Titin , also known as connectin, is a protein that in humans is encoded by the TTNgene. Titin is a giant protein, greater than 1 µm in length, that functions as a molecular spring which is responsible for the passive elasticity of muscle . It comprises 244 individually folded protein domains connected by unstructured peptide sequences. These domains unfold when the protein is stretched and refold when the tension is removed.
Titin is important in the contraction of striated muscle tissues. It connects the Z line to the M line in the sarcomere. The protein contributes to force transmission at the Z line and resting tension in the I band region. 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 muscle (e.g., cardiac or skeletal) have been correlated with differences in the mechanical properties of these muscles.
Titin is the third most abundant protein in muscle (after myosin and actin), and an adult human contains approximately 0.5 kg of titin. With its length of ~27,000 to ~35,000 amino acids (depending on the splice isoform), titin is the largest known protein. Furthermore, the gene for titin contains the largest number of exons (363) discovered in any single gene, as well as the longest single exon (17,106 bp).
Reiji Natori in 1954 was the first to propose an elastic structure in muscle fiber to account for the return to the resting state when muscles are stretched and then released. In 1977, Koscak Maruyama and coworkers isolated an elastic protein from muscle fiber which they called connectin. Two years later, Kuan Wang and coworkers identified a doublet band on electrophoresis gel corresponding to a high molecular weight, elastic protein which they named titin.
Siegfried Labeit in 1990 isolated a partial cDNA clone of titin. In 1995, Labeit and Bernhard Kolmerer determined the cDNA sequence of human cardiac titin. Labeit and colleagues in 2001 determined the complete sequence of the human titin gene.
The human gene encoding for titin is located on the long arm of chromosome 2 and contains 363 exons, which together code for 38,138 residues (4200 kDa). Within the gene are found a large number of PEVK (proline-glutamate-valine-lysine -abundant structural motifs) exons 84 to 99 nucleotides in length which code for conserved 28- to 33-residue motifs which 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.
A number of titin isoforms are produced in different striated muscle tissues as a result of alternative splicing. 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 Ig domains. a) Schematic of part of a sarcomere b) Structure of Ig domains c) Topology of Ig domains.
The titin protein is located between the myosin thick filament and the Z disk. Titin consists primarily of a linear array of two types of modules, also referred to as protein domains (244 copies in total): type I fibronectin type III domain (132 copies) and type II immunoglobulin domain (112 copies). 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 a PEVK region which is rich in proline (P), glutamate (E), valine (V) and lysine (K).
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.
The C-terminal region also contains a serine kinase domain that is primarily known for adapting the muscle to mechanical strain. It is "stretch-sensitive" and helps repair overstretching of the sarcomere. The N-terminal (the Z-disc end) contains a "Z repeat" that recognizes Actinin alpha 2.
Protein domains of Titin A-band. White boxes are Fn3 domains, red boxes are Ig domains, yellow boxes are Fn3 domains with the -AVNKYG- sequence, and the black box is protein kinase domain.
The titin domains have evolved from a common ancestor through many gene duplication events. 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 include obscurin and myomesin. Throughout evolution, titin mechanical strength appears to decrease through the loss of disulfide bonds as the organism becomes heavier.
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. 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.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.
Drosophila Titin, also known as Kettin or sallimus (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. There also exists a titin homologue, ttn-1, in C. elegans. It has a kinase domain, some Ig/Fn3 repeats, and PEVT repeats that are similarly elastic.
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. An N-terminal Z-disc region and a C-terminal M-line region bind to the Z-line and M-line of the sarcomere, 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 for chromosomes. 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.
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