Conformational Properties of Polypeptide Chains
Levels of Organization Primary structure Amino acid sequence of the protein Secondary structure H bonds in the peptide chain backbone α helix and β sheets Tertiary structure Non covalent interactions between the R groups within the protein Quanternary structure Interaction between 2 polypeptide chains
Why protein structure? In the factory of living cells, proteins are the workers, performing a variety of biological tasks. Each protein has a particular 3 D structure that determines its function. Structure implies function. Structure is more conserved than sequence. Protein structure is central for understanding protein functions. Sequence Structure Function
The basics of protein Proteins have one or more polypeptide chains Building blocks: 20 amino acids. Length range from 10 to 1000 residues. Proteins fold into 3 D shape to perform biological functions.
Anfinsen s thermodynamic hypothesis The three dimensional structure of a native protein in its normal physiological milieu (solvent, ph, ionic strength, presence of other components such as metal ions or prosthetic groups, temperature, etc.) is the one in which the Gibbs free energy of the whole system is lowest; that is, that the native conformation is determined by the totality of interatomic interactions and hence by the amino acid sequence, in a given environment. --- Anfinsen s Nobel lecture, 1972
Gibbs free energy A thermodynamic potential that measures the "useful" or process-initiating work obtainable from an isothermal, isobaric thermodynamic system. The maximum amount of non-expansion work that can be extracted from a closed system Gibbs free energy (ΔG) or available energy is the chemical potential that is minimized when a system reaches equilibrium at constant pressure and temperature.
Common structure of Amino Acid Cα is the chiral center R Side chain = H,CH 3, Atoms numbered β,γ,δ,ε,ζ.. Amino H H + N H C α H Atom lost during peptide bond formation Backbone C - O O Carboxylate
Amino Acids Hydrophilic Hydrophobic
The peptide bonds
Coplanar atoms
Primary structure The amino acid sequences of polypeptides chains.
Shape = Amino Acid Sequence Proteins are made of 20 amino acids linked by peptide bonds Polypeptide backbone is the repeating sequence of the N C C N C C in the peptide bond The side chain or R group is not part of the backbone or the peptide bond The amino acid sequence (the primary structure) of a protein determines its three dimensional structure, which in turn determines its properties.
1 structure = sequence of amino acids
Secondary structure Local organization of protein backbone: α helix β strand (which assemble into β sheet) turn and interconnecting loop.
Protein Folding The peptide bond allows for rotation around it and therefore the protein can fold and orient the R groups in favorable positions Weak non covalent interactions will hold the protein in its functional shape these are weak and will take many to hold the shape
Protein Folding Proteins shape is determined by the sequence of the amino acids The final shape is called the conformation and has the lowest free energy possible Denaturation is the process of unfolding the protein Can be down with heat, ph or chemical compounds If the chemical compounds are removed, the proteins can be renatured or refold
Refolding Molecular chaperones are small proteins that help guide the folding and can help keep the new protein from associating with the wrong partner
What drives protein folding? Hydrophobic effects Hydrophobic residues tend to be buried inside Hydrophilic residues tend to be exposed to solvent Hydrogen bonds help to stabilize the structure.
Non covalent Bonds in Proteins
Globular Proteins The side chains will help determine the conformation in an aqueous solution
Hydrogen Bonds in Proteins H bonds form between 1) atoms involved in the peptide bond; 2) peptide bond atoms and R groups; 3) R groups
Protein Conformation Framework Bond rotation determines protein folding, 3D structure
Bond Rotation
Bond Rotation Determines Protein Folding
Protein Conformation Framework Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle) τ Measures orientation of four linked atoms in a molecule: A, B, C, D
Protein Conformation Framework Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle) τ Measures orientation of four linked atoms in a molecule: A, B, C, D τ ABCD defined as the angle between the normal to the plane of atoms A-B- C and normal to the plane of atoms B- C-D
Ethane Rotation A B D C A B C D
Protein Conformation Framework Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle) τ Measures orientation of four linked atoms in a molecule: A, B, C, D τ ABCD defined as the angle between the normal to the plane of atoms A-B- C and normal to the plane of atoms B- C-D Three repeating torsion angles along protein backbone: ω, φ, ψ
Backbone Torsion Angles
Backbone Torsion Angles φ and ψ Dihedral angle φ : rotation about the bond between N and C α Dihedral angle ψ : rotation about the bond between C α and the carbonyl carbon
φ and ψ angles
Backbone Torsion Angles ω Dihedral angle ω : rotation about the peptide bond, namely C α 1 -{C-N}- C α 2 ω angle tends to be planar (0º - cis, or 180 º - trans) due to delocalization of carbonyl π electrons and nitrogen lone pair
Backbone Torsion Angles φ and ψ are flexible, therefore rotation occurs here However, φ and ψ of a given amino acid residue are limited due to steric hindrance Only 10% of the {φ, ψ} combinations are generally observed for proteins First noticed by G.N. Ramachandran
Consequences of double bond character in the peptide bond trans peptide bond cis peptide bond Still another consequence: in the cis form, the R groups in adjacent residues tend to clash. Hence almost all peptide bonds in proteins are in the trans configuration.
Planarity of the peptide bond Psi (ψ) the angle of rotation about the Cα-C bond. Phi (φ) the angle of rotation about the N-Cα bond. The planar bond angles and bond lengths are fixed.
Side chain properties Carbon does not make hydrogen bonds with water easily hydrophobic O and N are generally more likely than C to H bond to water hydrophilic General amino acids groups: Hydrophobic Charged (positive/basic & negative/acidic) Polar 37
Unlike the peptide bond, free rotation occurs about the other two backbone bonds, but steric interactions within the polypeptide still severely limit plausible conformations, so that only certain combinations of phi and psi angles are allowed and between carbonyls between R groups or combinations of an R group, carbonyl or amide group. 180 allowed Ψ 0 not allowed -180-180 0 φ 180
Steric Hindrance Interference to rotation caused by spatial arrangement of atoms within molecule Atoms cannot overlap Atom size defined by van der Waals radii Electron clouds repel each other
Steric Hindrance Most combinations of ψ and φ for an amino acid are not allowed because of steric collisions between the side chains and main chain. Theoretically if the torsion angles all are 180 o we have a purely trans system ( N, Cα, C ) if the torsion angles are all 0 we have the purely cis arrangement. Which is preferred? For the peptide bond (ω) [Ci Ni+1] The trans is preferred 1000:1
Certain side chain conformations are energetically favorable Ethane Valine Which valine stagger is energetically the best? The 1 st one Why?
G.N. Ramachandran Used computer models of small polypeptides to systematically vary φ and ψ with the objective of finding stable conformations For each conformation, the structure was examined for close contacts between atoms Atoms were treated as hard spheres with dimensions corresponding to their van der Waals radii Therefore, φ and ψ angles which cause spheres to collide correspond to sterically disallowed conformations of the polypeptide backbone
Ramachandran diagram Most angle combinations (75%) excluded by steric hindrance Dark green most favored Steric exclusion: powerful organizing principle Limited conformations favor protein folding, favorable entropy of too many conformations opposes folding
Ramachandran Plot QuickTime?and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime?and a TIFF (Uncompressed) decompressor are needed to see this picture. G.N. Ramachandran, 1963
φ, ψ and Secondary Structure Name φ ψ Structure alpha-l 57 47 left-handed alpha helix 3-10 Helix -49-26 right-handed. π helix -57-80 right-handed. Type II helices -79 150 left-handed helices formed by polyglycine and polyproline. Collagen -51 153 right-handed coil formed of three left handed helicies
The classical version of the Ramachandran plot for (a) alanine, (b) glycine. The fully allowed regions are shaded; the partially allowed regions are enclosed by a solid line. The connecting regions enclosed by the dashed lines are permissible with slight flexibility of bond angles (a,b). (c) for all 19 non-glycines and (d) for glycine. Most regions show a 45 degree slope rather than being parallel to any of the axes (c) For glycine (d), the beta sheet region is split into two distinct maxima and the two most populated regions (red), which were predicted to be only just permissible as shown in (b). There are five areas in the glycine plot; two with psi 0 and three with psi 180. Referenced from Hovmöller (2002)
Random Coil Following translation we have a primary amino acid sequence and it s random coil ~~~~~~~~~~~~ just flopping around folds to a 3D structure Uses van der waals interactions Hydrophobic interactions Salt Bridges H bonds Electrostatics forces
Motifs of Protein Structure Packing hydrophobic side chains into the interior of the molecule Creating a HYDROPHOBIC core and a HYDROPHILIC surface. The problem with creating such a hydrophobic core from a protein chain is to bring the side chains into the core the main chain must also come into the interior. The main chain is highly polar and therefore hydrophilic, with one hydrogen bond donor NH and one acceptor C = O for each peptide unit. In a hydrophobic environment these main chain polar groups must be neutralized by the formation of hydrogen bonds. This problem is solved very elegantly by the formation of regular secondary structure within the interior.
Secondary Structure The two very important secondary structures of proteins are: α helix β pleated sheet Both depend on hydrogen bonding between the amide H and the carbonyl O further down the chain or on a parallel chain. 5P2 49
Protein Folding 2 regular folding patterns have been identified formed between the bonds of the peptide backbone α helix protein turns like a spiral fibrous proteins (hair, nails, horns) β sheet protein folds back on itself as in a ribbon globular protein
α Helix 3.6 amino acids per turn of helix. The (p), distance per turn, is 0.54 nm, and the rise, distance between each atom is 0.15 (0.54/3.6). The N H group of an amino acid forms a hydrogen bond with the C=O group of the amino acid four residues earlier. This repeated i + 4 > i hydrogen bonding is the most prominent characteristic of an α helix
Characteristics of α Helix Although structurally ordered, isolated α helices are usually marginally stable in aqueous solution. A helix forms quickly (10 5 to 10 7 sec) but can unravel almost as quickly. Formation is generally independent of length, though unraveling isn t. The most common along the outside with one face out into solution and one into the hydrophobic core. Alpha helix have a hydrophobic and hydrophilic side Side chains to change from hydrophobic to hydrophilic with a periodicity of 3 4 residues.
α helix An α helix can, in theory, be right or left handed depending on the screw direction of the chain. Left handed ones don t exist!!! Because of steric interactions between side chains and CO groups ALWAYS right handed!
Properties of the alpha helix φ ψ 60 Hydrogen bonds between C=O of residue n, and NH of residue n+4 3.6 residues/turn 1.5 Å/residue rise 100 /residue turn 54
Fig. 4-2a, p.84
Helices have electric dipoles.
The dipole moment in the α helix All the hydrogen bonds in an α helix point in the same direction along the helical axis. The dipole moments are also aligned along the helical axis. A partial net positive charge at the amino end and a negative charge at C terminal end. The α helix has a macrodipole moment of ~n * 3.5 Debye units. n = # residues
Preferred amino acids in α helices Different side chains have a weak but definite preference either for or against being in an α helix. Good helix formers: Ala, Glu, Leu, Met Poor helix formers: Ser Gly small, flexible doesn t like to be in fixed position Try big and bulky Pro can t enter the i i + 4 groups because can t H bond
Proline and α helix Amino acid side chains project out from the α helix and don t interfere with it except for proline. The last atom of the proline side chain is bonded to the main chain N atom which forms a ring. This prevents the N from contributing to a hydrogen bond an adding steric hindrance to the α helix conformation. Proline fits very well into the first (N term) of an α helix Proline in long α helices distorts the local geometry causing a BEND. Pro s are often referred to as helix breakers.
Helical wheel α helices are amphipathic Non polar side chains along one side of the helical cylinder and polar residues along the remainder of its surface. Such helices often aggregate with each other or with other non polar surfaces. The view is down the helix with the hydrophobic core being in the middle. The helix repeats itself after 5 turns or 18 residues so the 19 21 residues are offset. Hydrophilic side Hydrophobic side
Variations on the classical α helix In natural proteins a slightly different geometry is seen. The CO groups tend to point out away from the helix axis and the H bonds are consequently not as straight So, φ ~ 62 o and ψ ~ 41 o instead of the classical 57 to 60 o and 47 to 50 o This geometry is more stable than the classical α helix because it permits each CO oxygen to H bond to the NH of the i + 4 residue. Occurs when the chain is more loosely or more tightly coiled With hydrogen bonds to residues i + 5 (π helix) or i + 3 (3 10 helix), respectively, NOT i + 4
3 10 helix and π helix Has 3 residues per turn and contains 10 atoms between the hydrogen bond donor and acceptor. Rare and usually occur at the ends of regular helices or as a single turn helices. Not energetically favorable for the most part The backbone atoms are packed too tight in the 3 10 or too loose in the π helix that there is a hole in the middle. Contrast of helix end views between α (squarish) vs 3-10 (triangular)
β sheet (pleated sheet) Two types of β sheet, parallel (same N to C direction) and anti parallel (opposite N to C direction) between polypeptide chains. β Sheet contains 2 amino acid residues per turn, and cannot form H bond between amide H and carboxyl O. Hydrogen bonds can only formed between adjacent polypeptide chains. Anti parallel form is more stable than parallel form. 64
The beta strand (& sheet) φ 135 ψ +135 65
H bond β pleated sheet
B Sheet: Lewis Structure CH C N HC C N CH C N O H H O H O N-term C-term C-term N-term CH C N HC C N CH C N O H H O H O Parallel sheet CH C N HC C N CH C N O H H O H O N-term C-term C-term N-term HC C N HC C N CH C N O H H O H O Antiparallel sheet
Topologies of β sheets
Properties of beta sheets Core of many proteins is the β sheet Form rigid structures with the H bond Formed of stretches of 5 10 residues in extended conformation Parallel/aniparallel, contiguous/non contiguous
Turns and Loops Secondary structure elements are connected by regions of turns and loops Turns short regions of non α, nonβ conformation Loops larger stretches with no secondary structure. Often disordered. Random coil Sequences vary much more than secondary structure regions Common AA found at turns are: glycine: small size allows a turn proline: geometry favors a turn
Proline C HN COOH H P
Globular Proteins Usually bind substrates (O2) within a hydrophobic cleft in the structure. Myoglobin and hemoglobin are typical examples of globular proteins. Both are hemoproteins and each is involved in oxygen metabolism. 5P2 72
Globular Protein Structure: Patterns of Folding Infinite number of globular protein folding, but some principles of protein folding are found. Made up of a number of domains a compact locally folded region of tertiary structure, which perform different functions of proteins. Two major kinds of folding patterns, α helic and β sheet. 73
Fig. 6.16
Myoglobin: 2 o and 3 o Aspects Globular myoglobin has 153 AA arranged in eight α helical regions labeled A H. The prosthetic heme group is necessary for its function, oxygen storage in mammalian muscle tissue. His E7 and F8 are important for locating the heme group within the protein and for binding oxygen. A representation of myoglobin follows with the helical regions shown as ribbons. 5P2 75
Myoglobin: 2 o and 3 o aspects Some helical regions Heme group with iron (orange) at the center 5P2 76
The Heme Group N of His F8 binds to fifth site on the iron. His E7 acts as a gate for oxygen. - OOC CH 2 CH 2 CH 2 CH 2 COO - H 3 C CH 3 N N H 2 Fe(II) C N N CH CH 3 Pyrrole ring CH 3 CH CH 2 5P2 77
Binding Site for Heme Lower His bonds covalently to iron(ii) Oxygen coordinates to sixth site on iron and the upper His acts as a gate for the oxygen. 5P2 78
3D Structure of Cytochrome c Cytochrome C 의 3차구조 Fig. 4-15, p.94
Fibrous Proteins Proteins with an elongated or filamentous form, often dominated by a single type of secondary structure over a large distance Fibrous proteins have a high concentration of α helix or β sheet. Examples include: a keratin collagen silk fibroin 5P2 80
Fibrous Proteins Structural Materials of Cells and Tissues More than 1/3 or more of the body protein in large vertebrates. Functions include: External protection such as skin, hair, feather and nail. Structure and support, shape and form, tendons, cartilage and bone. Native conformations of fibrous proteins are stable proteins after isolated and will not be denatured or unfolded. 81
Keratins Two classes of keratin, α and β keratins. α Keratins is the major fibrous proteins of hair and wool. Each α keratin molecule contains over 300 residues in length and all are α helical. Two α keratin helices bond together by their hydrophobic R side groups. The higher amounts of AAs are serine, glutamine, glutamic acid and cysteine. 84
Structure of α Keratin Adjacent polypeptide chains also crosslinked by disulfide bonds. Disulfide bond patterns between are what determine whether human hair is straight or curly.
Coiled Coil α Helical Dimer of α Keratin Amphipathic α helices: Residues a, d, a and d hydrophobic, other residues more hydrophilic
β-keratin Contains β-sheet structure. Are found mostly in birds and reptiles for feather and scales. 87
Fibroin They are all anti parallel pleated sheet structure. Comprise almost glycine, alanine and serine AA. Gly ala gly ala gly serine gly ala ala gly This alternation allows the β sheet to fit together. Strong and inextensible. No intrachain hydrogen bond, only interchain hydrogen bonds. No cystine cross linkage presence. The fibers are very flexible, e.g. silk. 88
Silk made by silkworms and spiders. Composed of microcrystalline array of antiparallel beta pleated sheets where each strand has alternating Gly and Ala or Ser residues. Structure of Silk Fibroin
Collagen Collagen is about one third of total protein in large animals, and is the major constituent of tendons and skin. Basic unit is tropocollagen which consists of three polypeptide chains in left handed helix tightly coiled into a three strand rope in right handed helix, arranged in head to tail. Tropocollagen have total about 3000 AA residues (each polypeptide has 1000 AA residues), having of molecule weight of 300,000. Every third residue can be only glycine because of the bulky effect. 91
Composition and structure of Collagen The tropocollagen subunit is a rod about 300 nm long and 1.5 nm in diameter, made up of three polypeptide strands, each of which is a left handed helix. Twisted together into a right handed coiled coil, a triple helix, a cooperative quaternary structure stabilized by numerous hydrogen bonds. Tropocollagen subunits spontaneously self assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues. There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices Prolines and hydroxyprolines cause it to be very tightly twisted and very rigid
Structure of Collagen Fibers Collagen is the most abundant vertebrate protein and the major stress-bearing component of connective tissue (bone, teeth, cartilage, tendon) and fibrous matrix of skin and blood vessels. 3 intertwined lefthanded helices 3.3 residues/turn Repeating Gly-X-Y (X often Pro, Y often Pro or hydroxypro)
Tropocollagen The 3 collagen molecules are crosslinked by : *H bonds very tight and do not stretch * Crosslinks between lysines, both interchain and intrachain, due to oxidation by enzyme LYSYL OXIDASE. Occurs only in collagen. Carbohydrates on hydroxylysines Tropocollagen molecules are head to tail and are staggered. More crosslinks occur with age.
Collagen Amino Acid Composition 1) Glycine (30%) 2) Proline 3) Modified amino acids hydroxyproline hydroxylysine 4) Glycosylation
Primary sequence of collagen Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Hyp Gly Pro Hyp Gly Ala Hyp Gly Pro Gln Gly Phe Gln Gly Pro Hyp Gly Glu Hyp Gly Glu Hyp Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Hyp Gly Pro Hyp Gly Lys Asn Gly Asp Asp
Glycosylation Sugars added to hydroxylysine or hydroxyproline Addition of disaccharides Glu Glu Gal Glu Gal Gal Glycosylation influences function Fibrils tendons low carbohydrate Sheets Lens cap, cornea High carbohydrate
Cross links between Tropocollagen 1) Between two lysine residues 1) Enzyme lysyl oxidase 2) Aldol condensation: enolate + carbonyl 3) Link Aldol cross link 2) Between two hydroxylysines and a lysine residue 1) Hydroxyprridinium cross link: increase heat stability Higher in older animals
Importance of Hydroxylation The principal residue to be hydroxylated in in proteins is proline. Lysine may also be hydroxylated. Catalyzed by prolyl 4 hydroxylase, prolyl 3 hydroxylase, and lysyl 5 hydroxylase. The reactions require iron (as well as molecular oxygen and α ketoglutarate) to carry out the oxidation Use vitamin C to return the iron to its oxidized state. Deprivation of ascorbate leads to deficiencies in proline hydroxylation Leads to less stable collagen, which can manifest itself as the disease scurvy.
Processes occuring within intracellular membrane of ER, golgi Synthesis of Pro α chain (RER) Hydroxylation of selected prolines and lysines Glycosylation of selected hydroxylysines Triple helix formation with 3 Pro α chains Secretion (collagen molecule)
Formation of Collagen Fiber