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Introduction

Titin, (TITN) also known as Connectin (PDB 3b43) is a human protein, unique for its large size. It is the largest known protein chain in the entire human body, comprising over 34,000 amino acids and holding a molecular weight of 3800 kD. Titin is found within muscle fibers and classified as a connectin protein. An adult human that weighs 80 kg may contain almost half a kilogram of Titin, making it extremely abundant as well. ^[1] Due to its abundance and overall importance to human musculature Titin is exceedingly important and vital to understand.

Discovery

The discovery of Titin first began in 1949 as the Australian scientists -- Draper and Hodge -- announced their findings of the first high resolution electron microscope images of striated muscle. ^[2] From this imaging it was theorized that there were three strands that comprise striated muscles, but after a multitude of papers began to be published on the two filament theory, it quickly became widely accepted. It took until 1986 for the advancement of Atomic Force Microscopy to fully differentiate and view the large protein which was measured to be greater than 10^6 Da and longer than 1 micrometer. ^[2] This idea is interesting although Titin had been imaged for more than 30 years before it was officially identified and named. It seemingly was ignored due to the popularity of a two filament system as the Actin and Myosin slide and pull over each other. It is now known that Titin is a structural aid that helps with elasticity, although it technically is the ‘third filament.’

Structure

Titin is the largest human protein, being greater than one micrometer in length and comprising more than 34,000 amino acids. Electron microscopy has revealed that the shape of the protein appeared rod-like and had a beaded substructure. ^[1] As found with most proteins, the structure is fundamental in the function of the protein itself. Being a long rod-like shape aids in the proteins main function of providing elasticity and unidirectional strength in muscle tissue.

Using monoclonal antibodies to map different parts of the protein, the discovery of the PEVK region was uncovered. This PEVK region is located near the I-Band (N-terminus of protein chain) composed mainly of Proline (P), Glutamate (E), Valine (V), and Lysine (K). ^[3] It is theorized that the length of the PEVK region is related to the elasticity as the PEVK region easily stretches. In Skeletal muscle, the PEVK region contains 2174 residues, while cardiac muscle contains a much shorter region; as short as 163 residues. ^[3] Within the PEVK region, there is a pattern of super-repeats containing both Immunoglobulins and Fibronectin Type 3 molecules. The N-terminus found in the I-band only had Immunoglobulins though the C-Terminus has both. ^[3]

Another unique aspect of Titin structure is the ability to uncoil itself aiding in pliability. The way that Titin can physically elongate itself is due to the immunoglobulin domains unfolding and extending. A complete uncoiling can increase peak length up to 29.7 ± 0.4 n. ^[4]This mechanism of extension is a result of disulfide isomerization reactions within the itself. ^[5] To complete these isomerization reactions, a sequence analysis ^[5] showed that up to 21% of Titin’s I-band immunoglobulin domains contained a conserved Cysteine triad enabling the engagement of disulfide isomerization reactions. The ability to unfold itself aids in elasticity, while protein folding will decrease the effective length and increase stiffness.

Titin interacts with other proteins as well, which can seem obvious as it is mainly encountered within muscle fibers which are full of protein. One such protein, (PDB 1ya5) , acts as a glue, holding two neighboring Titin particles together at their Ig domains. ^[6] Telethonin is important to the overall muscle fiber structure of the entire sarcomere, holding TItin molecules together linearly contributes to the parallel structure of striated muscle. The connection of the two Titin molecules are connected in a palindromic arrangement, using a 2:1 assembly of two Titin molecules to one Telethonin. ^[4] It is theorized through molecular dynamics that there is a series of hydrogen bonds that allow for stabilization of the Titin - Telethonin complex. These hydrogen bonds are very strong providing a tight attachment at the two N-terminals which line up nearest each other as previously stated, in a palindromic fashion. ^[4] An extremely high force is required to break these bonds which aids itself to the main function of Titin providing structure and strength to muscle sarcomeres.

Function

One of the main functions of Titin is to provide elasticity to the sarcomeres in our muscles. To understand the importance, it is first vital to have a basic understanding of human muscle structure.

Striated muscle is muscle tissue which is voluntarily used. It is normally attached to bones and deals with large skeletal movement. Striated muscle can also be found in the heart. It is called striated as when it is viewed under a microscope it appears to have a continuous series of stripes. These stripes are actually sarcomeres, which are the basic contractile unit of striated muscle. ^[7] Sarcomeres themselves are composed of smaller filaments of both actin and myosin that act together to create contractions in what is known as the sliding filament theory. These filaments ‘slide’ past each other when our muscles contract giving them elasticity as well as the ability to stretch with our movement. ^[7]

The ability to stretch is greatly impacted by Titin, which allows the sarcomere to increase in length under an applied force and then again shorten and return to the original length post stress. ^[8] The Titin molecule itself spans half of the sarcomere, lining the N-terminus with the Z-Line and C- terminus with the M-line. Closer to the N-terminus is the more elastic part of the protein that aids in stretching and is composed mainly of Immunoglobulin (Ig) domains, while the C-terminus end concerns more with binding to the thick filament comprising both immunoglobulins as well as Fibronectin type-3 (Fn3) domains. ^[8]

Medical Importance

One of the main concerns between Titin and diseases is Dilated Cardiomyopathy (DCM). Cardiomyopathy is characterized by systolic dysfunction, other associated risks include diastolic dysfunction and impaired right ventricular function. In some cases cardiomyopathy can be chronic leading to potential heart failure. ^[9] DCM itself can be genetic but the most prevalent cause is the TTN gene, which codes for Titin ^[10]. In a study performed in 2012 looking at 312 patients with end stage DCM, variants in the TTN gene were found in up to 27% of patients with DCM, which was significantly higher than the controls. These variants were found to yield a ‘truncated protein’ which was discovered as the proteins were transcribed and translated. ^[10]

Titin plays a role in Duchenne muscular dystrophy (DMD) as well. DMD is a fatal disease that is characterized by progressive muscle wasting starting in childhood. DMD is traditionally detected through increased Titin concentration in urine; the N-terminus fragment of Titin is the best-known biomarker. In DMD patients aged 3-29 years old, their urinary Titin concentrations were nearly 700 times higher than in healthy patients. ^[11] DMD mainly affects males, as females can be carriers but are rarely affected.