulphide bonds - Interacting amino acids are not necessarily next to each other in the primary sequence. - Two major classes → Fibrous and globular (water or lipid-soluble) - HYDROGEN BONDING - BETA SHEETS AND BETA STRANDS - METAL COORDINATION (BY POSITIVE AND NEGATIVE CHARGED RESIDUES) - HYDROPHOBIC RESIDUES - HYDROGEN BONDING (STOP FROM MOVING) - IONIC ELECTROSTATIC INTERACTION LINKAGE Quaternary Structure: - More than 1 polypeptide chain - If this protein forms a complex - Any polymerisation - adopts a quaternary structure - Haemoglobin - Results from interactions between 2+ polypeptide chains - Interactions = hydrogen bonding and disulphide bonds Advantages of quaternary structure: → Stability: reduction of surface-to-volume ratio → Genetic economy and efficiency → Bringing catalytic sites together → Cooperativity Week 2; Lecture 3: Folding and Unfolding; Globular and fibrous proteins; peptide sequencing - How do newly synthesised proteins fold - Explain how the process of folding is thermodynamically favourable and native conformation can be achieved from unfolding. - Understand the many biological functions of globular and fibrous proteins - Understand and apply the Methods for sequencing proteins - Sanger, Edman’s, MS No structural integrity and loss of activity = denaturation (unfolds and loses its function) ™ = melting temperature Melting curve → see if it affects tempertaure Proteins can be denatured by: → heat or cold → pH extremes → organic solvents → chaotropic agents: urea and guanidinium hydrochloride (suck up water) Melting curve: Protein + dye When temp starts to melt then more the dye INTENSIFIES - the more peptide bonds can be accessed How can proteins fold so fast?: - Levinthal paradox → if 1 conformation is attempted every 10^-13 seconds it will take over 10^30 years to randomly test all possibilities → RNAase folds completely in < 1 min Search for min is not random because Direction toward the native structure is thermodynamically most favourable. - SEQUENCE OF AMINO ACIDS INDICATES WHICH SECONDARY STRUCTURE Ken dill’s folding funnel: hypothesis Thermodynamics of protein folding: Proteins fold into 2^0 and 3^0 structures that possess the lowest possible free energy A protein’s internal residues direct its folding into native conformation - Hydrophobic Effect - Non polar groups aggregate and water molecules are released - Increase in entropy owing to the release of water molecules into bulk water. - Decrease in entropy of protein; increase in entropy of water Proteins folding follow a distinct path:  - EVERYTHING STARTS AS A SECONDARY STRUCTURE - HYDROPHOBIC RESIDUES INSIDE Ribonuclease refolding experiment: - Had to break bonds with a reducing agent  - Unfolded protein with urea - The protein spontaneously refolds to its native structure and disulphide bonds reform - The sequence alone determines the native conformation Protein misfolding: - Can get trapped in an energy state (if can't overcome energy barrier) - PrPsc acts as template for the transformation of normal protein into prion protein )pripn hypothesis) - Associate strongly with each other and forms insoluble protein aggregates in neutral cells - infectious , genetic or sporadic Alzheimer’s disease: - Beta-amyloid plaques found in the brain (protease-resistant structures characterised by a high content of β sheets) - Characterised by amyloid plaques surrounded by dying neurons; Neurofibrillary tangles Amyloid beta protein Ab, excised from APP (™ amyloid beta precursor protein) Has 2 stable conformations: native (embedded in membrane) and amyloid form The problem is Ab42 ; it has 2 extra hydrophobic residues Leaves the membrane, changes shape and aggregates into long fibrils Amyloid fibrils: neurotoxic agent Motifs (folds): Specific arrangement of several secondary structure elements – All alpha-helix – All beta-sheet – Both  Motifs can be found as reoccurring structures in numerous proteins Proteins are made of different motifs folded together Secondary Structures and Motifs: Groupings of secondary structural elements: -b-a-bmotif - b hairpin motif - a - a motif - Greek key motif (b hairpin folded over to form a 4 stranded antiparallel b sheet).  - Domains are specific areas with specific functions: Globular proteins: - Peptide chains satisfy the constraints inherent in their own structure - Ramachandran plot - Peptide chians fold to “bury” most hydrogphbic side chains, mining contact with water residues (Val, Leu, Met, Phe) - It is worse ot bury a charged residue tha expose a hydro - phoic one (Arg, His, Lys, Asp, Glu) - Charged and polar residues are found on the surface in contact with water - but may be buried if H-bonding and charge balance is satisfied (Ser, Thr, Asn, Gln, Tyr, Trp) Fibrous proteins: Collagen: ~ 12 major types; types I-III assemble in fibrils, type IV assembles in laminar network Elastin: crosslinked random coiled protein; gives elasticity to tissues Amino acid compositon: Four different fibrous proteins: High abundance of amino acids with non-bulky side chains (glycine, alanine, serine, glutamate, and glutamine); exception high amount of proline in collagen and elastin. COILED-COIL MOTIF PROTEINS: - Bundle of right-handed alpha helices wound into left-handed superhelix (rope like structure) - Right-handed α-helical rods consists of 7- residue repeats: (a-b-c-d-e-f-g)n, where a & d hydrophobic (nonpolar) residues: isoleucine, leucine or valine - 3.6 residues/turnà hydrophobic residues on one side of the helix - Important in formation of disulphide bonds - Pairs of helices twine about each other in left-handed coil (alpha helical coiled-coil) and bury the hydrophobic residues (stable structure) - Helps alpha helices stick together Alpha Keratin: A helix of helices - Mechanically durable and chemically nonreactive - Hard alpha keratin (occur in birds and reptiles) – shells, fingernails, claws - Soft alpha keratin (occur in mammals) – skin, hair, wool - High levels of cysteine and cross-links - Alpha-helical rod (300-314 residues) with non- helical N- and C-termini caps - Keratin fibers cross-link through disulphide bonds between cysteine residues on adjacent chains → forms a alpha helix → keratin - needs LOTS to form coil coil → N - C terminus → form head to tail interactions → start from protofilament to protofibril Silk Fibroin Structure: ONLY BETA STRANDS  - b-sheet protein - Alternating sequence: Gly-Ala-Gly-Ala.... - Gly on one side – Ala (or Ser) on the other – Gly on one sheet to meshes with Gly on an adjacent sheet (same for Ala/Ser) Spider silk: - Used for webs, egg sacks, and wrapping the prey - Extremely strong material – stronger than steel – can stretch a lot before breaking - A composite material – crystalline parts (fibroin-rich) – rubber-like stretchy parts - Composed of 1. Fibroin which gives structure 2. Seracin acts like glue or matrix Overall composite material like carbon fibre - higher tensile strength ELASTIN: - Major component of connective tissue of lung and arteries allows these to resume their shape after stretching or contracting - Hydrophobic , insoluble, forms 3 D elastic network; Formed from loose and unstructured polypeptide chains - Conformation that of random coil àpermits the protein to stretch and recoil. A variety of random coil conformations possible - Can stretch in any direction, structure more elastic than rubber Protein Sequencing: - It is essential to further biochemical analysis that we know the sequence of the protein we are studying - Actual sequence generally determined from DNA sequence - Edman Degradation (Classical method) – Successive rounds of N-terminal modification, cleavage, and identification – Can be used to identify protein with known sequence - Mass Spectrometry (Modern method) – MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence – Can be used to determine post-translational modifications Determining the amino acid sequence of a protein: 1. Separate chains. Cleavage of Disulfide Bridges → USE REDUCING AGENTS - (Performic acid oxidation; Sulfhydryl reducing agents (mercaptoethanol, dithiothreitol) Separation of chains: → exclusion chromatography → Subunit interactions depend on weak forces → Separation is achieved with (extreme pH; 8M urea; 6M guanidine HCl; high salt concentration -usually ammonium sulfate). Purify the chains 2. Acid hydrolysis + ion exchange chromatography - Determines the AA composition of the protein - Peaks indicate how many  1. Identify N- and C-terminal residues N-terminal analysis: Dinitrofluorobenzene (DNFB): Sanger reagent Colours will migrate Once reacted run TLC plate - each amino acid will form a different collour Ratio of how much the AA runs - tells you what you have Dansyl chloride forms a fluorescent derivative Edman's reagent (phenylisothiocyanate), – Cleaves one amino acid at N-terminus at a time. Helps to identify the number of distinct polypeptides eg Insulin has equal amounts of N-terminal residues (Phe and Gly). Two non identical polypeptide chains. DANSYL CHLORIDE: - Reacts with other amino acids C-terminal analysis: Hydrazine Reaction Treat intact polypeptide with hydrazine in mild acid Hydrolytically cleaves at the C=O of each peptide bond C Alpha at C terminus is untouched (free AA): No hydrazine Isolate and analyse by chromatography Edman’s Degradation: - Sequence amino acid 1 by 1 - Can only sequcne something up to 40 AA - You can separate into smaller fragments - Cleavage needs to be complete and highly specific  EDMUNS 1 BY 1 STRATEGY ecture 4: Protein synthesis and sorting; Collagen Summary / Outcomes: - chemical synthesis of peptides happens from C-terminal to N-terminal side. - amino acids are coded for on mRNA, carried by charged tRNA, and linked together by rRNA/ribosomes - there are five stages of protein biosynthesis - proteins are targeted to specific places through specific sequences - In co-translational translocation, the SRP binds the signal sequence on nascent peptide and subsequently is bound by the SRP receptor – The SRP and receptor mediate the hand-over of the nascent peptide into the translocon – Continued translation extrudes the peptide through the translocon without additional energy – The translocon has a central gated hydrophobic channel that allows passage of the peptide but not small ions proteins may be modified post-translationally