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Structure of protiens and the applied aspects
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- 2. CONTENTS • Overview ofProtein Structure • Primary Structure • Peptide bond • Secondary structure • Super-secondary structure • Tertiary and quaternary structure • Unstructured protein • Fibrous protein • Protein folding and disease associated with it • Denaturation of protein
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- 4. AMINO ACID, PEPTIDESAND PROTEINS Two amino acid molecules covalently joined through a substituted amide linkage peptide Bond • formed by – removal of the elements of water • from the α-carboxyl group of one amino acid • α-amino group of another
- 5. When afew amino acids are joined in this fashion, the structure is called an oligopeptide When many (10 to 50) amino acids are joined, the product is called a polypeptide molecules referred to as polypeptides generally have molecular weights below 10,000 Those called proteins (having >50 amino acids) have higher molecular weights.
- 6. OVERVIEW OF PROTEIN STRUCTURE •Configuration-geometric relationship between a given set of atoms – Ex- that distinguish L- from D-amino acids • Conformation – spatial arrangement of atoms in a protein. • Thermodynamically the most stable conformations exist. • Stabilized Largely by Weak Interactions
- 7. • Stability –tendency to maintain a native conformation. • Unfolded state of protein – high degree of conformational entropy • Native conformation stabilized by – Disulfide bonds – Noncovalent forces
- 8. NONCOVALENT FORCES ◦ Weak(non covalent) interactions Hydrogen bonds Hydrophobic interactions Ionic interactions Van der Walls forces Noncovalent forces leads to protein folding and contribute to a protein’s stability ◦ Cause a polypeptide fold into a unique native conformation ◦ Stabilizes the native structure against denaturation
- 9. Hydropho bic Interactio n Forces Electrostatic Interactions Vander Walls Forces Due to properties of water that surround the nonpolar group ◦ ( ionic or salt linkage ) ◦ Important in stabilization of protein structure ◦ Binding of charged ligand and substrate ◦ Weakest ◦ Van der Walls contact distance Distance of maximum favorable interaction between 2 atom Sum of Van der Walls radii of 2 atoms
- 10. HYDROGEN BOND – Whena hydrogen atom covalently bound to and electronegative atom • Donor atom – is shared with a second electronegative atom • Acceptor atom – For high bond energy • d= 2.7 – 3.1Å • Geometrically collinear H- bond O H N
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- 12. PRIMARY STRUCTURE OF PROTIEN Def. – Covalent structure, which includes number and sequence of amino acids. Importance of primary structure ◦ Required to understand its structure Mechanism of action ◦ Biosynthesis including post-translational modification ◦ Comparison between different animal species – shows essential and non-essential residues.
- 13. SEQUENCE COMPARISON • Usedto predict the similarity in structure and function of protein. 2 sequences are – – Homologous - when their sequence highly alignable • Protein that evolved from same gene – Analogy - structurally similar protein sequence, but no evolutionary relationship found. – Invariant residue - particular amino acid regularly found at same position. – Conservative substitution - by another amino acid with similar polarity – Nonconservative substitution – by another amino acid with different polarity
- 14. CLINICAL CORRELATION • Sicklecell anemia – HbS – a variant of normal hemoglobin • Nonconservative mutation – 6th position of β-globin gene – Glutamic acid(polar) → Valine(nonpolar) • So deoxy-HbS molecules get polymerized – Precipitation of Hb within RBC • Sickle shape and hemolysis – HbC – Lysine is substituted for glutamic acid at 6th position (A3 helix) in β chain – HbD – β121 (GH4) – Glu → Gln
- 15. PROTEOLYTIC CLEAVAGE OFINSULIN Proinsulin – ◦ Produced in pancreatic islet cells. ◦ Single polypeptide chain containing 86 amino acids 3 intra-chain cystine disulfide bond ◦ Cleaved by proteases present in islet cells ◦ Releases 2 molecule C-peptide – 35-residue fragments Insulin Insulin contain ◦ 2 polypeptide chain A – 21 AA B – 30 AA ◦ Covalently joined by the same disulfide bond
- 16. DIFFERENT TYPE OFINSULIN USED Porcine insulin ◦ More acceptable than bovine insulin As sequence is more similar to human insulin Bovine insulin Human insulin – ◦ Primary insulin in developed country ◦ Prepared from Genetically engineered bacteria Modifying pork insulin Majority of the population are able to utilize animal insulin without complication ◦ Because in amino acid sequence Small number and conservative nature of change They do not significantly change the 3-dimensional structure
- 17. PEPTIDE BOND ISRIGID AND PLANAR X-ray diffraction studies of crystals of amino acids – ◦ peptide C⎯N bond – somewhat shorter than the C⎯N bond in a simple amine atoms associated with the peptide bond are coplanar Partial double-bond in character cannot rotate freely ◦ Rotation is permitted about the N⎯Cα and the Cα⎯C bonds
- 18. Three bondsseparate sequential α-carbons in a polypeptide chain. The N⎯Cα and Cα⎯C bonds can rotate. The peptide C⎯N bond is not free to rotate Other single bonds in the backbone may also be rotationally hindered ◦ Depending on the size and charge of the R groups
- 19. Φ(phi) bond– between nitrogen and α-carbon Ψ(psi) bond – between α- carbon and carbonyl carbon
- 20. RAMACHANDRAN PLOT • Ramachandranplot for L-Ala residues. • Peptide conformations are defined by the values of Ψ and Φ • Conformations deemed possible are those that involve little or no steric interference, • The regions are Color Conformation allowed dark blue involve no steric overlap mediu m blue extreme limits for unfavorable atomic contacts lightes t blue permissible if a little flexibility is allowed yellow not allowed.
- 21. SECONDARY STRUCTURE OF PROTIEN particularly stable arrangements of amino acid residues giving rise to recurring structural patterns – Local spatial arrangement of its main-chain atoms – Confifurational relationship between tesidues which are about 3-4 amino acids apart in linear sequence. Contributed by ◦ each amino acids. ◦ Φ-bond ◦ Ψ-bond ◦ α-helix and β-strand conformations – most thermodynamically stable.
- 22. REGULAR SECONDARY STRUCTURE –Occurs in segments of a polypeptide chain in which – All Φ angles are equal. – All Ψ angles are equal. Helical structures are characterized by ◦ n – number of residues per turn of helix ◦ d – distance between α-carbon atoms of adjacent amino acids ◦ pitch – distance between repeating turn of helix on a line drawn parallel to helix axis p = n × d d p
- 23. Α- HELICAL STRUCTURE ◦Right-handed. ◦ n = 3.6 ◦ Peptide bond plane – parallel to the axis of helix. ◦ Each peptide form 2 hydrogen bonds peptide bond of 4th residue above and below ◦ Optimum geometry and distance for maximum hydrogen bond strength. H-bond
- 24. Β- STRUCTURE – Apolypeptide is hydrogen bonded to another polypeptide chain aligned in a parallel or antiparallel direction. – Hydrogen-bonded β- strands appear like a pleated sheet. Anti-parallel Parallel
- 25. IMPORTANCE OF SECONDARYSTRUCTURE Structure Characteristics Examples of occurrence α-helix, cross-linked by disulfide bonds Tough, insoluble protective structures of varying hardness and flexibility Keratin of hair, feathers, and nails β-Conformation Soft, flexible filaments Silk fibroin
- 26. SUPER-SECONDARY STRUCTURE Motif– recognizable folding pattern involving ◦ two or more elements of secondary structure ◦ connection(s) between them May or may not be independently stable Any advantageous folding pattern not a hierarchical structural element falling between secondary and tertiary structure
- 27. EXAMPLES OF DIFFERENTMOTIFS – Helix-turn-helix – • in many DNA-binding protein. – Strand-turn-strand – • in protein with antiparallel β-structure. – Alternating strand-turn- helix-turn-strand – • in many α/β-proteins Helix Helix turn
- 28. TERTIARY STRUCTURE Locationof each atoms in space. Includes ◦ geometric relationship between distant segments of primary and secondary structure and ◦ Positional relationship of the side chain with one another. ◦ Hydrophobic side chains are generally interior. ◦ Ionized side-chains are on the outside Stabilized by water of solvation.
- 29. • (a) Thepolypeptide backbone in a ribbon. • (b) Surface contour image; this is useful for visualizing pockets – Where other molecule might bind. • (c) Ribbon representation including side chains • (d) Space-filling model with all amino acid side chains. – Each atom depicted as size of its van der Waals radius
- 30. STRUCTURE OF MYOGLOBIN Hemeprosthetic group binds One molecule of oxygen 8 α helices, Rest forms turns and loops due to proline No β sheets
- 31. Structural domain– a compact globular structural unit formed within the polypeptide with Hydrophobic core Hydrophilic surface ◦ Typically contain 100 -150 amino acids ◦ Domains in multi-domain protein Connected by a segments that may lack secondary structure Fold – arrangement of secondary structure elements of a domain.
- 32. IMPORTANCE OF TERTIARYSTRUCTURE Trypsin contain ◦ 2 domain ◦ Cleft in between that contain Substrate-binding catalytic site An active site within an interdomain surface is characteristic of many enzyme In enzyme with more than one substrate or allosteric effector site ◦ Different site may be located in different domain
- 33. CALMODULIN AS EXAMPLE Calcium ion bind in calmodulin ◦ Within the loop of helix-turn-helix motif Called an EF-hand Fold of calmodulin domain ◦ Containing 2 EF-hand motifs ◦ Interconnected by an α-helical segment Addition of side chain group – ◦ Generate complete tertiary structure of domain
- 34. QUATERNARY STRUCTURE Arrangementof polypeptide chain in multi-chain protein. ◦ Subunits in a quaternary structure are associated non- covalently. Quaternary structure of de-oxy hemoglobin. • X-ray diffraction-analysis of de- oxy hemoglobin • (a) A ribbon representation. • (b) A surface contour model. • The α-subunits are shown in shades of gray • The β-subunits in shades of blue. • Heme groups (red) are relatively far apart.
- 35. STRUCTURE OF HEMOGLOBIN • TetramericProtein • 2 α Globin Chains and 2 β globin chains held by non- covalent interactions • Bind 4 molecules of oxygen • Cooperative binding
- 36. UNSTRUCTURED PROTEIN Intrinsicallyunstructured protein – protein with a non-folded conformation Partially unfolded conformation Ex –scaffold proteins, hormones, cyclin-dependant kinase and their inhibitors Functions –by binding to other protein or to DNA and RNA ◦ The property of weak binding often advantageous
- 37. NEWER ADVANCES ProteinComplexes ◦ Protein molecules in the cellular milieu are present in protein complexes containing multiple protein subunits. ◦ The complexes typically have 5-10 proteins. Network – proteins present in 2 or more different complexes can move between complexes to connect them into network.
- 38. • Hub –complex that interconnects with >3 other complexes in the network – Important target for drug therapies. • Interactom – functional network comprising interactive protein complexes Stem cell marker proteins Nanog and Rex1 (green) were used to pull down interacting proteins, including core (blue) and peripheral (red) targets
- 39. CLASSIFICATION OF PROTEIN ACCORDINGTO HIGHER LEVEL OF STRUCTURE• Globular Protein – – Spheroidal shape – Vary in size – Relatively high water solubility. – Function as • catalyst • Non-globular protein– • Low water solubility – Fibrous protein • Larger amount of regular secondary structure • Long cylindrical shape – Membranous protein – Lipoprotein – Glycoprotein
- 40. COLLAGEN ◦ Family ofextracellular proteins present in present in all tissues and organs ◦ Most prominent protein in human. ◦ Provides framework that give tissues form and strength. Amino acid composition ◦ Rich in Glycine Proline 4-hydroxyproline 5-hydroxylysine Amino acid sequence ◦ In all collagen type there are region with tripeptides repeats Gly-Pro-Y Gly-X-Hyp
- 41. COLLAGEN TRIPLE HELIX Tropocollagen consists of three fibers ◦ Three intertwined polypeptide strands twist to the left wrap around one another in a right- handed fashion Highly resistant to unwind ◦ n = 3.3 ◦ stabilized by hydrogen bonds between residues ◦ Additional stability covalent cross-links modified lysyl residues
- 42. Disease Menkes' syndrome Ehlers-Danlos syndrome scurvy etiology dietary deficiency ofthe copper Genetic disease dietary deficiency of vitamin C defect required by lysyl oxidase catalyzes a key step in formatio n of the covalent cross- links ◦ defects in the genes that encode collagen- 1 procollag en N- peptidase lysyl hydroxyla se •prolyl and lysyl hydroxylases •deficit in the number of hydroxyproline and hydroxylysine residues •undermines the conformational stability of collagen fibers Clinical feature kinky hair and growth retardation ◦ mobile joints ◦ skin abnormalitie s bleeding gums swelling joints poor wound healing ultimately death
- 43. ELASTIN Gives tissueand organ capacity to stretch without tearing Abundant in ◦ Ligaments ◦ Lungs ◦ Wall of arteries ◦ Skin Unordered coiled structure ◦ Amino acid residue within folded structure highly mobile ◦ Allysine form cross-links in elastin ◦ Form the heterocyclic structure of desmosine or hemidesmosine
- 44. KERATIN – in whicheach polypeptide is α- helical – Sequences in both proteins shows tandem repetition of 7 residue segments – Super twisted Coiled coil – Rich in hydrophobic residues – Left handed opposite in sense to α helix • Epidermal layer of skin • Nails • Hair
- 45. PROTEIN FOLDING Polypeptidesequence contain information for spontaneous folding Folding is under ◦ thermodynamic ◦ kinetic control Initiated by short-range noncovalent interaction. Partially folded structure intermediate ◦ Interact with each other to form a molten-globule state
- 46. CHAPERON PROTEIN Alsocalled ‘Heat shock protein’ Synthesis increased at high temperature Prevent protein aggregation prior to completion of folding Bind to polypeptides shielding the hydrophobic surface Also required for refolding of protein after they cross cellular membranes
- 47. PRION PROTEIN DISEASES Prion protein ◦ Infectious agent in absence of DNA or RNA ◦ Can occur Spontaneously Inheritance of mutated Prion protein ◦ Clinical features Ataxia Dementia Paralysis Almost always fatal. deposition of insoluble protein aggregates in neural cells include ◦ Creutzfeldt–Jakob disease in humans ◦ scrapie in sheep ◦ bovine spongiform encephalopathy (mad cow disease) in cattle ◦ vCJD younger patients
- 48. PrPc -highly soluble cellular conformation of prion protein ◦ glycoprotein ◦ short arm of chromosome 20 ◦ 3 α-helical ◦ 2 small β-strand ◦ rich in α-helix PrPsc - insoluble toxic conformation ◦ Conversion of α-helix → β-strand ◦ many hydrophobic aminoacyl side chains exposed to solvent ◦ accumulating PrPsc units coalesce insoluble protease-resistant aggregates ◦ serve as template for conformational transformation of PrPc molecules Many times of its number
- 49. BETA-THALASSEMIAS genetic defects ◦impair the synthesis of one of the polypeptide subunits of hemoglobin α-hemoglobin-stabilizing protein (AHSP) ◦ specific chaperone ◦ binds to free hemoglobin - subunits awaiting incorporation into the hemoglobin multimer absence of this chaperone ◦ free α-hemoglobin subunits aggregate ◦ role for AHSP in modulating the severity of -thalassemia in human subjects.
- 50. ALZHEIMER'S DISEASE Refoldingor misfolding ◦ protein endogenous to human brain tissue, β-amyloid Main cause remains elusive characteristic senile plaques and neurofibrillary bundles ◦ aggregates of the protein β-amyloid ◦ 4.3-kDa polypeptide ◦ Proteolytic cleavage of a larger protein amyloid precursor protein ◦ conformational transformation soluble α-helix–rich → rich in β-sheet ◦ prone to self-aggregation amyloid fibers in Alzheimer's - Crystal structure of a segment from the amyloid-beta protein Normally the two protein sheets are tightly associated in the spine of the fiber but in this case orange-G has wedged its way between the two sheets.
- 51. DENATURATION OF PROTEIN •Loss of three-dimensional structure sufficient to cause loss of function – without breakage of any peptide bond – rupture of ionic bond, hydrogen bond and hydrophobic bond. – ↓ solubility – ↑ precipitability – ↑ digestibility • COAGULATION – irreversible denaturation • FLOCCULATION- – Precipitation of proteins at iso-electric pH