Proteins Structure, Function, Folding PDF

Proteins: Structure, Function, Folding Marc A. Ilies, Ph. D. Lehninger - Chapter 4 (p115-155) [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas or recit...

Proteins: Structure, Function, Folding Marc A. Ilies, Ph. D. Lehninger - Chapter 4 (p115-155) [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas or recitation session ©MAIlies2024 1  Structure of Proteins Configuration: the fixed three-dimensional relationship of the atoms in a molecule, defined by the bonds between them Conformation: any of the spatial arrangements that the atoms in a molecule may adopt and freely convert between by rotation about individual single bonds - there are many conformations possible for one configuration (!): Unlike most organic polymers, protein molecules adopt a specific three- dimensional conformation. This particularly folded 3D structure is able to fulfill a specific biological function This structure is called the native fold The native fold has a large number of favorable interactions within the protein (if we change some of these interactions we can change the fold!) 2  Interactions in proteins that influence folding ΔG < 0 ΔG = ΔH - TΔS Hydrophobic effect – Association of hydrophobic residues with release of water molecules from the structured solvation layer around them increases the net entropy Hydrogen bonds – Interaction of N-H and C=O groups between different peptide bonds through H- bonding increases the stability of the fold and leads to local regular structures such as -helices and -sheets Electrostatic interactions – Long-range strong interactions between permanently charged groups, such as salt -bridges, especially if buried in the hydrophobic environment strongly stabilize the protein Covalent bonds - S-S binds between Cys residues 3 Protein structure: four levels of organization 6  Primary structure. The structure of the peptide bond Structure of the protein is partially dictated by the properties of the peptide bond The peptide bond is a resonance hybrid of two canonical structures A and B: The resonance causes the peptide bonds – to be less reactive compared to esters, for example – to be quite rigid and nearly planar (but flexible) – to exhibit a large dipole moment, in the favored trans configuration 7 The Rigid Peptide Plane and the Partially Free Rotations Rotation around the peptide bond is not permitted Rotation around bonds connected to the alpha carbon is permitted  (phi): angle around the -carbon—amide nitrogen bond  (psi): angle around the -carbon—carbonyl carbon bond In a fully extended polypeptide, both  and  are 180° The polypeptide is made up of a series of planes linked at α carbons 8  Distribution of  and  Dihedral Angles Some  and  combinations are very unfavorable because of steric crowding of backbone atoms with other atoms in the backbone or side chains Some  and  combinations are more favorable because of chance to form favorable H -bonding interactions along the backbone Some  and  values are impossible due to steric clash → Ramachandran plots:  L-Ala https://www.youtube.com/watch?v=Q1ftYq13XKk  and  are defined as 0o in this theoretical example 9  Secondary Structures Secondary structure refers to a local spatial arrangement of the polypeptide backbone Two regular arrangements are common: The  helix – stabilized by hydrogen bonds between nearby residues The  sheet – stabilized by hydrogen bonds between adjacent segments that may not be nearby Irregular arrangement of the polypeptide chain is called the random coil Can be determined experimentally through circular dichroism spectroscopy 10  The  Helix Helical backbone is held together by hydrogen bonds between the backbone amides of an n and n+4 amino acids (CO group of AA1 with NH group of AA5) Right-handed helix with 3.6 residues (5.4 Å) per turn Peptide bonds are aligned roughly parallel with the helical axis Side chains point out and are roughly perpendicular with the helical axis ϕ = - 57 o ψ = - 47 o 11  The  Helix - α-helix sequence can be represented through the helix wheel, which provides a top view, indicating connectivity but also the positioning of the AA residues along the helix; can be color-coded to indicate polarity of different regions, etc: - amino acids have different propensities to form α-helices, depending on their R group properties: 12  Sequence affects helix stability Not all polypeptide sequences adopt -helical structures 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 Gly acts as a helix breaker because the tiny R-group supports other conformations Attractive or repulsive interactions between side chains 3–4 amino acids apart will affect formation → the position of an AA relative to its neighbors is important! 13  -Sheets The planarity of the peptide bond and tetrahedral geometry of the -carbon create a pleated sheet-like structure, at high ψ and ϕ values Sheet-like arrangement of backbone is held together by hydrogen bonds between the backbone amides in different strands (in the plane of the sheet) Side chains protrude from the sheet alternating in up and down direction 14  Parallel and Antiparallel  Sheets Parallel or antiparallel orientation of two chains within a sheet are possible In parallel  sheets the H-bonded strands run in the same direction – Resulting in bent H-bonds (weaker); short repeat period (6.5 Å) In antiparallel  sheets the H-bonded strands run in opposite directions – Resulting in linear H-bonds (stronger); longer repeat period (7 Å) 15   Turns are common into proteins -turns occur when  sheets change directions They are 180o turns that involve 4 AAs, with AA1 and AA4 being H-bonded Gly and Pro are common participants at positions 2 and 3 (type I contains Pro at position 2, type II contains Gly in position 3)  Gly is small and Pro can form a cis conformation easier than most AAs.  turns are commonly found at the surface of a protein, where the peptide groups of AA2 and 3 can H-bond with water. 16  Protein tertiary structure The overall 3D arrangement of all atoms in a protein is referred as the protein’s tertiary structure Includes long-range aspects of amino acid sequence (AA that are far apart in polypeptide sequence may interact within the completely folded 3D structure of the protein) Interacting segments of polypeptide chains are held in their characteristic tertiary positions by weak interactions and by disulfide covalent bonds: Ionic Salt bridge A globular protein 17  Protein quaternary structure Some proteins contain two or more separate polypeptide chains The arrangement of these protein subunits in 3D complexes constitutes quaternary structure Exemplified for fibrous proteins: - have structural role in organism - Consist usually largely of a single type of secondary structure - E. g. α-keratin, collagen, silk fibroin  Fibrous proteins: Structure of α-keratin evolved for strength; found in hair, wool, nails, claws, quills, horns, hooves, outer layer of skin two α-helices assemble to form a coiled coil; further assemble into protofilaments, protofibrils: strength enhances by covalent cross-links made out of disulfide bonds that stabilize quaternary structure 19  Structure of α-keratin Permanent waiving as biochemical engineering: reduce, moist heat (shaping i.e. straightening or curling), oxidize: 20  Structure of collagen Collagen evolved to provide strength too, in connective tissue, cartilages, tendons, organic matrix of the bone, cornea Collagen contains a very high proportion of Gly (35%) and Pro (21%); also 4-OH Pro, Ala)  left-handed helix, with 3 AA per turn (usually Gly-Pro-4OH-Pro) Three separate polypeptides are super-twisted about each other in a unique tertiary and quaternary structure: 4OH-Pro biosynthesized from Pro by proline hydroxylase; requires ascorbic acid (vitamin C) collagen fibrils cross-linked by imine bonds between modified Lys and 5-OH Lys 21  Globular proteins: Structure of myoglobin Myoglobin is a globular protein containing a single polypeptide (153 AAs) and a single iron protoporphirin (heme) group: Contains 8 α-helices, interrupted by bends, some of which are β-turns Abundant in muscles, has oxygen transport role through coordination of O2 molecule by Fe2+ (ferrous form): Heme placed in a hydrophobic crevice created by α-helices to prevent oxidation of Fe2+ to Fe3+ (does not bind O2), which occurs in presence of water 22  Approximate Proportion of α Helix and β Conformation in TABLE 4-4 Some Single-Chain Proteins Residues (%)a Protein (total residues) α Helix β Conformation Chymotrypsin (247) 14 45 Ribonuclease (124) 26 35 Carboxypeptidase (307) 38 17 Cytochrome c (104) 39 0 Lysozyme (129) 40 12 Myoglobin (153) 78 0 SOURCE: Data from C. R. Cantor and P. R. Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological Macromolecules, p. 100, W. H. Freeman and Company, 1980. aPortions of the polypeptide chains not accounted for by α helix or β conformation consist of bends and irregularly coiled or extended stretches. Segments of α helix and β conformation sometimes deviate slightly from their normal dimensions and geometry. 23  Globular proteins Human serum albumin, another globular protein: - Important protein in blood plasma, with role in maintaining osmotic pressure, and transport of fatty acids, steroids, thyroid hormones - Very high α-helix content Hemoglobin, a globular protein (multimer); quaternary structure has 4 almost identical subunits (oligomers), each sub-unit similar to myoglobin - Main transporter of O2 in blood (red blood cells) 24  Special Ternary Structures of DNA Binding Proteins Helix-Turn-Helix Zinc Fingers Leucine Zipper motif Figs 28-11,12,14 Motif: a supersecondary structure defining a recognizable folding pattern involving two or more elements of secondary structure and the connection between them Domain: part of polypeptide chain that is independently stable or could undergo movements as a single entity with respect to the entire protein  Protein denaturation and folding Protein native conformation maintenance is essential for its function → proteostasis  requires permanent protein synthesis and folding, refolding Folding can occur spontaneously or can involve specialized proteins called chaperones (e.g. heat shock proteins HSP, chaperonins) Chaperonins 26  Protein misfolding and formation of amyloid fibrils Misfolding can trigger aggregation → fibrils (accumulation can trigger diabetes, Parkinson’s Alzheimer’s diseases): 27  Protein denaturation and renaturation Loss of protein structure results in loss of function in a process called denaturation of proteins Proteins can be denatured by: - heat - extreme pH, - addition of amphiphilic solvents such as EtOH, acetone, - addition of certain solutes such as urea and guanidine hydrochloride - addition of detergents Tertiary structure of proteins is encoded in the AA sequence  direct proof is the renaturation of proteins (return to native conformation once denaturant is removed) 28  Goals and Objectives Upon completion of this lecture at minimum you should be able to answer the following: ► What is the structure of proteins, configuration, conformations, native fold and biological function; ► Which are the interactions in proteins that influence folding ► What is the protein primary structure and the structure of peptide bond; which are the protein secondary structures, what constitutes tertiary and quaternary structure of proteins ► What is the structure of fibrous proteins (keratin, collagen), globular proteins (myoglobin, human serum albumin, hemoglobin) ► Which are the special ternary structures of DNA-binding proteins ► What is protein denaturation and how can occur, what is protein renaturation and how protein folding can occur 29