Paper 4. Biomolecules and their interactions MODULE 6: Structures of -keratin, silk fibrin and collagen OBJECTIVES To learn about the structure and assembly of fibrous proteins. To learn structural details of α-keratin, silk fibrin and collagen. Introduction Fibrous proteins are also known as Scleroproteins. These form one branch of the three main types of the proteins present in nature. 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 fiber 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 helix. 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 myosin • The triple helix in collagen • Beta-sheets in amyloid fibers and silks 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 spider 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 prion disease as a result of protein 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 skin. 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 mammals. In general keratin filaments are abundant in keratinocytes and they are also found in epithelial cells. α-keratins 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 turn linker regions. Keratins or cytokeratins 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 cysteine 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 intermediate filament 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 hydrogen 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 mutations. 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 mutation in Keratin 1 gene. Large cell lung carcinoma with rhabdoid phenotype is a rare histological form of lung cancer, occurring mainly due to missense mutation in cytokeratin-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 matrix of many connective tissues, thus making it the most abundant protein in animal bodies. It is commonly found in tendons, ligaments and skin as elongated fibrils. Many other areas in the body also contain abundant amounts of keratin like corneas, bones, cartilage 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 tissue, and accounts for 6% of the weight of strong, tendinous muscles. The fibroblast is the most common cell that creates collagen. The food industry puts collagen to its use in an irreversibly hydrolysed form of gelatin. In the field of medicine, collagen is used for treatment of skin and bone 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 amino acid residues present in collagen are different from the others found in rest of the proteins like hydroxyproline. The common motifs found in collagen are glycine-proline-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 hydroxylysine. 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-collagens with globular extensions about 200 amino acids at both ends, that contain signal sequences. They are transported to rough endoplasmic reticulum, where they are hydroxylated and then assembled into triple helix. Then the signal peptides are cleaved off releasing procollagen. Procollagen peptidase processes procollagen and forms tropocollagen. Three polypeptide strands, each has the conformation of a left-handed helix (not be confused with the right-handed alpha helix) 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 hydrogen bond 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-translation modifications and cross links in collagen which includes formation of hydroxyproline and hydroxylysine.