4. THE THREE-DIMENSIONAL STRUCTURE OF PROTEINS

Transcription

1 4. THE THREE-DIMENSIONAL STRUCTURE OF PROTEINS

2 4.1 Proteins Structures and Function Levels of Structure in Proteins Native conformation - Biological activity - Random structure: no obvious regular repeating structure

3 4.1 Proteins Structures and Function Levels of Structure in Proteins Four levels of structure 1 Primary structure - The order in which the amino acids are covalently linked together. 2 Secondary structure - The arrangement in space of the atoms in the peptide backbone - α-helix and β-sheet - Domains or supersecondary structure: independently folded portions of proteins 3 Tertiary structure - Three-dimensional arrangement of all the atoms in the protein 4 Quaternary structure - Arrangement of subunits with respect to one another

4 4.2 Primary Structure of Proteins The amino acid sequence - Three dimensional structure, its properties, and correct functioning - The effects of changes in primary structure: site-directed mutagenesis - Importance: hemoglobin associated with sickle-cell anemia Determining the sequence of amino acids - classical biochemistry operation - routine, but not trivial

5 Biochemical Connections: Complete proteins and nutrition RDA RDA Arg* Unknown Met 0.70g His* Unknown Phe 1.12g (includes Tyr) Ile 0.84g Thr 0.56g Leu 1.12g Trp 0.21g Lys 0.84g Val 0.96g Rice & corn: poor in lys Bean: poor in met RDA (recommended dietary allowances) PER (protein efficiency ratio) Protein PER %Protein Whole egg Beef muscle Cow's milk 66 4 (largely H2O) Peanuts Corn 32 9 Wheat 26 12

6 4.3 Secondary Structure of Proteins The secondary structure - Conformation of the backbone Two bonds with free rotation 1 Bond between the α-carbon and the amino nitrogen (angle φ) 2 Bond between the α-carbon and the carboxyl carbon (angle ψ) - proposed by G. N. Ramachandran

7 4.3 Secondary Structure of Proteins α-helix and β-pleated sheet - Occur frequently in proteins - The Φ and Ψ angles repeat themselves in contiguous amino acids. Periodic Structures in Protein Backbones Rodlike α-helix and two-dimensional β-sheet -Their features repeat at regular intervals.

8 4.3 Secondary Structure of Proteins The α-helix Helical conformation - stabilized by hydrogen bonds parallel to the helix axis residues for each turn of the helix - pitch of the helix: 5.4A

9 4.3 Secondary Structure of Proteins The α-helix Model of the hemoglobin

10 4.3 Secondary Structure of Proteins The α-helix Two proteins with a high degree of α-helical content

11 4.3 Secondary Structure of Proteins The α-helix Several factors can disrupt the α-helix. - proline 1) restricted rotation around the bond between the nitrogen and the α-carbon 2) Proline s α-amino group cannot participate in intrachain hydrogen bonding. - Other localized factors: 1) electrostatic repulsion (lys & arg, glu & asp) 2) steric repulsion (bonded to β-carbon: val, ile, thr)

12 4.3 Secondary Structure of Proteins The β-sheet The peptide backbone - Almost completely extended - parallel pleated sheet: The peptide chains run in the same direction. - antiparallel pleated sheet: Alternating chains run in opposite directions.

13 4.3 Secondary Structure of Proteins The β-sheet Repeated zigzag structure - The hydrogen bonds are perpendicular to the direction of the protein chain.

14 4.3 Secondary Structure of Proteins Irregularities in Regular Structures Other helical structures - Shorter stretches - Most common 3 10 : three residues per turn and 10 atoms in the ring (cf. 3.6 residues per turn) Nonrepetitive irregular antiparallel β-sheets - β-bulge

15 4.3 Secondary Structure of Proteins Irregularities in Regular Structures Reverse turns - Ability to change directions - Steric reasons (glycine, proline) Type I Type II Any amino acid Glycine Proline + Glycine

16 4.3 Secondary Structure of Proteins Supersecondary Structures and Domains The combination of α- and β-strands - βαβ unit

17 4.3 Secondary Structure of Proteins Supersecondary Structures and Domains - αα unit

18 4.3 Secondary Structure of Proteins Supersecondary Structures and Domains - β-meander

19 4.3 Secondary Structure of Proteins Supersecondary Structures and Domains - Greek-key

20 4.3 Secondary Structure of Proteins Supersecondary Structures and Domains Motif - Repetitive supersecondary structure

21 4.3 Secondary Structure of Proteins Supersecondary Structures and Domains - β-barrel in the tertiary structure of the protein

22 4.3 Secondary Structure of Proteins Supersecondary Structures and Domains Domains - independently folded portions of proteins - similar conformation associated with the particular function

23 4.3 Secondary Structure of Proteins The collagen Triple Helix Collagen - A component of bone and connective tissue - Three polypeptide chains wrapped around each other in a ropelike twist - Repeating sequence X Pro Gly or X Hyp Gly (more stable)

24 4.3 Secondary Structure of Proteins The collagen Triple Helix Triple helical molecule: tropocollagen nm long and 1.5 nm in diameter - Hydrogen bonds involving the hydroxyproline and hydroxylysine residues - cross linking, increase with age (meat from older animals, tougher) - vitamin C: help hydroxylation of proline and lysine more stable

25 4.3 Secondary Structure of Proteins Two types of Protein Conformations: Fibrous and Globular The nature of the side chains can influence the folding Fibrous proteins - Silk: largely b-sheets - Wool: largely a-helical

26 4.3 Secondary Structure of Proteins Two types of Protein Conformations: Fibrous and Globular The nature of the side chains can influence the folding Globular proteins - More or less spherical shape

27 4.4 Tertiary Structure of Proteins Noncovalent interactions - More stable structure, lowest energy - In secondary, tertiary and quaternary structure - 1 Complexed to a metal ion, 2 hydrophobic interaction, 3 hydrogen bonding, 4 electrostatic attraction Covalent links - 5 Disulfide bonds (cys-cys)

28 4.4 Tertiary Structure of Proteins

29 4.4 Tertiary Structure of Proteins Tertiary structure - Three-dimensional arrangement of all the atoms - Result of the interplay of all the stabilizing forces X-ray crystallography - Perfect crystals can be grown under controlled conditions. - Only from proteins of very high purity crystals - Fourier series for analysis

30 4.4 Tertiary Structure of Proteins Tertiary structure of α-lactalbumin

31 4.4 Tertiary Structure of Proteins Nuclear magnetic resonance (NMR) spectroscopy - 2-D NMR - Use a Fourier series to analyze results - Samples in aqueous solution

32 4.4 Tertiary Structure of Proteins Myoglobin: An Example of Protein Structure Globular protein, complete tertiary structure aa residues and the heme group - 8 a-helix (A through H) - polar exterior - nonpolar interior - 2 his inside - heme s porphyrin ring: hydrophobic attraction - Fe(II) in heme group (cf. Fe(II), ferrous; Fe(III), feric)

33 4.4 Tertiary Structure of Proteins Myoglobin: An Example of Protein Structure Heme group - Metal ion, Fe(Ⅱ) and an organic part, protoporphyrin Ⅸ - Fe(II): 6 sites for binding

34 4.4 Tertiary Structure of Proteins Myoglobin: An Example of Protein Structure The oxygen binding site of myoglobin

35 4.4 Tertiary Structure of Proteins Myoglobin: An Example of Protein Structure (153 residues) Oxygen and carbon monoxide binding to the heme group

36 4.4 Tertiary Structure of Proteins Denaturation and Refolding Denaturation - Unfolding of a protein - Primary structure determines tertiary structure.

37 4.4 Tertiary Structure of Proteins Denaturation and Refolding Cause of denaturation - Heat - Extremes of ph - Binding of detergents (SDS) - Reagents (urea, guanidine hydrochloride) - Reduction of disulfide bridges: β-mercaptoethanol and urea Refolding - AA sequence contains information for complete 3-D structure.

38 4.5 Quaternary Structure of Proteins Quaternary structure - A property of proteins - More than one polypeptide chain (subunit) - Dimers, trimers, and tetramers (up to more than a dozen): oligomer - Allosteric: noncovalent interactions subtle changes at one site drastic changes at a distant site

39 4.5 Quaternary Structure of Proteins Hemoglobin Tetramer, two α-chains (141 residues) and two β-chains (146 residues)

40 4.5 Quaternary Structure of Proteins Hemoglobin - Oxygen-binding curve of myoglobin: hyperbolic - Oxygen-binding curve of hemoglobin: sigmoidal Functions of two proteins - Myoglobin: oxygen storage in muscle - Hemoglobin: oxygen transport in blood

41 4.5 Quaternary Structure of Proteins Hemoglobin Positive cooperativity - Cooperative binding of oxygen to hemoglobin - When one oxygen molecule is bound, it becomes easier for the next to bind.

42 4.5 Quaternary Structure of Proteins Hemoglobin The structures of deoxyhemoglobin and oxyhemoglobin

43 4.5 Quaternary Structure of Proteins Conformational Changes That Accompany Hemoglobin Function Other ligands when oxygen binds to hemoglobin - H + and CO 2 - Altering hemoglobin s three-dimensional structure

44 4.5 Quaternary Structure of Proteins Conformational Changes That Accompany Hemoglobin Function Bohr effect - Effect of H + - Lowering of the ph reduces the oxygen affinity of hemoglobin. - Myoglobin is not affected by H + or CO 2.

45 4.5 Quaternary Structure of Proteins Conformational Changes That Accompany Hemoglobin Function - Oxygen saturation curves at different ph values

46 4.5 Quaternary Structure of Proteins Conformational Changes That Accompany Hemoglobin Function Effect of CO 2 - CO 2 H 2 CO 3 HCO H + The affinity of hemoglobin for oxygen is lowered. - CO 2 can combine with the free α-amino groups to form carbamate. R NH 2 + CO 2 R NH COO - + H + Transport of oxygen 1 Large amounts of oxygen in the lungs 2 Hemoglobin binds to O 2. 3 The oxygenated hemoglobin transport oxygen to the tissues.

47 4.5 Quaternary Structure of Proteins Conformational Changes That Accompany Hemoglobin Function 2,3-bisphosphoglycerate (BPG), another ligand

48 4.5 Quaternary Structure of Proteins Conformational Changes That Accompany Hemoglobin Function - electrostatic interaction

49 4.5 Quaternary Structure of Proteins Conformational Changes That Accompany Hemoglobin Function - Stripped hemoglobin when BPG has been removed. - BPG controls oxygen-binding capacity.

50 4.5 Quaternary Structure of Proteins Conformational Changes That Accompany Hemoglobin Function - Supply a growing fetus with oxygen - Fetus (Hb F, a 2 g 2 ): more affinity, but adult (Hb A, a 2 b 2 )

51 4.6 Protein Folding Dynamics Bioinformatics - Prediction of protein structure - comparison of base sequence in nucleic acid The several steps in predicting protein architecture - comparative modeling: if greater than 25-30% homology - fold recognition algorithm - de novo prediction

52 4.6 Protein Folding Dynamics A comparison of the predicted structures of two proteins - Protein Data Bank ( Crystal structure Predicted Crystal structure Predicted DNA repair protein Bacterial protein

53 4.6 Protein Folding Dynamics Hydrophobic Interactions: A Case Study in Thermodynamics Major factor in the folding of proteins Liposomes - Phospholipids: polar head groups and long nonpolar tails - Double layer arrangement

54 4.6 Protein Folding Dynamics Hydrophobic Interactions : A Case Study in Thermodynamics Major factor in the folding of proteins The three dimensional structure of the protein cytochrome c - hydorphobic side chains: in red - hydrophilic side chains: in green

55 4.6 Protein Folding Dynamics Hydrophobic Interactions : A Case Study in Thermodynamics ΔS universe > 0 Lipid hydrocarbon hexane (C 6 H 14 ) with water - hard to form a mixed solution thermodynamically

56 4.6 PROTEIN FOLDING DYNAMICS The importance of correct folding The problem of protein aggregation - In the protein-dense environment of the cell: 1 Proteins may begin to fold incorrectly as they are produced. 2 Proteins may begin to associate with other proteins before completing their folding process.

57 4.6 PROTEIN FOLDING DYNAMICS Protein-Folding Chaperones Chaperones - tricky folding process in vivo - aid in the correct and timely folding of other proteins - exist in organisms from prokaryotes through humans - example: hsp70 (70,000 MW heat-shock protein)

58 4.6 PROTEIN FOLDING DYNAMICS Protein-Folding Chaperones Balancing the components of hemoglobin - Excess α-chains can form aggregates, which could lead to damaged red blood cells and a disease called thalassemia

59 Biochemical Connections: Prions Cause of mad-cow disease (variant Creutzfeldt-Jakob disease) Found by Stanley Prusiner (1997 Novel Prize winner) Propagation of abnormal prions Propagated in nervous tissue

60 The end! Hope costs nothing. By Colette