Amino Acids and Proteins structure (2 nd -3th part)

Transcription

1 Amino Acids and Proteins structure (2 nd -3th part) Medical students 90- IB of GUMS By Dr. Aghajany Nasab- Monireh

2 Oligopeptide Polypeptide- Protein When a few amino acids are joined,the structure is called an oligopeptide. When many amino acids are joined, the product is called a polypeptide. Molecules referred to as polypeptides generally have molecular weights below 10,000, and those called proteins have higher molecular weights.

3 Peptides & Proteins Like free amino acids, peptides have characteristic titration curves characteristic isoelectric ph (pi) at which they do not move in an electric field.

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5 Multisubunit proteins Some proteins consist of a single polypeptide chain, but others, called multisubunit proteins, have two or more polypeptides associated noncovalently. The individual polypeptide chains in a multisubunit protein may be identical or different.

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9 Polypeptide chain,φ and Ψ angle Φ (Phi) angel: N-Cα Ψ(Psi) angel: C-Cα

10 Levels of Protein Structure Secondary structure elements combine to form tertiary structure Quaternary structure occurs in multienzyme complexes

11 Protein structures Primary structure :the sequence of the amino acids in a polypeptide chain secondary structure, the folding of short (3- to 30-residue), contiguous segments of polypeptide into geometrically ordered units tertiary structure, the assembly of secondary structural units into larger functional units such as the mature polypeptide and its component domain Quaternary structure, the number and types of polypeptide units of oligomeric proteins and their spatial arrangement

12 Insulin structure

13 Clinical correlation Insulin used in treatment of Diabetes Mellitus Pig Insulin Cow Insulin Human Insulin Differences in sequence Initial allergic response due to produced anti-insulin antibodies

14 α- Helix

15 α- Helix

16 α- Helix

17 α- Helix

18 Properties of -helices residues in length Often amphipathic or dual-natured Half hydrophobic and half hydrophilic If we examine many -helices, we find trends Helix formers: Ala, Glu, Leu, Met Helix breakers: Pro*, Gly, Tyr, Ser

19 Ferritin : Alpha helix

20 Beta Sheets Are Stabilized by Hydrogen Bonding BetweenPolypeptide Strands

21 Beta Sheets in Fatty acid-binding protein

22 Properties of beta sheets Formed of stretches of 5-10 residues in extended conformation Pleated each C a bit above or below the previous Parallel/aniparallel, contiguous/non-contiguous 22

23 Tertiary Structure Water-Soluble Proteins Fold into Compact Structures with Nonpolar Cores

24 Tertiary structure of proteins The entire three-dimensional conformation of a polypeptide

25 Quaternary Structure Polypeptide Chains Can assemble into Multisubunit Structure

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27 Protein denaturation

28 PROTEIN FOLDING Proteins are conformationally dynamic Molecules Can fold into their functionally competent conformation in a time frame of milliseconds, and oftentimes can refold if their conformation becomes disrupted, or denatured HOW????????

29 Chaperone participate in the folding of over half of mammalian proteins Chaperones prevent aggregation, thus providing an opportunity for the formation of appropriate secondary structural element

30 The folding of many proteins is protected by chaperonin proteins

31 Chaperone chaperone proteins can "rescue" unfolded proteins providing a second chance to fold productively

32 Clinical correlation Prion diseases PrPc:Normal protein-highly soluble PrPsc :Abnormal protein- insoluble toxic conformation

33 Clinical correlation Prion diseases Ataxia, dementia, paralysis and almost always fatal Pathological examination: amyloid plaques and spongiform degeneration Normal protein: 3α helix, 2 β strand Amyloid protein: 2 α helix, 3 β strand

34 Overview of Protein structure Amino Acids and Peptids PEPTID BANDS.swf 4 STEP FOLDING.swf Folding protein folding.swf

35 PROTEINS CAN BE STUDIED According to : Solubility Size Charge Binding Affinity

36 Proteins Can Be Separated and Purified The solubility of proteins is generally lowered at high salt concentrations, an effect called salting out. Ammonium sulfate ((NH4)2SO4) is often used for this purpose because of its high solubility in water.

37 Centrifugation

38 Column chromatography of proteins The stationary phase: small spherical beads of modified cellulose, acryl amide, or silica surface coated with chemical functional group interact with proteins based on their charge, hydrophobicity,and ligand-binding properties.

39 Column chromatography

40 Ion exchange chromatography Proteins interact with the stationary phase by charge-charge interactions. Cation exchangers :Proteins with a net positive charge at a given ph adhere to beads with negatively charged functional groups Anion exchangers :proteins with a net negative charge adhere to beads with positively charged functional groups

41 Ion exchange chromatography

42 Affinity chromatography high selectivity of proteins for their ligands Enzymes may be purified by affinity chromatography using immobilized substrates, products, coenzymes, or inhibitors

43 Affinity chromatography

44 Size-exclusion chromatography, gel filtration or

45 HPLC High-pressure liquid chromatography (HPLC) employs incompressible silica or alumina micro beads as the stationary phase and pressures of up to a few thousand pressure (psi) Incompressible matrices permit both high flow rates and enhanced resolution.

46 HPLC output Gel filtration by HPLC defines the individual proteins because of its greater resolving power: (1) thyroglobulin (669 kd), (2)catalase (232 kd), (3) bovine serum albumin (67 kd) (4) ovalbumin (43 kd), (5) ribonuclease (13.4 kd)

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48 Gel electrophoresis Electrophoresis allows determination of properties of a protein such as its isoelectric point and approximate molecular weight gels made up of the cross-linked polymer polyacrylamide the migration of proteins approximately in proportion to their charge-to-mass ratio.

49 SDS- PAGE polyacrylamide gel electrophoresis (PAGE) in the presence of the anionic detergent sodium dodecyl sulfate (SDS) SDS denatures and binds to proteins at a ratio of one molecule of SDS per two peptide bonds.

50 SDS SDS binds to most proteins in amounts roughly proportional to the molecular weight of the protein, about one molecule of SDS for every two amino acid residues.

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54 Clinical application Blood protein electrophoresis Alpha 1-Antitrypsin Alpha2-Macroglobulin, Haptoglobin, Ceruloplasmin Beta-lipoprotein, Transferrin Fibrinogrn Ig A, IgG

55 Isoelectric focusing

56 Two-dimensional electrophoresis

57 Two-dimensional electrophoresis

58 Cleavage of polypeptides

59 Cleavage of polypeptides

60 Edman reaction for protein sequencing

61 Proteins acts in all biological processes Material transport (hemoglobin, transferrin) Motion (myosin, actin, etc) proteins Defense (antibodies, toxins) Mechanical support (collagen) Metabolism (enzymes) Replication and repairing of genetic information (DNA and RNA polymerases)

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63 FIBROUS PROTEIN Globular proteins

64 OVERVIEW Collagen, elastin, Fibroein and Creatin are examples of common, well-characterized fibrous proteins that serve structural functions in the body. collagen and elastin are found as components of skin, connective tissue, blood vessel walls, and sclera and cornea of the eye.

65 COLLAGEN Collagen is the most abundant protein in the human body. A typical collagen molecule is a long, rigid structure in which three polypeptides " chains" are wound around one another in a rope-like triple-helix

66 Collagen

67 Types of collagens

68 Structure of collagen Amino acid sequence: Collagen is rich in proline and glycine, both of which are important in the formation of the triplestranded helix.

69 Structure of collagen The glycine residues are part of a repeating sequence. Gly X Y, where X is frequently proline and Y is often hydroxyproline or hydroxylysine Most of the.- chain can be regarded as a polytripeptide whose sequence can be represented as ( Gly X Y ) 333

70 Structure of collagen

71 Prolin hydroxylation These hydroxylation reactions require molecular oxygen and the reducing agent vitamin C the hydroxylating enzymes: prolyl hydroxylase and lysyl hydroxylase, are unable to function without vit. C

72 Collagen defect due to Vit C. deficiency In the case of vit.c deficiency (therefore, a lack of prolyl and lysyl hydroxylation), collagen fibers cannot be cross-linked, greatly the tensile strength of the assembled fiber. Vit.C deficiency disease known as scurvy. Patients with vit.c deficiency often show capillary fragility

73 Glycosylation: The hydroxyl group of the hydroxylysine residues of collagen may be enzymatically glycosylated. Most commonly, glucose and galactose are sequentially attached to the polypeptide chain prior to triple-helix formation

74 Collagen synthesis

75 Collagen synthesis

76 Elastin is Structure of elastin an insoluble protein polymer synthesized from a precursor, tropoelastin, ( a linear polypeptide composed of about 700 amino acids that are primarily small and nonpolar) (e.g. glycine, alanine, and valine). rich in proline and lysine, contains a little hydroxyproline contains no hydroxylysine.

77 Desmosine in Elastin

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79 HEMOGLOBIN Myoglobin

80 The Backbone structure of Myoglobin

81 Structure of Haem

82 Synthesis of globin

83 Alpha & beta chains

84 Adult hemoglobin Hb A Hb A 2 Hb F structure 2 b 2 2 d 2 2 g 2 Normal % % % % Methemoglobinmia: Tyr

85 Hemoglobin

86 Oxy hemoglobin (Red)- Desoxy hemoglobin (blue)

87 Oxygenation of hemoglobin

88 Quaternary structure of deoxy- and oxyhemoglobin T-state R-state

89 Oxygen-haemoglobin dissociation curve O2 carrying capacity of Hb at different Po2 Sigmoid shape Binding of one molecule facilitate the second molecule binding P 50 (partial pressure of O2 at which Hb is half saturated with O2) 26.6mmHg

90 Oxygen-haemoglobin dissociation curve

91 Oxygen-haemoglobin dissociation curve

92 Oxy & deoxyhaemoglobin

93 Hb-oxygen dissociation curve The normal position of curve depends on Concentration of 2,3-DPG H + ion concentration (ph) CO 2 in red blood cells Structure of Hb

94 Hb-oxygen dissociation curve

95 Hb-oxygen dissociation curve Right shift (easy oxygen delivery) High 2,3-DPG High H + High CO 2 HbS Left shift (give up oxygen less readily) Low 2,3-DPG HbF

96 Hemoglobinopathy definition An inherited mutation of the globin genes leading to a qualitative or quantitative abnormality of globin synthesis

97 Structural hemoglobinopathy Amino acid substitution in the globin chain e.g. sickle hemoglobin (HbS)

98 Sickle Cell Anemia blood film Sickle Cells Erythroblasts

99 The Thalassemias Syndromes in which the rate of synthesis of a globin chain is reduced beta thalassemia - reduced beta chain synthesis alpha thalassemia reduced alpha chain synthesis

100 Laboratory diagnosis Beta thalassaemia relies on raised F and A2 Alpha thalassaemia F and A2 normal gene analysis

101 Glycosylated hemoglobin HbA1c: 5% HbA In Normal Individuals More than 5%: use for blood Glu. Control in diabetic patients since 120 past days