Individual globular domains and domain unfolding visualized in overstretched titin molecules with atomic force microscopy.
Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin's globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme's ATP-binding site and hence contributing to the molecule's mechanosensory function.
Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
Zsolt
Z
Mártonfalvi
Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary ; MTA-SE Molecular Biophysics Research Group, Semmelweis University, Budapest, Hungary.
Miklós
M
Kellermayer
D000818:Animals; D064211:Connectin / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D007136:Immunoglobulins / Q000378:metabolism*; D008297:Male; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000378:metabolism; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011817:Rabbits
D064211:Connectin; D005353:Fibronectins; D007136:Immunoglobulins