Structural Proteins

Characteristics of Structural Proteins:

  • Long, filamentous.

  • Generally insoluble.

  • Contains unusual amino acids.

  • Often contain cross-linked polypeptide chains.

Cellular:

  • Filamentous muscle protein.

  • Cytoskeletal proteins.

  • Keratin - epithelial cells.

Extracellular:

  • Collagen - connective tissue.

  • Elastin - yellow connective tissue (blood vessels).

  • Resilin - insect connective tissue.

  • Fibroin - silkworm silk.

  • Spidroin - spider silk.

Intracellular Structural Proteins:

The cytoskeleton is composed of 3 types of structural protein:

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Three Types of Cytoskeleton:

The overall function of the cytoskeleton is: endocytosis, cell division, intracellular transport, motility, reaction to external forces etc.

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Actin Structure:

Two molecular types:

  • Globular (G-actin) - monomeric.

  • Filamentous (F-actin) - polymeric.

Filament formation:

  • G-actin + ATP - dimers or trimers (nucleation).

  • Elongation by monomer addition (treadmilling).

    • + = barbed end.

    • - = pointed end.

Example of Actin Polymerisation and Regulation by ARP.

Binding of WCA (Wiskott–Aldrich syndrome protein (WASP)-homology-2, central, acidic) domain promotes a conformational change that primes the complex for activation, which occurs upon binding of the WCA–actin–ARP2/3 assembly to the mother filament, preferentially near the barbed end.

  • WCA domain presents an ATP–actin monomer to the complex and/or possibly to the barbed end of the mother filament.)

    • ATP is hydrolysed on ARP2 concomitant with or shortly after nucleation of the daughter filament.

      • The WCA dissociates, although the trigger for this is unknown.

        • Phosphate is released from ARP2. Mother and daughter filaments elongate and age by ATP hydrolysis and phosphate release.

          • Phosphate release from ARP2 and filament ageing weaken the interactions between ARP2/3 and the daughter and/or mother filament allowing branch disassembly and release of the ARP2/3 complex, presumably in an inactive, ADP-bound conformation.

            • Nucleotide exchange on ARP2 (and possibly on ARP3) occurs and the cycle begins again.

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Microtubules:

Microtubules consist of α and β-tubulin monomers, and heterodimers.

Elongation:

  • Addition of heterodimers, forming a GTP-cap at the (+)-end, protecting microtubules from shrinkage.

Shrinkage:

  • If the (+)-end loses its GTP-cap, it induces microtubule shrinkage.

A single microtubule contains 10 to 15 protofilaments that wind together to form a 24nm wide hollow cylinder.

They are fueled by GTP hydrolysis.

Roles:

  • Major components of the cytoskeleton.

  • Found in all eukaryotic cells.

  • Involved in mitosis, cell motility, intracellular transport and maintenance of cell shape.

  • Microtubules tend to grow out from the centrosome to the plasma membrane.

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Intermediate Filaments:

The intermediate filament (IF) supergene family are ubiquitous structural components that comprise the cell type-specific cytoskeleton of animal tissues.

  • All IF proteins show an organised, extended α-helical conformation prone to form two-stranded coiled coils.

    • Highly flexible, stress-resistant cytoskeletal filaments.

  • IF proteins are highly charged.

Keratin is the most common IF protein.

  • If has α-helices and β-pleated sheets.

  • α-keratins are intermediate filament proteins playing an important role in nuclei, cytoplasm, and cell surfaces.

  • E.g. in wool, they are mostly made of:

    • Glu/Gln - contribute to the hydrophilic properties.

    • Cys - essential for disulfide bond formation.

    • Ser - contains -OH groups making it hydrophilic.

α-keratin contains a coiled-coil (distorted right handed α-helix), a double α-helical coiled-coil with left handed supercoiling (dimer), a protofilament, protofibril, and an IF.

  • They contain cysteine bridges to stabilise the structure through covalent interactions.

  • They have a heptad structure.

    • I.e. repeats of seven amino acids in positions labelled a-g. a-d are hydrophobic interactions, e-g are ionic H interactions.

    • a and d are often Leu, Ile, Ala.

    • e and g are often Glu and Gln.

  • Electrostatic interactions and salt bridges can form to enhance stability.

β-keratin is harder than its alpha counterpart.

  • Its’ monomer has hydrophobic interactions between 4β-strands, producing anti-parallel β-sheets.

Extracellular Matrix (ECM) Structural Proteins:

Collagen:

  • Makes up ~25-35% of all protein in the body.

  • Major component of connective tissues such as skin, tendons, cartilage, ligaments and bones.

  • Has a generalised amino acid composition:

    • 33% Gly

    • 11% Ala

    • 10% Pro

    • 5% Hydroxyproline (Hyp)

    • 0.6% Hydroxylysine (Hyl)

    • 0% Trp

Standard Structure of Collagen:

Molecular Structure of Collagen:

  • Glycine residues line the interior of the triple helix.

    • A lack of sidechains allows for tighter packing.

  • Bulkier side chains position on the outer faces of the helices.

  • Type I

    • Tendons, ligaments, bones.

    • Contains two α1 chains and one α2 chain.

  • Type II

    • Cartilage.

    • Contains three α1 chains (all three are identical).

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FACIT and Related Collagen Structures:

Fibril-associated collagens with interrupted triple helices (FACIT) and related collagens have a different structure to standard fibrillar collagen; they contain non-collagenous regions—that is, non-triple helical sequences.

  • These lead to kinks in the resulting macromolecular structure that straighten under small strains.

  • They associated with standard collagen, forming a higher-order structure.

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Post-Translational Modifications:

Collagens are glycoproteins that are modified post-translationally.

  • Hydroxylation of Pro and Lys.

  • Glycosylation.

They are exported to the ECM (~1000 amino acids).

  • Deamination of Lys to aldehydes takes place, as well as cross-linking*.

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*Cross-Linking of Collagen:

Collagens cross-link extensively post-translation (type I).

  • Lys is oxidised to ε-aldehyde (an Allysine intermediate), which is catalysed by LOX.

  • Condensation reactions take place, generating covalent cross-bridging between allysine:

    • Allysine;

    • Lysine (10)

    • Products: stable covalent bonds between LNL, DHLNL and HLNL.

  • Covalent cross-linking continues with time, which makes collagen more brittle with age.

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Higher-Order Collagen Structures:

  • Triple stranded (3 separate chains) intertwined with a right-handed helical structure.

  • Staggered molecules.

  • Characteristic covalent cross-linking.

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Collagen Defects:

Mutations in the collagen genes (~30) lead to severe disorders:

  • Osteogenesis imperfecta.

  • Osteoporosis.

  • Ehlers-Danlos syndrome.

  • Familial aortic aneurism.

Mutations at Gly-Xaa-Yaa lead to collagen misfolding, increased Lys hydroxylation, improper processing of protofilaments by enzymes and chaperones, and weakening of the collagen protofilaments.

General Structural Similarities between Silk and Spider Silk:

  • Relatively weak Van der Waals forces between sheets allowing for high flexibility.

  • The Gly-linked interface lacks large side chains and presents little resistance to bending.

  • Fibroin and spidroin also contain repeat polyalanine motifs.

    • These form crystalline domains which are held together by sericins.

      • This gives the fibre the strength of composites, and the flexibility of polymers.

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Fibroin Compositions:

  • Fibrous protein secreted by insects and arachnids.

  • Amino acid composition of:

    • 45% Gly, 30% Ala, 12% Ser, 5% Tyr.

  • Anti-parallel β-sheet.

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Fibroin Structure:

  • Sheets, stabilised by hydrogen bonds, are stacked with side chains perpendicular to the structural plane.

  • Ala and Gly side chains are separated on opposite sides of the sheets.

  • Extended covalent structures allow little elongation and provide high tensive strength.

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