Lect. 6-2 Fibrous vs. Globular Proteins 1. Compact protein structureExtended protein structure 2. Soluble in water (or in lipid Insoluble in water (or in lipid bilayers) bilayers) 3. Secondary structure is complex Secondary structure is simple with a mixture of -helix, -sheet based on one type only and loop structures 4. Quaternary structure is held Quaternary structure is usually together by noncovalent forcesheld together by covalent bridges 5. 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) Globular Fibrous
Lect. 6-3 Fibrous Proteins Fibrous proteins have high -helix or -sheet content. Most are structural proteins. Examples include: Collagen Elastin Keratin Fibroin
Lect. 6-4 Fibrous Proteins Much or most of the polypeptide chain is parallel to a single axis Fibrous proteins are often mechanically strong & highly cross-linked Fibrous proteins are usually insoluble Usually play a structural role
Lect. 6-5 COLLAGEN What makes collagen a strong tensile protein?
Lect. 6-6 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?
Lect. 6-7 Collagen Background The 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: (a)The triple helix secondary structure (b)The assembly of tropocollagen subunits into a fibre (c)Chemical cross linking to strengthen the fibre
Lect. 6-8 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 -helix is right-handed). (2) 3.3 residues per turn (3) Each chain forms hydrogen bonds with the other two: STRENGTH!
Lect. 6-9 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 = 285,000 300 nm long, 1.4 nm diameter Unique amino acid composition
Lect. 6-10 Collagen Amino Acid Composition Nearly one residue out of three is Gly Proline content is unusually high Many modified amino acids present: –4-hydroxyproline –3-hydroxyproline –5-hydroxylysine Pro and HyPro together make 30% of res.
Lect. 6-11 Collagen Amino Acid Sequence AA sequence of C-terminal region of bovine type-I collagen
Lect. 6-12 Hydroxylated residues found in collagen
Lect. 6-13 Biosynthesis of hydroxyPro and hydroxyLys requires O 2 and ascorbic acid (vitamin C). Vit. C deficiency leads to disorders in bone, skin and teeth.
Lect. 6-14 The Collagen Triple Helix The unusual amino acid composition of collagen is not favorable for helices OR - sheets But it is ideally suited for the collagen triple helix: three intertwined helical strands Much more extended than helix, with a rise per residue of 2.9 Angstroms 3.3 residues per turn Long stretches of Gly-Pro-Pro-HyPro
Lect. 6-15 In collagen triple helix H- bonds form between separate chains. In -helix H-bonds formed between residues of the same chain.
Lect. 6-16 Collagen Fibers Fibers are formed by staggered arrays of tropocollagens Banding pattern in EMs with 68 nm repeat Since 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
Lect. 6-17 Electron micrographs of colagen fibers showing band pattern
Lect. 6-18 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 1.Synthesis on ribosome. Entry of chains into lumen of endoplasmic reticulum occurs with the first processing reaction removing signal peptide 2.Collagen precursor with N and C terminal extensions 3.Hydroxylation of selected protein and lysines
Lect. 6-20 Biosynthesis and assembly of collagen (Con’t) 4.Addition of Asn-linked oligosaccharides to collagen 5.Initial glycosylation of hydroxylyine residues 6.Alignment of three polypeptide chains and formation of inter- chain disulfide bridges 7.Formation of triple helical procollagen 8.Transfer by endocytosis to transport vesicle 9.Exocytosis transfers triple helix to extracellular phase 10.Removal of N and C terminal propeptides by specific peptidase 11.Lateral association of collagen molecules coupled to covalent cross linking creates fibril
Lect. 6-21 Structural Basis of Collagen Triple Helix Every third residue faces the crowded center of the helix only Gly fits Pro and HyPro suit the constraints of phi and psi Interchain H-bonds involving HyPro stabilize helix Fibrils are strengthened by intrachain lysine- lysine and interchain hydroxypyridinium cross links
Lect. 6-22 Biosynthesis of Aldol Cross-links in Collagen
Lect. 6-23 Biosynthesis of cross links between Lys, His, and hydroxy- Lys residues in collagen.
Lect. 6-24 The Major Collagen Groups In humans at least there are 19 different collagens. Within these 19 structural types four major classes are generally identified.
Lect. 6-25 Classification of Collagens TypeChainsTissue FoundCharacteristic s Ia1(I)2, a2(I)Bone, skin, tendons Low carbohydrate; <10%Hydrox ylysines per chain IIa1(II)3Cartilage, vitreous 10% carbohydrate; >20 hydroxylysine s per chain IIIa1(III)3Blood vessels, scar tissue, uterine wall
Lect. 6-26 Classification Continued IV[a1(IV)3 a2(IV)3] Basement membrane lens capsule High carbohydrate, >40 hydroxylysines per chain V[a1(V)2a2(V)] [a1(V)3] [a1(V)a2(V)a3( V)] Cell surface and exoskeleton High carbohydrate, high glycine and hydroxylysine VIAortic intima, placenta, kidney Low mol.weight, equal amounts of hydroxylysine and hydroxyproline
Lect. 6-27 Thermal Denaturation Curve In normal collagens the transition midpoint temperature or Tm is related to the normal body temperature of the organism and for animal is above 40 o C as shown in blue line in the graph.. Tm Triple helix stabilization is through HyPro and formation of H bonds with neighboring chains.
Lect. 6-29 Disorders of Collagen Deposition Disorders of collagen deposition –insufficient collagen content –presence of chemically and/or morphologically abnormal collagen –excessive collagen content –insufficient collagen resorption –excessive collagen resorption
Lect. 6-30 Disorders of Collagen Deposition Genetic abnormalities of collagen –mutations that lead to aminoacid deletions or additions –deficient synthesis of a portion –disorders in post-translational modification (hydroxylation of lysine, hydroxylation of proline) –defects in enzymes essential for post-translational modification
Lect. 6-31 Disorders of Collagen Deposition Collagen is the building block; thus, its disorders lead to significant deterioration in the mechanical integrity of tissues Several disorders –Ehlers-Danlos syndrome –Osteogenesis Imperfecta –Marfan syndrome
Lect. 6-32 ELASTIN Three factors make it stretchy and elastic
Lect. 6-33 Elastin Elastin can stretch several times - then return to the original starting size Elastin 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
Lect. 6-34 Desmosine is formed from 4 lysines, 3 of which are oxidised. Desmosine
Lect. 6-35 (2) Amino acid composition of elastin 33% Gly 10% Pro and Hyp 23% Ala 13% Val Hence 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. Elastin (3) Secondary structure of elastin A 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 -spiral.
Lect. 6-36 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.
Lect. 6-37 Elastin Cross-links formed from allyysine (aldehyde derivative of Lys) Extracellular Lys oxidase specific for Lys-Ala-Ala-Lys and Lys-(Ala) 3 -Lys sequences Lys + 3 allysine combine to from desmosine or isodesmosine cross-links responsible for yellow color of elastin Also forms lysinorleucine cross-links from 2 allysine, as in collagen. Cross-links responsible for elasticity & insolubility
Lect. 6-38 Lysine amino oxidase Aldol condensations Desmosine cross-link Biosynthesis of desmosine and isodesmosine cross-links unique to elastin
Lect. 6-39 KERATIN -Keratins are found in mammals -Keratins are found as a left-handed super helix -Keratins are found in birds and reptiles -Keratins are analogs to the silk fibroin structures produced by spiders and silkworms
Lect. 6-40 -KERATIN Two reasons why this is a tough protective fibrous protein
Lect. 6-41 -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 -helices. It is rich in amino acids that favours -helix formation (Phe, Ile, Val, Met, Ala) These hydrophobic side chains are on the -helix surface-explaining its insolubility. It is also rich in Cys residues.
Lect. 6-42 Structure of dimer of two -helices.
Lect. 6-43 Proposed structure for -keratin intermediate filaments Two monomers (a) pair via a parallel coiled-coil to form 50- nm-long dimer (b) These then associate to form 1 st protofilament (c) These then associate to form protofibril (d) Regular spacing of 25 nm along the fibers is accounted for by overlap
Lect. 6-44 Disulphide bridges and toughness in -keratin Disulphide bridges are also frequently used to stabilise the interior of a globular protein. (2) Cys residues form disulphide bridges in -keratin, and link the -helices together. The more disulphides, the stronger the - keratin. Cys
Lect. 6-45 Quaternary structure -keratin of The association of long parallel a-helices also gives toughness to -keratin. UP-TO- DATE MODEL Proto- filament of anti- parallel dimers The up-to-date view is that TWO parallel -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 -keratin is such a tough protein. Clinical relevance in skin diseases: psioriasis – the overproduction of -keratin The incorrect explanation of -keratin structure states that THREE -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!
Lect. 6-47 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 other Fibroins 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
Lect. 6-48 Structure of silk fibroin (a)Three dimension view of the stacked -sheets (b) Interdigitation of Al or Ser and Gly side chain The plane of the section is perpendicular to the folded sheets