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UNIT 2. Structure and function of proteins.

UNIT 2. Structure and function of proteins. OUTLINE. 2.1. Amino acids. Structure. Ionic properties/Acid-base properties Uncommon amino acids. 2.2. Peptides. Primary structure determination. Peptide bond. Nomenclatures of the peptides. Characteristics of the peptides.

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UNIT 2. Structure and function of proteins.

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  1. UNIT 2. Structure and function of proteins.

  2. OUTLINE • 2.1. Amino acids. • Structure. • Ionic properties/Acid-base properties • Uncommon amino acids. • 2.2. Peptides. Primary structure determination. • Peptide bond. • Nomenclatures of the peptides. • Characteristics of the peptides. • Analysis of the primary structure of a protein • Protein sequencing. • Peptides of biological interest.

  3. OUTLINE • 2.3. Three-Dimensional structure and function of proteins. • Proteins classification. • Secondary structure: Ramachandran Diagram. -Helix. -pleated sheet. -loops. • Motives or super secondary structures. • Tertiary structure. • Denaturation and renaturation. • Quaternary structure. • Fibrous proteins: -keratins. Fibroin. Collagen.

  4. 2.1. Amino acids. • STRUCTURE: • 20 -amino acids = 20 common amino acids • Uncommon amino acids Carboxyl group Proline (a-imino acid) Amino group Side Chain

  5. 2.1. Amino acids. • STRUCTURE: • WHAT DO YOU HAVE TO KNOW? • Name of the 20 common amino acids • Chemical composition of the 20 common amino acids • Three-letter code used to represent the amino acids • Amino acids classification • - Main properties of the amino acids grouped into each category.

  6. 2.1. Amino acids. • STRUCTURE: • You should know names, structures, pKa values, 3-letter and 1-letter codes • Non-polar amino acids • Polar, uncharged amino acids • Acidic amino acids • Basic amino acids

  7. 2.1. Amino acids. • STRUCTURE: • pH of the cells  7,4: Zwitterion = ionic forms of the amino acid (neutral=net charge 0). Soluble in water Zwitterion (neutral)

  8. 2.1. Amino acids. • STRUCTURE: • Disulfide bridges between cysteine residues (S-S) Thiol Intrachain Interchain

  9. 2.1. Amino acids. • STRUCTURE: • Asymmetric/chiral carbon. Amino acids show optical and stereochemical properties. All but glycine are chiral • Stereoisomers: same chemical composition, different spatial organization. • Enantiomers: type of steroisomers. Nonsuperimposable mirror-image (L and D). Dextrorotatory behaviour Levorotatory behaviour

  10. 2.1. Amino acids. • STRUCTURE: • D,L-nomenclature is based on D- and L-glyceraldehyde • L-amino acids predominate in nature

  11. 2.1. Amino acids. • IONIC PROPERTIES/ACID-BASE PROPERTIES: • Amino Acids are Weak Polyprotic Acids. All the amino acids contain at least two dissociable hydrogens.

  12. 2.1. Amino acids. • IONIC PROPERTIES/ACID-BASE PROPERTIES: • Isoelectric point (pI) = pH where the amino acids have a net charge of 0. • Simple amino acid (no dissociable hydrogens in the side chain): Titration of Glycine pI = ½ (pK1 + pK2)

  13. 2.1. Amino acids. • IONIC PROPERTIES/ACID-BASE PROPERTIES: • Amino acid with dissociable hydrogens in the side chain Acidic amino acids (net negative charge at neutral pH): pI = ½ (pK1 + pKR)

  14. 2.1. Amino acids. • IONIC PROPERTIES/ACID-BASE PROPERTIES: • Amino acid with dissociable hydrogens in the side chain Basic amino acids (net positive charge at neutral pH): pI = ½ (pKR + pK2) Titration of Histidine

  15. 2.1. Amino acids. • IONIC PROPERTIES/ACID-BASE PROPERTIES: You should know these numbers and know what they mean! Alpha carboxyl group  pKa = 2 Alpha amino group  pKa = 9 These numbers are approximate, but entirely suitable for our purposes.

  16. 2.1. Amino acids. • UNCOMMON AMINO ACIDS: • They are produce by modifications of one of the 20 amino acids already incorporated into a protein :

  17. 2.1. Amino acids. • UNCOMMON AMINO ACIDS: • Amino acids with specific biological functions. They occur only rarely in proteins: Dopamine: Neurotransmitter Histamine: Allergy reactions Tiroxine: Hormone GABA (-aminobutyric acid): Neurotransmitter Citrulline: Urea cycle intermediate L-ornithine: Urea cycle intermediate

  18. 2.2. Peptides. Primary structure determination. • PEPTIDE BOND: • Peptide bond: covalent amide bond establish between the -COOH and the -NH3+ groups of two amino acids. • One water molecule is eliminated during this reaction. • It allows the polymerisation of the amino acids to form peptides and proteins.

  19. 2.2. Peptides. Primary structure determination. • PEPTIDE BOND: • Properties of the peptide bond: • - It is usually found in the trans conformation • - It has partial (40%) double bond character • - It is about 0.133 nm long - shorter than a typical single bond but longer than a double bond • - N partially positive; O partially negative Peptide bond is best described as a resonance hybrid f these two structures

  20. 2.2. Peptides. Primary structure determination. O O H C • PEPTIDE BOND: C N C N H C C C Trans Cis Due to the double bond character, the six atoms of the peptide bond group are always planar! Geometry of the peptide backbones.

  21. 2.2. Peptides. Primary structure determination. • PEPTIDES CLASSIFICATION ACCORDING TO THE NUMBER OF AMINO ACIDS : • Dipeptide (2) • Tripeptide (3) • Oligopeptide (more than 12 and less than 20) • Polipeptide (many) Serylglicylthyrosylalanylleucine Ser-Gly-Tyr-Ala-Leu SGYAL

  22. 2.2. Peptides. Primary structure determination. • PEPTIDES PROPERTIES: • Peptides show polarity (direction).

  23. (Cys-Ala)   Cationic form (pH ) Zwitterion Anionic fom (pH ) H H H + + H3N-CH-C-N-CH-COO- H3N-CH-C-N-CH-COOH H2N-CH-C-N-CH-COO- ‖ ‖ ‖ CH3 HS-H2C HS-H2C HS-H2C O O CH3 O CH3 2.2. Peptides. Primary structure determination. • PEPTIDES PROPERTIES: • Peptides ionic forms: • Minimal peptide solubilisation at pH= pI • No migration (no movement) in an electrical field.

  24. 2.2. Peptides. Primary structure determination. • PEPTIDES PROPERTIES: • Titration curve: Amphoteric behaviour Tetrapeptide (Glu-Gly-Ala-Lys) WHAT DO YO HAVE TO KNOW: - How to calculate the isoelectric point related to a peptide

  25. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE: • Amino acids sequence comparison (haemoglobin from human beings and sperm whale) : •  84% identical amino acids (They determine the biological role of the protein). •  94% homologous.

  26. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE: • Acid hydrolysis liberates the amino acids of a protein • Thin layer chromatography. • Ion exchange chromatography. • Reverse-phase high-performance liquid chromatography (HPLC). 10-100 horas a 105-110 ºC

  27. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE: • Chromatographic methods used to separate amino acids: • Ion exchange chromatography: the charged molecules of interest (amino acids) are exchanged for another ion (salt ion) on a charged solid support (resins). Resins containing negatively charged groups interact with positive charge molecules, which elute from the resins by changing the pH buffer or the salt ion. • Thin layer chromatography: amino acids absorbed on a thin layer of silica gel are separated thanks to the solvent migration (buthanol: water: acetic acid 4:1:1) by capillarity. • Reverse-phase high-performance liquid chromatography (HPLC): amino acids are separated on the base of their polarity by the used of a column having a nonpolar liquid immobilised on an inert matrix (stationary phase). A more polar liquid serves as the mobile phase. Amino acids are eluted in proportion to their solubility in this more polar liquid.

  28. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE: • Ion exchange chromatography:

  29. Ninhidrine Amino acid Hidrantine Purple max = 570 nm 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE: • Methods for amino acids identification: 1. UV absorbance 2. Ninhidrine reaction Proline: yellow complex able to absorb at 440 nm

  30. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE: • Methods for amino acids identification: 3. Fluorescence (Edman degradation): Phenylisothiocyanate (=Edman reagent) combines with the free amino terminus of a protein. Not only identifies the N-terminal residue of a protein. Successive reaction cycles can reveal the amino acid sequence of a peptide

  31. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE • Amino acid sequence: • 1. If the protein contains more than one polypeptide, the chains are separated and purified. • 2. Cleavage of disulfide bridges (intrachain). • 3. Determination of the N-terminal and C-terminal. • 4. The polypeptide chain is cleaved into smaller fragments (proteolytic enzymes). • 5. Analysis of the amino acid composition and sequence of each fragment (Edman degradation). • 6. The overall amino acid sequence of the protein is reconstructed from the sequences in overlapping fragments.

  32. O ‖ HC – O - OH 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE • Cleavage of disulfide bridges. or 2-mercaptoethanol ( ICH2COO- ) Met interferences

  33. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE • Identification of the N-terminal residue: • 1. Sanger reagent: FDNB (Sanger reagent) peptide (FDNB) Acid hydrolysis 2-dinitrophenyl-peptide 2-dinitrophenyl-N-terminal residue

  34. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE • Identification of the N-terminal residue: • 2. Edman reagent:

  35. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE • Identification of the N-terminal residue: • 2. Edman reagent: Phenylisothiocyanate Peptide Peptide-PTC (phenylthiocarbamil) Smaller peptide (one amino acid residue is released) PTH-alanine (PTH derivative)

  36. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE • Identification of the C-terminal residue: • 1. Carboxipeptidases: • - Carboxipeptidase A: Hydrolyses the C-terminal peptide bond of all amino acids except Pro, Arg and Lys. • - Carboxipeptidase B: Hydrolyses the C-terminal peptide bond of the basic amino acids residues (Arg or Lys).

  37. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE • Fragmentation of the polypeptide chain:

  38. 2.2. Peptides. Primary structure determination. • ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE

  39. 2.2. Peptides. Primary structure determination. Methods to fragmentise the polypeptide chains in order to analyse de amino aid sequence of a protein MethodCleavage targetSpecificity A. Terminal fragmentation: 1. Sanger reagent C-side of the N-terminal Rn = all aa 2. Edman Degradation idem idem 3. Carboxipeptidase A N-side of the C-terminal Rn Arg, Lys, Pro Rn-1 Pro 4. Carboxipeptidase B N-side of the C-terminal Rn = Arg, Lys Rn-1 Pro B. Intrachain cleavage: 1. Cyanogen bromide C-side of the Rn Rn = Met 2. Trypsin C-side of the Rn Rn = Lys, Arg Rn+1 Pro 3. Chymotrypsin C-side of the Rn Rn = Phe, Tyr, Trp, Leu Rn+1 Pro 4. Thermolysin N-side of the Rn Rn = Phe, Tyr, Trp, Leu, Ile, Val Rn-1 Pro 5. Pepsin N-side of the Rn Rn = Phe, Tyr, Trp, Leu, Asp, Glu Rn-1 Pro

  40. 2.2. Peptides. Primary structure determination. • OTHER METHODS OF PROTEIN SEQUENCE ANALYSIS: • Amino acid sequence determined by the analysis of the gene sequence (nucleotides). It is possible to obtain the sequence of the protein directly produced during the translation process, but not the post-translational modifications

  41. 2.2. Peptides. Primary structure determination. • PEPTIDES OF BIOLOGICAL INTEREST:

  42. 2.3. Three-Dimensional structure and function of proteins. • PROTEIN STRUCTURE: LEVELS OF ORGANIZATION:

  43. 2.3. Three-Dimensional structure and function of proteins. • PROTEINS CLASSIFICATION: • Biological role: • Catalysis: enzymes. • Structural role (protection and support): collagen, fibroin, elastin. • Movement: actin, tubulin. • Defence: keratin (against mechanical or chemical damage), fibrinogen and thrombin (avoid blood loosing), immunoglobulins (immunosytem proteins). • Regulation: hormones, growth factors. • Transport: membrane transporters, haemoglobin, lipoproteins. • Storage: ovalbumin, casein (from milk), ferritin. • Adaptations to environmental changes: cytochrome P450, heat chock proteins.

  44. 2.3. Three-Dimensional structure and function of proteins. • PROTEINS CLASSIFICATION: • On the basis of the shape and solubility: • Fibrous proteins • Globular proteins • Membrane proteins • On the basis of the chemical composition: • Simples • Conjugates: (it contains non peptidic component: prosthetic group) • Apoprotein: protein without prosthetic group. • Holoprotein: protein + prosthetic group. - Glucoproteins - Lipoproteins - Methaloproteins - Phosphoproteins - Haemoproteins

  45. 2.3. Three-Dimensional structure and function of proteins. • PROTEINS CLASSIFICATION: Conformation: Overall three-dimensional architecture of a protein (the radicals can modified their spatial position by rotation. Bonds are not cleavage during this process. Configuration: Geometric possibilities fro a particular ser of atoms. In going from one configuration to another, covalent bonds must be broken ant rearranged.

  46. 2.3. Three-Dimensional structure and function of proteins. • SECONDARY STRUCTURE. RAMACHANDRAN DIAGRAM:

  47. 2.3. Three-Dimensional structure and function of proteins. • SECONDARY STRUCTURE. RAMACHANDRAN DIAGRAM: The reasonable conformations are those avoiding steric crowding Ramachandran diagram corresponding to L-Ala residues.  and  angles = 0º, no favourable conformation in proteins.

  48. 2.3. Three-Dimensional structure and function of proteins. • SECONDARY STRUCTURE. -HELIX: n = 3.6 residues (single turn) nº atoms/single turn = 13 d = 0.15 nm = 1.5 Å Travel along the helix axis per turn (pitch of the helix) (v) = 0,54 nm = 5,4 Å (v = n·d)

  49. Hydrogen bonds COO- H  C + O CH2 H2N ‖ C – N - C H CH2 H2C C O H ‖ C - C CH2 N H2C CH2 2.3. Three-Dimensional structure and function of proteins. • SECONDARY STRUCTURE. -HELIX: Right- hand twists Left-hand twists proline

  50. 2.3. Three-Dimensional structure and function of proteins. • SECONDARY STRUCTURE. b-PLEATED SHEET: Strands run in opposite directions 7 Å

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