Presentation on theme: "BCH 443 Biochemistry of Specialized Tissues"— Presentation transcript:
1 BCH 443 Biochemistry of Specialized Tissues 2. Fibrous Proteins
2 Fibrous vs. Globular Proteins Globular Fibrous1. Compact protein structure Extended protein structure2. Soluble in water (or in lipid Insoluble in water (or in lipid bilayers) bilayers)3. Secondary structure is complex Secondary structure is simplewith a mixture of a-helix, b-sheet based on one type onlyand loop structures4. Quaternary structure is held Quaternary structure is usually together by noncovalent forces held together by covalent bridges5. Functions in all aspects of Functions in structure of the body metabolism (enzymes, transport, or cell (tendons, bones, muscle, immune protection, hormones, etc). ligaments, hair, skin)
3 Fibrous ProteinsFibrous proteins have high a-helix or b-sheet content. Most are structural proteins.Examples include:CollagenElastinKeratinFibroin
4 Fibrous ProteinsMuch or most of the polypeptide chain is parallel to a single axisFibrous proteins are often mechanically strong & highly cross-linkedFibrous proteins are usually insolubleUsually play a structural role
6 Questions?1. How would you define the structure of a collagen molecule?2. What are the dimensions of a collagen molecule?3. What are the dimensions of a collagen fibril?4. State the most important amino acids in collagen and explain their importance.5. What is the periodicity of collagen? Why does it happen?
7 Collagen BackgroundThe collagens are the most abundant proteins in the body.They occur in connective tissues where tensile strength is needed.Examples: skin, tendons, cartilage, bones.Tensile strength results from the use of:The triple helix secondary structureThe assembly of tropocollagen subunits into a fibreChemical cross linking to strengthen the fibre
8 Secondary structure - the triple helix Collagen is formed from tropocollagen subunits. The triple helix in tropocollagen is highly extended and strong.Features:(1) Three separate polypeptide chains arranged as a left-handed helix (note that an a-helix is right-handed).(2) 3.3 residues per turn(3) Each chain forms hydrogen bonds with the other two: STRENGTH!
9 Collagen A Triple Helix Principal component of connective tissue (tendons, cartilage, bones, teeth)Basic unit is tropocollagen:Three intertwined polypeptide chains (1000 amino acid residues each)MW = 285,000300 nm long, 1.4 nm diameterUnique amino acid composition
10 Collagen Amino Acid Composition Nearly one residue out of three is GlyProline content is unusually highMany modified amino acids present:4-hydroxyproline3-hydroxyproline5-hydroxylysinePro and HyPro together make 30% of res.
11 Collagen Amino Acid Sequence AA sequence of C-terminal region of bovine type-I collagen
13 Biosynthesis of hydroxyPro and hydroxyLys requires O2 and ascorbic acid (vitamin C). Vit. C deficiency leads to disorders in bone, skin and teeth.
14 The Collagen Triple Helix The unusual amino acid composition of collagen is not favorable for a-helices OR b-sheetsBut it is ideally suited for the collagen triple helix: three intertwined helical strandsMuch more extended than a-helix, with a rise per residue of 2.9 Angstroms3.3 residues per turnLong stretches of Gly-Pro-Pro-HyPro
15 In collagen triple helix H-bonds form between separate chains In collagen triple helix H-bonds form between separate chains. In a-helix H-bonds formed between residues of the same chain.
16 Collagen FibersFibers are formed by staggered arrays of tropocollagensBanding pattern in EMs with 68 nm repeatSince tropocollagens are 300 nm long, there must be 40 nm gaps between adjacent tropocollagens (5 x 68 = 340 Angstroms)40 nm gaps are called "hole regions" - they contain carbohydrate and are thought to be nucleation sites for bone formation
17 Electron micrographs of colagen fibers showing band pattern
18 Structure of collagen fibers (a) and (b) the primary and secondary structure(c) lower magnification emphasizes the triple-helix(d) tropocollagen molecules align side by side to form collagen fiber
19 Biosynthesis and assembly of collagen Synthesis on ribosome. Entry of chains into lumen of endoplasmic reticulum occurs with the first processing reaction removing signal peptideCollagen precursor with N and C terminal extensionsHydroxylation of selected protein and lysines
20 Biosynthesis and assembly of collagen (Con’t) Addition of Asn-linked oligosaccharides to collagenInitial glycosylation of hydroxylyine residuesAlignment of three polypeptide chains and formation of inter-chain disulfide bridgesFormation of triple helical procollagenTransfer by endocytosis to transport vesicleExocytosis transfers triple helix to extracellular phaseRemoval of N and C terminal propeptides by specific peptidaseLateral association of collagen molecules coupled to covalent cross linking creates fibril
21 Structural Basis of Collagen Triple Helix Every third residue faces the crowded center of the helix only Gly fitsPro and HyPro suit the constraints of phi and psiInterchain H-bonds involving HyPro stabilize helixFibrils are strengthened by intrachain lysine-lysine and interchain hydroxypyridinium cross links
23 Lecture 9: Fibrous Proteins Biosynthesis of cross links between Lys, His, and hydroxy-Lys residues in collagen.
24 Lecture 9: Fibrous Proteins The Major Collagen GroupsIn humans at least there are 19 different collagens. Within these 19 structural types four major classes are generally identified.
25 Classification of Collagens Lecture 9: Fibrous ProteinsClassification of CollagensTypeChainsTissue FoundCharacteristicsIa1(I)2, a2(I)Bone, skin, tendonsLow carbohydrate; <10%Hydroxylysines per chainIIa1(II)3Cartilage, vitreous10% carbohydrate; >20 hydroxylysines per chainIIIa1(III)3Blood vessels, scar tissue, uterine wall
26 Classification Continued Lecture 9: Fibrous ProteinsClassification ContinuedIV[a1(IV)3a2(IV)3]Basement membranelens capsuleHigh carbohydrate,>40 hydroxylysines per chainV[a1(V)2a2(V)][a1(V)3][a1(V)a2(V)a3(V)]Cell surface and exoskeletonHigh carbohydrate, high glycine and hydroxylysineVIAortic intima, placenta, kidneyLow mol.weight, equal amounts of hydroxylysine and hydroxyproline
27 Lecture 9: Fibrous Proteins Thermal Denaturation CurveIn normal collagens the transition midpoint temperature or Tm is related to the normal body temperature of the organism and for animal is above 40 oC as shown in blue line in the graph..TmTriple helix stabilization is through HyPro and formation of H bonds with neighboring chains.
29 Disorders of Collagen Deposition insufficient collagen contentpresence of chemically and/or morphologically abnormal collagenexcessive collagen contentinsufficient collagen resorptionexcessive collagen resorption
30 Disorders of Collagen Deposition Genetic abnormalities of collagenmutations that lead to aminoacid deletions or additionsdeficient synthesis of a portiondisorders in post-translational modification (hydroxylation of lysine, hydroxylation of proline)defects in enzymes essential for post-translational modification
31 Disorders of Collagen Deposition Collagen is the building block; thus, its disorders lead to significant deterioration in the mechanical integrity of tissuesSeveral disordersEhlers-Danlos syndromeOsteogenesis ImperfectaMarfan syndrome
32 Three factors make it stretchy and elastic ELASTINThree factors make it stretchy and elastic
33 ElastinElastin can stretch several times - then return to the original starting sizeElastin is found in large arteries (the aorta), ligaments, and the lung wall.(1) The subunits of elastin are called tropoelastin – molecules 1, 2, 3 and 4.The crosslinking of tropoelastin via lysine residues results in a stable starting network of elastin (i.e. when not stretched).Either desmosine (4 Lys) links 4 molecules of tropoelastin, or lysinonorleucine (2 Lys) links 2 tropoelastin molecules.It is clinically relevant in cardiovascular disease and lung emphysema
34 DesmosineDesmosine is formed from 4 lysines, 3 of which are oxidised.
35 Elastin (2) Amino acid composition of elastin 33% Gly 10% Pro and Hyp 23% Ala 13% ValHence 79% of the residues come from 4 amino acids.There are large hydrophobic peptides rich in Ala, Val, Ile and Leu.As these sidechains do not interact with each other by hydrogen bonds, they enable the core of elastin to separate and stretch easily.(3) Secondary structure of elastinA different type of helix structure from those in the a-helix is present. This is able to stretch and relax like a coiled spring. So elastin is elastic!This is constructed from a helix of repeated b-turns based on the sequence Val.Pro.Gly.Val, and is called the b-spiral.
36 Elastin Abundant in ligaments, lungs, artery walls, skin. Provides tissues with ability to stretch in all directions without tearing.Contains predominantly small hydrophobic residues: 1/3 Gly, 1/3 Ala + Val, many Pro but no hydroxyPro or hydroxyLys.Lacks regular secondary structure.Has unordered coil structure that is highly cross-linked into 3-dimensional network of fibers to provide rubber-like elasticity.
37 Elastin Cross-links formed from allyysine (aldehyde derivative of Lys) Extracellular Lys oxidase specific for Lys-Ala-Ala-Lys and Lys-(Ala)3-Lys sequencesLys + 3 allysine combine to from desmosine or isodesmosine cross-links responsible for yellow color of elastinAlso forms lysinorleucine cross-links from 2 allysine, as in collagen.Cross-links responsible for elasticity & insolubility
38 Lysine aminooxidaseAldol condensationsDesmosinecross-linkBiosynthesis of desmosine and isodesmosine cross-links unique to elastin
39 KERATIN a-Keratins are found in mammals a-Keratins are found as a left-handed super helixb-Keratins are found in birds and reptilesb-Keratins are analogs to the silk fibroin structures produced by spiders and silkworms
40 Two reasons why this is a tough protective fibrous protein a-KERATINTwo reasons why this is a tough protective fibrous protein
41 a-Keratina-keratin is found in hair, nails, outer layer of skin. It forms almost the entire dry weight of these materials.(1) The entire secondary structure is a dimer of two a-helices.It is rich in amino acids that favours a-helix formation (Phe, Ile, Val, Met, Ala)These hydrophobic side chains are on the a-helix surface-explaining its insolubility.It is also rich in Cys residues.
43 Proposed structure for a-keratin intermediate filaments Two monomers (a) pair via a parallel coiled-coil to form 50- nm-long dimer (b)These then associate to form 1st protofilament (c)These then associate to form protofibril (d)Regular spacing of 25 nm along the fibers is accounted for by overlap
44 Disulphide bridges and toughness in a-keratin (2) Cys residues form disulphide bridges in a-keratin, and link the a-helices together.The more disulphides, the stronger the a-keratin.CysDisulphide bridges are also frequently used to stabilise the interior of a globular protein.Cys
45 Quaternary structure a-keratin of The association of long parallel a-helices also gives toughness to a-keratin.The incorrect explanation of a-keratin structure states that THREE a-helices supercoil around each other to form a protofibril, and that the association of 2 and 9 protofibrils forms a hair microfibril.Lippincott’s Fig 3.31 on page 45 is wrong!UP-TO-DATE MODELProto-filament of anti-parallel dimersThe up-to-date view is that TWO parallel a-helices supercoil around each other to form a dimer.Then each dimer associates antiparallel with two other dimers to form the protofibril.The association of four protofibrils forms a four-stranded rope. These successive overlaps explain why a-keratin is such a tough protein.Clinical relevance in skin diseases: psioriasis – the overproduction of a-keratin
47 Fibroin Fibroins are the silk proteins. They also form the spider webs Made with a -sheet structures (M6.12) with Gly on one face and Ala/Ser on the otherFibroins contain repeats of [Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Gly-(Ser-Gly-Ala-Gly-Ala-Gly)8]The -sheet structures stack on top of each other (M6.12b)Bulky regions with valine and tyrosine interrupt the -sheet and allow the stretchiness
48 Structure of silk fibroin (a)Three dimension view of the stacked b-sheets(b) Interdigitation of Al or Ser and Gly side chainThe plane of the section is perpendicular to the folded sheets