We use single molecule techniques to study the mechanical design of the giant muscle protein titin. Titin spans half the length of a muscle sarcomere and can be over a micrometer long. The region of titin that overlaps with the sarcomeric I band determines muscle elasticity. I band titin has a characteristic modular design composed of tandem repeats of immunoglobulin (Ig) type domains, interrupted by a region rich in P, E, V and K residues and, in the case of cardiac muscle, another region made of a unique sequence named N2B. Our long-term aim is to understand the molecular mechanisms underlying titin elasticity in normal and diseased states. Detailed single molecule studies of a few modules of the constitutively expressed regions of titin have revealed a complex structure-dependent mechanical design. However, titin elasticity is finely regulated through alternative splicing of its I band by a stunning number of exons, 106 exons coding for Ig modules and 114 exons coding for PEVK sequences. Nothing is known of the mechanical motifs encoded by the alternatively spliced regions of titin or how they govern titin elasticity. Also, a large number of titin Ig domains have been shown to have a potential for the formation of mechanically stabilizing disulfide bridges. Moreover, titin molecules have been shown to interact suggesting that each elastic filament may be composed of several titin molecules, which may form elastic quaternary structures. Hence, titin elasticity might be finely modulated by several novel mechanisms. We combine single molecule AFM and protein engineering techniques to study the individual building blocks of titin mechanics. We will examine the mechanical features of the different Ig modules and the PEVK sequences in the alternatively spliced regions of titin. We will examine the role of disulfide bridge formation on the mechanical properties of Ig domains. We will examine the mechanical stability of other protein folds such as the helical coiled-coil topology of the muscle protein utrophin and the alpha-beta topology of the highly conserved protein ubiquitin. We will study the origin of their mechanical design and compare it to that of the titin immunoglobulin modules. Through the use of protein engineering and mutagenesis, we will examine the role played by prolines (as well as E, V and K residues) in controlling the elasticity of the PEVK regions. Using a variety of oligomerization domains we will assemble bundles of titin based polyproteins to examine the effect of supra-molecular arrangements on titin mechanics. We expect that in addition to uncovering new mechanisms of regulating titin elasticity, our studies will contribute to further develop the new field of research on single molecule force spectroscopy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL061228-06
Application #
6571358
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Evans, Frank
Project Start
1999-07-01
Project End
2008-05-31
Budget Start
2003-06-01
Budget End
2004-05-31
Support Year
6
Fiscal Year
2003
Total Cost
$447,991
Indirect Cost
Name
Columbia University (N.Y.)
Department
Biology
Type
Other Domestic Higher Education
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027
Rivas-Pardo, Jaime Andrés; Badilla, Carmen L; Tapia-Rojo, Rafael et al. (2018) Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins. Proc Natl Acad Sci U S A 115:9222-9227
Eckels, Edward C; Tapia-Rojo, Rafael; Rivas-Pardo, Jamie Andrés et al. (2018) The Work of Titin Protein Folding as a Major Driver in Muscle Contraction. Annu Rev Physiol 80:327-351
Giganti, David; Yan, Kevin; Badilla, Carmen L et al. (2018) Disulfide isomerization reactions in titin immunoglobulin domains enable a mode of protein elasticity. Nat Commun 9:185
Echelman, Daniel J; Lee, Alex Q; Fernández, Julio M (2017) Mechanical forces regulate the reactivity of a thioester bond in a bacterial adhesin. J Biol Chem 292:8988-8997
Valle-Orero, Jessica; Rivas-Pardo, Jaime Andrés; Tapia-Rojo, Rafael et al. (2017) Mechanical Deformation Accelerates Protein Ageing. Angew Chem Int Ed Engl 56:9741-9746
Haldar, Shubhasis; Tapia-Rojo, Rafael; Eckels, Edward C et al. (2017) Trigger factor chaperone acts as a mechanical foldase. Nat Commun 8:668
Valle-Orero, Jessica; Tapia-Rojo, Rafael; Eckels, Edward C et al. (2017) Proteins Breaking Bad: A Free Energy Perspective. J Phys Chem Lett 8:3642-3647
Valle-Orero, Jessica; Rivas-Pardo, Jaime Andrés; Popa, Ionel (2017) Multidomain proteins under force. Nanotechnology 28:174003
Popa, Ionel; Rivas-Pardo, Jaime Andrés; Eckels, Edward C et al. (2016) A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics. J Am Chem Soc 138:10546-53
Rivas-Pardo, Jaime Andrés; Eckels, Edward C; Popa, Ionel et al. (2016) Work Done by Titin Protein Folding Assists Muscle Contraction. Cell Rep 14:1339-1347

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