Fibrous Proteins Fibrous proteins have high α-helix or β -sheet content. Most are structural proteins. Examples include: Collagen Elastin Keratin

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

Fibrous Proteins Collagen and Elastin are the examples of fibrous proteins. These are basic structural elements. These proteins have special mechanical properties. They are found as components of skin, connective tissue, blood vessels, sclera and cornea of eye. Each fibrous protein exhibits special mechanical properties, resulting from its unique structure, which is obtained by combining specific amino acids into regular, secondary structural elements.

Collagen

Collagen Background Collagen is the main component of connective tissue, and is the most abundant protein in mammals, making up about 25% to 35% of the whole-body protein content A typical collagen molecule is a long, rigid structure in which three polypeptides ( called as α chains) are wound around one another in a rope-like triple helix The final cooperative quaternary structure is stabilized by numerous hydrogen bonds. It occurs in connective tissues where tensile strength is needed. Examples: skin, tendons, cartilage, bones.

Tensile strength results from the use of: The triple helix secondary structure The assembly of tropocollagen subunits into a fibre Chemical cross linking to strengthen the fibre

Collagen: A Triple Helix The collagen molecule, also known as the “tropocollagen”, is part of larger collagen aggregates such as fibrils.

Types of Collagen The collagen superfamily of proteins includes more than 25 collagen types, as well as additional proteins that have collagen-like domains The three α chains are held together by hydrogen bonds between the chains Variations in the amino acid sequence of these chains result in structural components that are about the same size (approximately 1000 aa long) but with slightly different properties These α chains are combined to form the various types of collagen found in the tissues. For example, the most common collagen, Type I includes two chains α1 and one chain α2 (α1 2 α2 ) whereas Type II collagen includes three α1 chains (α1 3 )

Types of Collagen The collagens can be organized into three groups, based on their location and functions in the body 1. Fibril Forming Collagens. They have rope like structure 2. Network Forming Collagens. They form a three dimensional mesh, rather than distinct fibrils 3. Fibril-associated Collagens. They bind the surface of collagenfibrils, linking these fibrils to one another and to other components in the extracellular matrix.

Fibril Forming Collagens Types I,II and III are the fibrillar collagens They have the typical rope-like structure described for a collagen molecule Type I collagen fibers are found in supporting elements of high tensile strength is needed. E.g. Tendon, cornea, skin, bone etc Type II collagen molecules are found in cartilageneous tissues like cartilage, intervertebral disk etc. Type III collagen molecules are prevalent in more distensible tissues like blood vessels.

Network Forming Collagens Types IV and VII are the network forming collagens They have a mesh like structure Type IV molecules are an important part of basement membranes Basement membranes are thin, sheet-like structures that provide mechanical support for neighboring cells They function as a semipermeable filtration barrier to macromolecules in organs such as kidney and the lung

Classification of Collagens TYPETISSUE DİSTRIBUTION FIBRIL FORMING Type ISkin, bone, tendon, cornea Type IICartilage, intervertebral disk, vitreous body Type IIIBlood vessels, fetal skin NETWORK FORMING Type IVBasement membrane Type VIIStratified squamous epithelia FIBRIL ASSOCIATED Type IXCartilage Type XIITendon, ligaments, other tissues

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 residues Lect. 6-14

Collagen Amino Acid Composition  Collagen is a glycoprotein containing galactose and glucose as the carbohydrate content.  Glycine is one - third of total amino acid content of collagen followed by hydroxyproline and proline account for another one-third of amino acid content of collagen.  Proline - facilitate the formation of helical conformation of α- chain, because its ring structure causes a kink in the peptide chain.  Glycine- found in every third position of the polypeptide chain. It fits into the restricted spaces where the three chains of the helix come together.

Collagen Amino Acid Composition Glycine is the part of the repeating sequence. Gly- X-Y X- is frequently proline Y- hydroxy proline or hydroxylysine. Lect. 6-16

Collagen Amino Acid Sequence

Triple- helix structure Amino acids side chains are on the surface of the triple helical molecule. This allows bond formation between the exposed R- groups of neighboring collagen monomers- This leads to aggregation into fibrils.

Biosynthesis of hydroxyPro and hydroxyLys requires O 2 and ascorbic acid (vitamin C). Vit. C deficiency leads to disorders in bone, skin and teeth.

In collagen triple helix H-bonds form between separate chains. In α-helix H-bonds formed between residues of the same chain.

22 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

23 Biosynthesis and assembly of collagen 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

24 Biosynthesis and assembly of collagen 7. Formation of triple helical procollagen 8.Exocytosis transfers triple helix to extracellular phase

Biosynthesis and assembly of collagen 10.Removal of N and C terminal propeptides by specific peptidase 11.Lateral association of collagen molecules coupled to covalent cross linking creates fibril

Structural Basis of Collagen Triple Helix Every third residue faces the crowded center of the helix only Gly fits Interchain H-bonds involving HyPro stabilize helix Fibrils are strengthened by intrachain lysine-lysine and interchain hydroxypyridinium cross links

Biosynthesis of Aldol Cross-links in Collagen Lysyl oxidase is a cupper (Cu) containing enzyme and oxidatively deaminates some of the lysyl and hydroxylysyl residues in collagen

Biosynthesis of Aldol Cross-links in Collagen Lysine- and hydroxylysine-derived aldehydes can react with corresponding aldehydes on neighboring polypeptide chains forming aldol condensation products, or with unmodified lysine and hydroxylysine

Biosynthesis of cross links between Lys, His, and hydroxy-Lys residues in collagen.

Degradation of Collagen Normal collagens are highly stable molecules, having half lives as long as several years However, connective tissue is dynamic and constantly being remodeled, often in response to growth or injury of the tissue Breakdown of collagen fibers is dependent on the proteolytic action of collagenases For type I collagen, the cleavage site is specific, generating three- quarter and one-quarter length fragments These fragments are further degraded by other matrix proteinases to their constituent amino acids.

DISORDERS OF COLLAGEN DEPOSITION

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

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

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

Ehlers- Danlos Syndrome -Cutis hyperelastica- Ehlers–Danlos syndrome is a group of inherited connective tissue disorders, caused by a defect in the synthesis of collageninheritedconnective tissuecollagen EDS can result from a deficiency of collagen processing enzymes (e.g. Lysyl hydroxylase or procollagen peptidase) or from mutations in the amino acid sequences of collagen types I, III or V The most clinically important mutations are found in the gene for type III collagen

Ehlers- Danlos Syndrome -Cutis hyperelastica- Because type III collagen is an important component of the arteries, potentially lethal vascular problems occur. Collagen plays a very significant role in the skin, joints, muscles, ligaments, blood vessels and visceral organs; abnormal collagen leads to increased elasticity within these structures.

Osteogenesis Imperfecta This disease, known as “brittle bone syndrome” is a heterogeneous group of inherited disorders distinguished by bones that easily bend and fracture The most common mutations cause the replacement of glycine residues (Gly-X-Y) by amino acids with bulky side chains. The resulting structurally abnormal pro alpha chains prevent the formation of required triple-helical conformation

Osteogenesis Imperfecta Type I. Osteogenesis imperfecta tarda Decreased production of α1 and α2 chains. Fractures secondary to minor trauma Type II. OI congenita Most severe form Patient die due to pulmonary hypoplasia in utero or during the neonatal period

Elastin

Structure of Elastin It is a connective tissue protein Rubber like properties Elastin & glycoprotein microfibrils are present in lungs, walls of large arteries, elastic ligaments. Can be stretched to several times their normal length, but recoil back.

Structure of Elastin Insoluble protein polymer Precursor is Tropoelastin--- it is a linear polypeptide composed of about 700 amino acids – small and non –polar AA. Rich in Proline and Lysine Very little hydroxy proline & hydroxy lysine.

Tropoelastin is secreted by the cells into the extracellular matrix. There it interacts with specific glycoprotein microfibrils – called fibrillin. Fibrillin acts as a scaffold on which tropoelastin is deposited. Elastin

Some of the lysyl side chains of the tropoelastin polypeptides are oxidatively deaminated by lysyl oxidase and forms allysine residues Elastin

Three of the allysine side chains and one lysine residue form a desmosine cross link Desmosine

Desmosine cross-links produces elastin- an extensively interconnected, rubbery network that can strech and bend in any direction when stressed, giving the connective tissue elasticity Elastin

DISORDERS OF ELASTIN

Mutations in fibrillin are responsible for Marfan’s syndrome. Connective tissue supports the tendons, ligaments, blood vessels, cartilage and heart valves in the body. Affects three major organ systems of the body: the heart and circulatory system, the bones and muscles, and the eyes. Marfan Syndrome

Fibrillin is the primary component of the microfibrils that allow tissues to stretch repeatedly without weakening. If fibrillin is abnormal, connective tissues are looser than usual, which weakens or damages the support structures of the entire body. The most common external signs associated with Marfan syndrome include excessively long arms and legs Patients with Osteogenesis imperfecta, Ehlers Danlos and Marfan Syndrome may have blue sclera due to tissue thinning that allows underlying pigment to show through. Marfan Syndrome

Blood and other body fluids contain a protein, α-1 antitrypsin that inhibits a number of proteolytic enzymes that hydrolyze and destroy proteins. α-1 AT comprises more than 90% of the α1 globin fraction of the normal plasma. α-1 AT has the most important physiological role of inhibiting neutrophil elastase- a powerful protease that is released into the extracellular space, and degrades elastin of alveolar walls Role of α-1 antitrypsin in elastin degradation

In the normal lung, the alveoli are chronically exposed to low levels of neutrophil elastase released from activated and degenerating neutrophils This proteolytic activity can destroy the elastin in alveolar walls if is not protected by the activity of α-1 AT Because lung tissue cannot regenerate, emphysema results from the destruction of the connective tissue of alveolar walls Role of α-1 antitrypsin in elastin degradation

α-Keratin

Keratin Fibrous (structural) proteins. Individual molecules combine to form insoluble structures. Keratins are the molecular basis for hair, nails, wool, feathers, claws and horns. Keratin is also found in the cytoskeleton of cells. Keratin clusters are found on chromosomes 12 and 17.

 - Keratin  -Keratin is found in hair, nails, outer layer of skin. It forms almost the entire dry weight of these materials. 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 Cysteine residues.

Structure of dimer of two  - helices.

Proposed structure for  - keratin intermediate filaments Two monomers (a) pair 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

 - Keratin Structure

Lect Disulphide bridges and toughness in  -keratin Two Cys residues form disulphide bridges in  - keratin, and link the  - helices together. The more disulphides, the stronger the  - keratin. Cys

Psoriasis - a keratin over production disorder- In psoriasis, an activated immune system triggers the skin to reproduce every three to four days, building up on the outer layers (epidermis and keratin). The epidermis thickens, blood flow increases and reddens the skin, and silver-gray scales cover it.

Psoriasis - a keratin over production disorder-

Perming and Relaxing Process in Hair When the hair is permed (and sometimes when straightened) the disulfide bonds of the hair are broken through ‘reduction’. A reduction reaction involves either the removal of oxygen or the addition of hydrogen. In the case of permanent waving, the reduction is due to the addition of hydrogen.

Perming and Relaxing Process in Hair The disulfide bonds join one sulfur atom on one polypeptide chain to another sulfur atom on different polypeptide chain. Perms use reducing agents called thiol compounds, which break the disulfide bonds by adding a hydrogen atom to each of the sulfur atoms in the disulfide bonds. With the disulfide bonds broken, the polypeptide chains are able to slip into their new shape.

Perming and Relaxing Process in Hair The broken disulfide bonds are reformed through the neutralization process The most common neutralizer is hydrogen peroxide and the chemical process that removes the hydrogen atoms and reforms the disulfide bonds is called “oxidation”. The removal of the hydrogen atoms from the sulfur atoms forces them to reform their disulfide bonds in the new shape (around the perm rods).

Perming and Relaxing Process in Hair The process is the same for relaxers and straighteners that use thio compounds, except that these are removing curl rather than creating it.

Fibroin Fibroin, is the fibrous protein that makes up silk cloth and spider webs. Made with a  -sheet structures with Gly on one face and Ala/Ser on the other The high glycine (and, to a lesser extent, alanine) content allows for tight packing of the sheets, which contributes to silk's rigid structure that can't be stretched.