Cardiac sarcomere structure, featuring titinReconstruction of the thin (green) and thick filament from mammalian cardiac tissue. Myosin is in blue, MyBP-C is in yellow, and titin is in two shades of red (dark red for titin-alpha and light red for titin-beta).
Titin[5] (/ˈtaɪtɪn/; also calledconnectin) is aprotein that in humans is encoded by theTTNgene.[6][7] The protein, which is over 1μm in length,[8] functions as a molecularspring that is responsible for the passive elasticity ofmuscle. It comprises 244 individually foldedprotein domains connected by unstructuredpeptide sequences.[9] These domainsunfold when the protein is stretched andrefold when the tension is removed.[10]
Titin is important in the contraction ofstriated muscle tissues. It connects theZ disc to theM line in thesarcomere. The protein contributes to force transmission at the Z disc and resting tension in theI band region.[11] It limits the range of motion of the sarcomere in tension, thus contributing to the passive stiffness of muscle. Variations in the sequence of titin between different types of striated muscle (cardiac orskeletal) have been correlated with differences in the mechanical properties of these muscles.[6][12]
Titin is the third most abundant protein in muscle (aftermyosin andactin), and an adult human contains approximately 0.5 kg of titin.[13] With its length of ~27,000 to ~35,000amino acids (depending on thesplice isoform), titin is the largest knownprotein.[14] Furthermore, the gene for titin contains the largest number ofexons (363) discovered in any single gene,[15] as well as the longest single exon (17,106bp).
In 1954, Reiji Natori proposed the existence of an elastic structure in muscle fiber to account for the return to the resting state when muscles are stretched and then released.[16] In 1977, Koscak Maruyama and coworkers isolated an elastic protein from muscle fiber that they called connectin.[17] Two years later,Kuan Wang and coworkers identified a doublet band onelectrophoresis gel corresponding to a high molecular weight, elastic protein that they named titin.[5][18]
In 1990, Siegfried Labeit isolated a partialcDNA clone of titin.[7] Five years later, Labeit and Bernhard Kolmerer determined the cDNA sequence of human cardiac titin.[9] In 2001, Labeit and colleagues determined the complete sequence of the human titin gene.[15][19]
The human gene encoding for titin is located on the long arm of chromosome 2 and contains 363 exons, which together code for 38,138amino acidresidues (4200 kDa).[15] Within the gene are found a large number of PEVK (proline-glutamate-valine-lysine -abundantstructural motifs) exons 84 to 99 nucleotides in length, which code for conserved 28- to 33-residue motifs that may represent structural units of the titin PEVK spring. The number of PEVK motifs in the titin gene appears to have increased during evolution, apparently modifying the genomic region responsible for titin's spring properties.[20]
A number of titinisoforms are produced in different striated muscle tissues as a result ofalternative splicing.[21] All but one of these isoforms are in the range of ~27,000 to ~36,000 amino acid residues in length. The exception is the small cardiac novex-3 isoform, which is only 5,604 amino acid residues in length. The following table lists the known titin isoforms:
Titin is the largest known protein; its human variant consists of 34,350amino acids, with themolecular mass of the mature "canonical" isoform of the protein being approximately 3,816,030.05Da.[22] Its mouse homologue is even larger, comprising 35,213 amino acids with a molecular weight of 3,906,487.6Da.[23] It has a theoreticalisoelectric point of 6.02, and itschemical formula is C169,719H270,374N45,688O52,238S911.[22] It has a theoreticalinstability index (II) of 42.38, classifying the protein as unstable.[22] The protein'sin vivohalf-life, the time it takes for half of the amount of protein in a cell to break down after its synthesis in the cell, is predicted to be approximately 30 hours (inmammalianreticulocytes).[21]
Titin Ig domains. a) Schematic of part of a sarcomere b) Structure of Ig domains c) Topology of Ig domains.[24]
The Titin protein is located between themyosin thick filament and the Z disk.[25] Titin consists primarily of a linear array of two types of modules, also referred to asprotein domains (244 copies in total): type Ifibronectin type III domain (132 copies) and type IIimmunoglobulin domain (112 copies).[13][9] However, the exact number of these domains is different in different species. This linear array is further organized into two regions:
N-terminal I-band: acts as the elastic part of the molecule and is composed mainly of type II modules. More specifically the I-band contains two regions of tandem type II immunoglobulin domains on either side of aPEVK region that is rich inproline (P),glutamate (E),valine (V) andlysine (K).[25]
C-terminal A-band: is thought to act as a protein-ruler and is composed of alternating type I (Fn3) and II (Ig) modules with super-repeat segments. These have been shown to align to the 43 nm axial repeats of myosin thick filaments with immunoglobulin domains correlating to myosin crowns.[26]
The C-terminal region also contains a serinekinase domain[27][28] that is primarily known for adapting the muscle to mechanical strain.[29] It is “stretch-sensitive” and helps repair overstretching of the sarcomere.[30] The N-terminal (the Z-disc end) contains a "Z repeat" that recognizesActinin alpha 2.[31]
The elasticity of the PEVK region has bothentropic andenthalpic contributions and is characterized by a polymerpersistence length and astretch modulus.[32] At low to moderate extensions PEVK elasticity can be modeled with a standardworm-like chain (WLC) model ofentropic elasticity. At high extensions PEVK stretching can be modeled with a modified WLC model that incorporates enthalpic elasticity. The difference between low-and high- stretch elasticity is due to electrostatic stiffening andhydrophobic effects.
Embedded between the PEVK and Ig residues are N2A domains.[33]
The titin domains have evolved from a common ancestor through many gene duplication events.[34] Domain duplication was facilitated by the fact that most domains are encoded by single exons. Other giant sarcomeric proteins made out of Fn3/Ig repeats includeobscurin andmyomesin. Throughout evolution, titin mechanical strength appears to decrease through the loss of disulfide bonds as the organism becomes heavier.[35]
Titin A-band has homologs in invertebrates, such as twitchin (unc-22) and projectin, which also contain Ig and FNIII repeats and a protein kinase domain.[30] The gene duplication events took place independently but were from the same ancestral Ig and FNIII domains. It is said that the protein titin was the first to diverge out of the family.[28]Drosophila projectin, officially known as bent (bt), is associated with lethality by failing to escape the egg in some mutations as well as dominant changes in wing angles.[36][37][38]
Drosophila Titin, also known as Kettin orsallimus (sls), is kinase-free. It has roles in the elasticity of both muscle and chromosomes. It is homologous to vertebrate titin I-band and contains Ig PEVK domains, the many repeats being a hot target for splicing.[39] There also exists a titin homologue,ttn-1, inC. elegans.[40] It has a kinase domain, some Ig/Fn3 repeats, and PEVT repeats that are similarly elastic.[41]
Sliding filament model of muscle contraction. (Titin labeled at upper right.)
Titin is a large abundant protein of striated muscle. Titin's primary functions are to stabilize the thick filament, center it between the thin filaments, prevent overstretching of the sarcomere, and to recoil the sarcomere like a spring after it is stretched.[42] An N-terminal Z-disc region and a C-terminal M-line region bind to the Z-line and M-line of thesarcomere, respectively, so that a single titin molecule spans half the length of a sarcomere. Titin also contains binding sites for muscle-associated proteins so it serves as an adhesion template for the assembly of contractile machinery in muscle cells. It has also been identified as a structural protein forchromosomes.[43][44] Considerable variability exists in the I-band, the M-line and the Z-disc regions of titin. Variability in the I-band region contributes to the differences in elasticity of different titin isoforms and, therefore, to the differences in elasticity of different muscle types. Of the many titin variants identified, five are described with complete transcript information available.[6][7]
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