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Home , Alpha helix, Collagen, Collagen helix, Fibroin

Paper 4. and their interactions MODULE 6: of -, fibrin and

OBJECTIVES To learn about the and assembly of fibrous .

To learn structural details of α-keratin, silk fibrin and collagen.

Introduction

Fibrous proteins are also known as . These form one branch of the three main types of the proteins present in . Many super families of proteins, including keratin and collagen fall under this class of proteins. Fibrous proteins particularly form long filaments, generally shaped like rods and wires. They are typically water-insoluble and inert proteins. They serve the cell as structural or storage proteins. Some of the functions include formation of connective tissues, muscle etc. Due to the hydrophobic side chains that protrude from the surface of the molecule, fibrous proteins form aggregates. When compared to globular proteins, scleroproteins are resistant to denaturation.

Fibrous proteins generally contain few amino acids that are often present as repeat sequences. They can form unusual secondary structures, for example: collagen . Keratin helix on the other hand displays cys-cys disulphide cross-linking between chains.

Fibrous proteins are usually divided into three different groups

• Coiled-coil -helices present in keratin and

• The in collagen

• Beta-sheets in amyloid and

Coiled alpha helices can be seen in wool fibers giving elasticity and flexibility to it, whereas collagen fibers are relatively rigid and strong. Beta-sheet fibers are both strong and flexible. A classic example to quote would be that of web, some of which are even stronger than the same dimensional steel and highly flexible at the same time. Fibrous beta-sheet structures can be seen in various diseases like Alzheimers and disease as a result of misfolding, where the protein forms aggregate like structures, which are resistant to protease digestion.

α-KERATIN

Keratin is a fibrous structural protein that protects epithelial cells from potentially lethal damage or stress. Hence, it makes up the outer layer of the human . Keratin is also the key

component of nails and hair. Tongue and the hard palate get the necessary strength for food mastication due to presence of keratin.

Keratin monomers assemble in to bundles to form intermediate filaments that are tough and strong. These filaments make up the most of hard tissues found in reptiles, birds, amphibians, and also . In general keratin filaments are abundant in keratinocytes and they are also found in epithelial cells.

α- are found in hair, hoofs, claws and horns of mammals, whereas β-keratins can be seen in nails and scales of reptiles and feathers, beaks and claws of birds. β-keratins are primarily composed of beta sheets and α-keratins mostly contain  – helices.

Hanukoglu and Fuchs determined the first sequences of keratins revealing two distinct but homologous keratin families namely Type I and Type II. From the sequence, it was proposed the presence of central 310 amino acids as 4 helices separated by 3 beta linker regions.

Keratins or form type III and IV intermediate filaments that are found in chordates only.

Keratin monomers left-handedly supercoiled in order to form a very stable super helical or coil- coiled structure. Along the length of the filament there are hydrophobic interactions between the non-polar amino acids and thus maintains the coiled-coil structure. For the structural details of coil-coiled structures, see the module super-secondary structures in module 9. Permanent rigidity is achieved by thermally stable cross-linking between residues present in the keratin filaments. This extensive disulphide bonding makes keratin insoluble.

Keratins in hair are more flexible Monomer of Keratin molecule has about 310 central amino than those present in nails and acids, flanked by two globular domains. These individual hoofs due to presence of less molecules become dimer as coiled-coils. These dimers inter-chain disulphide bonds in assemble to form tetramer and octamer. The bundle of eight the former. Hair is composed of chains forms a or protofilamet. dead cells, each cell packed with (Figure adopted from Wikipedia) keratin macrofibrils. α-keratins are α- helically coiled single protein strands that contain regularly arranged intra-chain bonds. Keratin monomers form long intermediate filaments

beginning with the dimerisation, continue to oligomerise till the octamers are available for end- to-end joining to yield a long filamentous form. This can be viewed in the accompanying slides.

By the process of cornification, stratified squamous epithelial cells form the skin barrier. Cells undergo programmed cell death and become fully keratinized.

Clinical manifestations

Since keratin is the building block of hair and nails, its disorders lead to significant deterioration in the mechanical integrity of these tissues.

The disease and disorders could be a result of either gene . Some of the disorders are:

Monilethrix (also referred to as beaded hair) is a rare autosomal dominant hair disease that results in short, fragile, broken hair.

Epidermolytic hyperkeratosis, (also known as Bullous congenital) is a rare skin disease affecting around 1 in 250,000 people. It is caused due to in Keratin 1 gene.

Large cell carcinoma with rhabdoid phenotype is a rare histological form of lung cancer, occurring mainly due to missense mutation in -8 gene.

Keratin is also helpful in elucidating the epithelial origin of anaplastic cancers. Keratin, if ingested is highly resistant to digestive acids present in the gut. This could be the result of trichophagia, is the compulsive eating of hair. In some people trichophagia could also lead to a potentially fatal Rapunzel syndrome in humans, where small or large bowel obstruction by hair.

COLLAGEN

It is the main structural protein present in the extracellular of many connective tissues, thus making it the most abundant protein in bodies. It is commonly found in , and skin as elongated . Many other areas in the body also contain abundant amounts of keratin like , , etc. Collagen fibers in bones are arranged at an angle to each other to give strength from all directions. Collagen constitutes one to two percent of muscle , and accounts for 6% of the weight of strong, tendinous muscles. The is the most common cell that creates collagen.

The food industry puts collagen to its use in an irreversibly hydrolysed form of . In the field of medicine, collagen is used for treatment of skin and complications.

Tensile strength of collagen results due to

(a) The triple helix secondary structure

(b) The assembly of tropocollagen subunits into a fibre

(c) Chemical cross linking to strengthen the fibre

(d) Collagen is formed from tropocollagen subunits.

(e) The triple helix in tropocollagen is highly extended and strong.

The residues present in collagen are different from the others found in rest of the proteins like . The common motifs found in collagen are --X and glycine-X-hydroxyproline (X could be any amino acid other than glycine, proline or hydroxylproline). This shows presence of unusually high proline content nearly 17% of collagen. Another atypical amino acid that is seen is .

Types of collagen: Collagen is classified in to many groups on the basis how their structures are formed. Variations in the amino acid sequences of protein chains (generally called as  chains) in collagen lead to slightly different properties, but they generally have same size.

 Fibrillar (Type I, II, III, V, XI)  Non-fibrillar (Type IX, XII, XIV, XVI, XIX, VIII, X, IV, XV, XVIII, XIII, XVII, VI, VII)

Collagen formation

Collagen proteins longer precursors about 1000 amino acids, called pro- with globular extensions about 200 amino acids at both ends, that contain signal sequences. They are transported to rough , where they are hydroxylated and then assembled into triple helix. Then the signal are cleaved off releasing procollagen. processes procollagen and forms tropocollagen.

Three polypeptide strands, each has the conformation of a left-handed helix (not be confused with the right-handed ) to form collagen fiber. These three left- handed helices are twisted together into a right-handed triple helix or "super helix", a cooperative quaternary structure stabilized by many hydrogen bonds. Each collagen fiber has several micrometers long and 50 to Structure of Collagen with (Gly-Pro- 200 nanometers in diameter. The extended left hand Hyp)3 as sequence. (The figure is helix has 3.3 residues / turn with rise per each turn of adopted from Bhattacharjee and Bansal, 9.6 Å; rise per each amino acid is 2.9 Å. The triple IUBMB life, 2005.) helix has a repeat axis of 100 Å. Triple helical structure of collagen has one inter-chain per tripeptide.

In collagen triple helix H-bonds form between separate chains. In alpha-helix H-bonds are formed within the same chain. In this triple helix, every 3rd amino acid is close to the central axis, where there is no space and only Glycine can accommodate there. Any other residue deforms the triple helix. Hydrogen bonds are formed between NH groups of Glycine with CO groups of Proline of different chains.

The adjoining slides depict the structural representation of collagen molecule. Hydroxy Pro or Pro containing triple helix of collagen structures are almost same. The combination of these amino acids (Gly-Pro-hydroxy Pro) prefers this conformation. In the triple helix structure hydroxyl Pro is always comes on the surface of the structure. Water mediated hydrogen bonds between inter and intra chain triple helices will differ and thus stability of triple helices must be different. There are different post- modifications and cross links in collagen which includes formation of hydroxyproline and hydroxylysine.

Clinical manifestations:

As the collagen is building block thus any mutation or disorders lead to significant loss in mechanical strength of tissue. The synthesis pathogenesis includes C deficiency called as during which formation is impaired due to defective collagen. Autoimmune diseases like Systemic lupus erythrematosus or rheumatoid arthiritis may attack collagen fibres.

Ehlers-Danlos syndrome – It includes muscle, skin and junction disorders.

Osteogenesis Imperfecta (brittle bone disease or Lobstein syndrome: Usually cause because of deficiency of ), can also cause short height, hearing loss etc.

SILK FIBRIN

Fibrin is an insoluble protein that is found in all types of silk. The source of silk could be , Bombyx mori and many moth families. Silk contains two proteins, and .

Fibroin protein is composed of anti-parallel beta sheets. The amino acid sequence is mainly a repeat of Gly-Ser-Gly-Ala-Gly-Ala sequence. Presence of high Glycine helps in tighter packing of the silk fibers, gives silk its enormous tensile strength and rigidity. Fibroin protein can opt three structural conformations thus yielding Silk I, II and III.

Silk I: This form of silk is naturally formed in the glands of infamous silkmoth larvae Bombyx mori.

Silk II: The fibroin arranges itself into silk II conformation in the case of spun silk. It has greater strength and is used commercially as well.

Silk III: This is relatively new type of fiber found at fibroin solution present at various phase interfaces such as water-oil, air-water etc.

Spiders generate seven distinct fibers by drawing liquid crystalline proteins from a set of separate gland called as spinneret complexes. The silk fibroin is produced inside these glands. The proteins are stored in highly concentrated (~50%) solution in the glands and are mostly in alpha- helical conformation. This solution is passed through spinning machinery and mixed with other components; producing a variety of different fibers and beta-sheet structures. The process of spinning converts the silk proteins from soluble -helical protein to fibrous form containing - sheet structures. Spiders can produce different types of silk with different strength. Dragline fiber is used by the spider to hide and has remarkable mechanical properties. Dragline fiber is stronger than steel but can bend, contract and stretch. Silk fibroin genes code for similar sequence pattern and with N and C- terminal variable domains. Most of the central part has large repetition of – 8 residues of poly and Gly-Gly- X (X can be Ser, Thr or Gln). Bulky regions with and interrupt the -sheet and allow the stretchiness. This middle region varies in length in different genes; it can be up to 800 amino acid long.

Spider silk fibrin arrangement:

Spider silk is formed mainly by large silk fibroin polypeptide chains. Some of the parts are formed into micro-crystal structure and other regions form the less ordered mesh like structure, where crystals are embedded. The crystal part gives strength and mesh like structure gives flexibility. This can be clearly viewed in the The silk fibrin is made up of several silk fibrin accompanying slides which gives a three polypeptide chains. Some parts form dimensional overview of the fiber. microcrystalline structures and other parts form studies indicated that mesh like structure. The crystal form is made-up some proteins in spider silk fibers adopt by stacking of beta-sheets and also contains beta-sheet structure, similar to the . (Figure adopted from Wikipedia) common silk. Fibers contain small micro-crystals of ordered regions inserted in the matrix of less ordered regions as mentioned above (from EM & NMR studies). Understanding and manipulating structural details of these regions might result in fascinating new materials. The micro-crystals comprise about 30% of the protein in the fibers arranged as beta-sheets and contain trace amounts of Calcium and are about 70 to 100 nanometers in size. Prof. Lynn Jelinski, Cornell University, established that poly-Ala repeats are involved in micro-crystals. They fed spiders with C13 labeled Ala and studied spider chains by NMR.

SUMMARY

Fibrous proteins are structural proteins that could be made up of helical or extended structures. Alpha-Keratins are coiled-coil proteins, consisting of alpha-helices. These are structural proteins,

building blocks for hair and nails. Coiled-coil structures are cross linked through disulfide bridges. Higher the cross-linking results in more rigid structures.

Collagen is made up of triple helix structure. Three proteins form left-handed helices and are twisted together into a right-handed triple helix or "super helix", a cooperative quaternary structure stabilized by many hydrogen bonds. Almost every third amino acid is Glycine, which comes in the center of triple helix enabling the tighter packing. Collagen is a building block for skin, bone and junctions. Any mutation in collagen genes causes several diseases. There are different post-translation modifications and cross links in collagen fibers, which gives strength to fibers.

Silk fibrins are mostly structures. The beta sheet structures form micro crystalline structures which also contains calcium , mostly build by poly-Ala sequences. Other regions form less ordered mesh like structure, where crystals are embedded. The crystal part gives strength and mesh like structure gives flexibility.