Topic 3.pptx

Objectives (week 3 & 4) Chapter 11.6-11.7 [Denniston 7th Edition] Proteins •Molecular structure of amino acids •Classification of amino acids Dipeptides & Polypeptides •Peptide bond •Structure of a dipeptide, tripeptide, etc •Skeletal structure of polypeptides (1st,2nd,3rd ,4th) •Haemoglobin struc...

Objectives (week 3 & 4) Chapter 11.6-11.7 [Denniston 7th Edition] Proteins •Molecular structure of amino acids •Classification of amino acids Dipeptides & Polypeptides •Peptide bond •Structure of a dipeptide, tripeptide, etc •Skeletal structure of polypeptides (1st,2nd,3rd ,4th) •Haemoglobin structure •Denaturation Proteins monomers are amino acids condensation reaction water lost α N-terminal C-terminal peptide bond Biosynthesis proceeds from N- to C- terminal AA Peptide & Proteins Read from left to right (amino to carboxyl end) *hydrolysis of peptide is exergonic *peptide bonds are quite stable *half-life is 7 years Proteins • condensation leads to an unbranched polypeptide • peptide – fewer than 50 AA residues; dipeptide, tripeptide, etc. • oligopeptide – a few AA joined together • polypeptide – many AA joined together (MW<1000) • protein – more than 50 AA residues • most proteins contain many hundreds of AA ribonuclease (103 AA) degrades RNA (9 AA) Love hormone induces labour apamin (18 AA) bee venom Amino acids diversity of proteins from only 20 AA keratin luciferin + ATP by luciferase enzyme lens protein Amino acids 1806 - 1938 names of AA: glutamate asparagine “glykos” – sweet glycine “tyros” -cheese tyrosine Amino acids *chiral carbon allows D and L *enantiomers *optically active *AA in proteins are exclusively L *D has been found in some peptides in bacterial cell wall and peptide antibiotics Amino acids AA are characterized by R group grouped based on polarity (interaction with water at pH 7.0): *nonpolar/hydrophobic (water-insoluble) to polar/hydrophilic (watersoluble) hard to group: glycine, histidine, cysteine Amino acids Nonpolar, Aliphatic R groups: *their R groups tend to cluster in proteins stabilizing protein structure through hydrophobic effects *glycine – simplest structure; no real hydrophobic effect *methionine – S containing; slightly nonpolar *proline – cyclic; reduces flexibility Amino acids Aromatic R groups: *relatively nonpolar/hydrophobic effect *tyrosine – in enzymes; H bonding *tyrosine & tryptophan – more polar than phenylalanine due to OH and N in indole ring *They can absorb UV light at 280 nm (characterisation of proteins) Amino acids Polar, Uncharged R groups: *hydrophilic due to H bond *serine & threonine – hydroxyl (OH) *asparagine & glutamine – amide (NH); easily hydrolysed by acids or base to aspartate & glutamate *cysteine – sulfhydryl (SH) is a weak acid and makes weak H bonds; readily oxidized to cystine Amino acids *cysteine oxidized to cystine = disulphide bridge (nonpolar) Important in linking polypeptides in protein structure Amino acids Positively charged (basic) R groups: *most hydrophilic along with negatively charged groups. *lysine – second primary amino group *arginine – guanidium group *histidine – aromatic imidazole group; at pH 7.0 is the only AA that may be positively charged or uncharged Amino acids Negatively charged (acidic) R groups: *most hydrophilic along with negatively charged groups. *both have a second carboxyl group 300 additional AA have been found in cells *various functions, not all are constituents of proteins. Amino acids Uncommon AA also have important functions 6-N-methyllysine *found in myosin in muscle γ-carboxyglutamate *found in blood clotting protein prothrombin 4-hydroxyproline and 5hydroxylysine *both found in collagen – a connective tissue; the former in plant cell wall proteins Amino acids Uncommon AA also have important functions desmosine *found in elastin pyrrolysine *found in methaneproducing archaebacteria 300 additional AA have been found in cells *various functions, not all are constituents of proteins. Amino acids AA can act as (weak) acids and bases *due to the amino, carboxyl, and ionisable R groups *Zwitterion (dipolar ion) is formed when dissolved in water (pH 7.0), and acts as an acid or a base amphoteric ampholytes acid yields proton base gains proton Amino acids AA residues (approx.) = Molecular weight (MW) 110 e.g. haemoglobin 64,500 / 110 = 586 AA The avg. AA is 138 but smaller AA dominate a chain (128). Water molecules lost are 18. 128 – 18 = 110 average of 1000 proteins The structure of proteins primary structure quaternary structure secondary structure tertiary structure α helix amino acid residue w/ peptide bonds polypeptide chain assembled subunits *E. coli – 3000 unique proteins *humans – >20,000 proteins primary structure *important: determines the folding of the 3-D structure and thus the function of the enzyme. Defects can lead to diseases. The structure of proteins: 1ry structure The function of a protein depends on its AA sequence *one difference or a chunk missing from a polypeptide can lead to diseases. sickle – cell anemia polymorphic proteins – the AA for a protein is not fixed; some differences may exist but it does not disrupts its function. The structure of proteins: 1ry structure The AA sequence of millions of proteins have been determined *Sanger determined the AA sequence in bovine insulin Fredrick Sanger 1953 *soon it was realized that AA sequence and DNA sequence are related The structure of proteins: 1ry structure 1) Edman’s degradation - two-step chemical sequencing process; labels and removes only the amino-terminal residue from a peptide; leaving all other peptides in tact. PTC formed AA identified peptide bond cleaved The structure of proteins: 1ry structure 2) For larger proteins: *disulphide bonds must be eliminated by performic acid or dithiothreitol (DTT) *proteins must be cleaved into smaller polypeptides in a predictable and reproducible way by enzyme proteases The structure of proteins: 1ry structure 3) Mass spectrometry offers an alternative method to determine AA sequences: *provide accurate MW of protein *provide short sequence (20-30) in a short time. • the analyte (molecule in gas phase to be analysed) is ionized in a vacuum • then passed through a magnetic field • their paths are a function of their mass-to-charge ratio (m/z) • mass (m) is deduced high voltage vacuum interface The structure of proteins: 1ry structure 3) Mass spectrometry offers an alternative method to determine AA sequences: *cannot work for large macromolecules like protein. In gas phase it decomposes *2 techniques developed to solve this problem A. • • • MALDI MS (matrix-assisted laser desorption/ionization mass spec) proteins placed in a light absorbing matrix short pulse of laser to ionize proteins Ionized proteins are desorbed from matrix into vacuum system B. ESI MS (electrospray ionization mass spec) • proteins solution force from liquid to gas • passed through a charged needle that is kept at high electrical potential • solution forms a mist of charged microdroplets • solvent around the macromolecules rapidly evaporates • protons are added during the passage of the needle The structure of proteins: 1ry structure ESI Each successive peak corresponds to a species that differs from that of its neighbouring peak by a charge difference of 1 and a mass difference of 1 (one proton). What is tandem MS or MS/MS? The structure of proteins: 1ry structure Protein sequences help to elucidate the history of life on earth Study families of closely related proteins homologous proteins (homologs) – members of a protein family Paralogs – two proteins in the same family (homologs) are in the same species Orthologs – homologs in different species The process of tracing evolution involves first identifying suitable families of homologous proteins and then using them to reconstruct evolutionary paths. Questions Review Amino acids Aromatic R groups: *relatively nonpolar/hydrophobic effect *tyrosine – in enzymes; H bonding *tyrosine & tryptophan – more polar than phenylalanine due to OH and N in indole ring * They can absorb UV light at 280 nm (characterisation of proteins) Determine protein concentration UV spectrophotometry Maximum light absorbance at A280 Beer-lambert equation to calculate sample concentration c = (A*ɛ)/b • • • • c is concentration (ng/uL) A is absorbance ɛ is wavelength coefficient (ng-cm/uL) b is pathlength (cm) transmits light of a particular wavelength UV spectrophotometer Ultraviolet-visible spectrophotometer schematics: Io I I – light reflected by sample Io – light reflected by reference (blank) Transmittance (%T) = I/ Io Absorbance = -log(%T) Absorbance = log(Io/I) Absorbance and concentration are directly proportional VIDEO *UV-vis simplified https://www.youtube.com/watch?v=wxrAELeXlek Determining protein concentration *tyrosine and trypthophan in protein absorb UV at 280 nm i.e. Protein [ ] at absorbance of 1.0 and wavelength 280nm = 1 mg/mL Bradford assay: uses a standard calibration curve of bovine serum albumin (BSA) *Coomassie brilliant blue binds to protein and absorbs UV at 595 nm Ultraviolet-visible spectrophotometer *plot absorbance against concentration *determine unknown concentration at known absorbance x is concentration and y is absorbance. R2 is the extinction coefficient. What does it tell you? How to prepare stock solutions M1V1 = M2V2 1- initial molarity and volume 2- final molarity and volume e.g. prepare 1 mL of the following protein standards of BSA if you have a stock solution of 1 mg/mL. 0 (dH2O), 200, 400, 600, 800, 1000 µg/mL (stock BSA). Conversion factors: 1000 µg = 1 mg; 1000 µL = 1 mL M1V1 = M2V2 1 mg/mL x ? = 0.2 mg/mL x 1 mL x = 0.2 mg/mL x 1 mL 1 mg/mL x = 0.2 mL or 200 µL Mix 200 µL of stock BSA solution in 800 µL of water to get 1mL of 200 µg/mL. Complete ACHIEVE HW 2: Intro to Biochemistry END protein secondary structure Types: *α-helix *β conformations *β-turn, *random coil protein secondary structure The alpha helix is a common protein secondary structure Types: α-helix, β conformations; β turn, and random coil. * α-helix was observed in hair and porcupine quills using X-ray *exist because of H bonding *tightly wound around an imaginary axis *R groups protrude outward *the turn is 5.4 Å long and includes 3.6 AA protein secondary structure Question 1: Question 2: Hexokinase, a protein in yeast, comprises of two polypeptide chains and a total number of 972 amino acid residues. What is the length of a polypeptide of hexokinase? protein secondary structure AA sequence affects stability of the alpha helix The twist of alpha helix ensures critical interactions *the AA R group interacts with another AA R group three residues away *Positively charge AA found 3 residues away from a negatively charged AA Amino terminal has negatively charged AA Carboxyl terminal has positively charged AA This maintains the stability protein secondary structure AA sequence affects stability of the alpha helix proline and glycine are rarely found in the alpha helix NH2+ has no substituent H to participate in H bonding take up coiled structures quite different from an α helix protein secondary structure The β Conformation Organizes Polypeptide Chains into Sheets β conformations *second type of repetitive structure *Backbone of the chain is zig zag *R groups protrude alternately from the sides of the zig zag pattern protein secondary structure Antiparallel Parallel β sheet – several segments side by side *H bonding forms between each segment *may be near each other or far apart on the same chain or on different polypeptide chains The amino-terminal to carboxyl terminal orientations of adjacent chains (arrows) can be the opposite or the same, forming (b) an antiparallel β sheet or (c) a parallel β sheet. protein secondary structure β turns are common in proteins β turns In globular proteins, which have a compact folded structure, some amino acid residues are in turns or loops where the polypeptide chain reverses direction. *connect the ends of two adjacent segments of an antiparallel β sheet. *180° turn involving four amino acid residues *the carbonyl oxygen of the first residue forming a hydrogen bond with the amino-group hydrogen of the fourth. protein secondary structure β turns cis isomer small & flexible Beta turns are often found near the surface of a protein, where the peptide groups of the central two amino acid residues in the turn can hydrogen-bond with water. protein secondary structure γ turns Considerably less common is the γ turn, a three-residue turn with a hydrogen bond between the first and third residues. tertiary & quaternary structure tertiary structure – 3-D arrangement of all atoms in a protein *active site – where reactions occur *domain – stable parts of a protein that evolved function quaternary structure – two or more polypeptides joined together to form a protein *subunits – one polypeptide (same or different) *domain swapping – results in quaternary structure protein tertiary & quaternary structure quaternary structure - two or more separate polypeptide chains two groups of proteins: fibrous proteins – polypeptide chains arranged in long strands or sheets *consist of a single type of secondary structure *provide support, shape, and external protection to vertebrates globular proteins – polypeptide chains folded into a spherical or globular shape. *contain several types of secondary structure. *enzymes and regulatory proteins protein tertiary & quaternary structure Fibrous proteins are adapted for a structural function *properties that give strength and/or flexibility to the structures *insoluble in water *high concentration of hydrophobic amino acid residues both in the interior of the protein and on its surface protein tertiary & quaternary structure Example 1 of fibrous proteins: α-keratin evolved for strength Intermediate filament (IF) proteins family constitutes the entire dry weight of: protein tertiary & quaternary structure α-keratin *right handed α helix *a coiled coil of two parallel strands (amino terminus are at the same end) which are lefthanded supertwists *each turn is 5.15 to 5.2 Å *held together by hydrophobic AA: Ala, Val, Leu, Ile, Met, and Phe supramolecular complex protein tertiary & quaternary structure α-keratin strength is due to disulphide bonds in between coiled coils to form supramolecular complex. Four protofibrils make up one intermediate filament. How many keratin strands are in one intermediate filament? Rhinocerous horn Toughest & hardest α-keratin due to 18% of residues are cysteines involved in S-S bonds. protein tertiary & quaternary structure α-keratin What is happening at the molecular level in permanent waving (curling hair)? Straightening hair with heat or perm? protein tertiary & quaternary structure Example 2 of fibrous proteins: collagen *also evolved for strength *found in connective tissues: protein tertiary & quaternary structure collagen *left-handed *3 AA residues per turn *3 separate α polypeptide chains *right-handed twisting repeating tripeptide unit, Gly–X–Y *vertebrate collagen: 5% Gly, 11% Ala, and 21% Pro and 4-Hyp (4-hydroxyproline) Gelatin Little nutritional value tight turn due to Gly protein tertiary & quaternary structure Scurvy, collagen and ascorbic acid (vitamin C) *lack of vitamin C leads to instability in collagen which leads to deterioration of connective tissues and diseases like scurvy protein tertiary & quaternary structure collagen fibrils *supramolecular structures of collagen triple helix *cross-linked by Lys or His residues *30 collagen variants in mammals abnormal bone formation in babies Ehlers-Danlos syndrome protein tertiary & quaternary structure collagen *M 300,000 *rod-shaped *3000 Å long; 15 Å thick *3 α chains w/ 1000 residues each *collagen fibrils are made up of collagen in a staggered fashion and cross-linked for strength protein tertiary & quaternary structure Example 3 of fibrous proteins: silk fibroin Silk protein produced by insects and spiders Predominantly the β conformation (antiparallel) rich in Ala and Gly residues H bonding and Van der Waals between each sheet protein tertiary & quaternary structure Silk fibroin spinnerets interdigitate protein tertiary & quaternary structure Globular proteins *segments of the polypeptide chain (or multiple polypeptide chains) fold back on each other *enzymes, transport proteins, motor proteins, regulatory proteins, immunoglobulins Protein data bank & JSmol software protein tertiary & quaternary structure Example of a globular protein: hemoglobin *4 polypeptide chains – 2α and 2β RBCs haemoglobin heme group protein tertiary & quaternary structure Myoglobin *first globular protein studied by x-ray diffraction *reddish brown color to muscles *protein w/ heme group (green) *oxygen (red) storage in muscles *backbone of 8 α helix w/ β turns. *right-handed helices *longest α helix has 23 AA residues *shortest has 7 one polypeptide of 153 AA protein tertiary & quaternary structure Globular Proteins Have a Variety of Tertiary Structures folding or structural patterns: motif or fold - a recognizable folding pattern involving two or more elements of secondary structure and the connection(s) between them β-α-β loop simple motif β barrel elaborate motif globin fold In all globins domain- a part of a polypeptide chain that is independently stable or could undergo movements as a single entity with respect to the entire protein Factors that cause protein denaturation Denaturation - a loss of 3-D structure sufficient to cause loss of function. *precipitation – protein aggregates as hydrophobic parts become exposed *heat affects H bond; thermophilic bacteria have heat-stable proteins *pH by organic solvents, urea, and detergents *disrupts the hydrophobic aggregation of nonpolar AA side chains; H bonds Complete ACHIEVE test 1: Intro to Biochem to Proteins END