The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy.
The domains of the giant muscle protein titin (connectin) provide interaction sites for other sarcomeric proteins and fulfill mechanical functions. In this paper we compare the unfolding forces of defined regions of different titin isoforms by single-molecule force spectroscopy. Constructs comprising six to eight immunoglobulin (Ig) domains located in the mechanically active I-band part of titin are compared to those containing fibronectin III (Fn3) and Ig domains from the A-band part. The high spatial resolution of the atomic force microscope allows us to detect differences in length as low as a few amino acids. Thus constructs of different lengths may be used as molecular rulers for structural comparisons with other modular proteins. The unfolding forces range between 150 and 300 pN and differ systematically between the constructs. Fn3 domains in titin exhibit 20% lower unfolding forces than Ig domains. Fn3 domains from tenascin, however, unfold at forces only half those of titin Fn3 domains. This indicates that the tightly folded titin domains are designed to maintain their structural integrity, even under the influence of stretching forces. Hence, at physiological forces, unfolding is unlikely unless the forces are applied for a long time (longer than minutes).
Rief
M
M
Lehrstuhl für Angewandte Physik, Ludwig Maximilians Universität M unchen, 80799 München, Germany.
Gautel
M
M
Schemmel
A
A
Gaub
H E
HE
D001696:Biomechanical Phenomena; D055592:Biophysical Phenomena; D001703:Biophysics; D064211:Connectin; D004355:Drug Stability; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics; D066298:In Vitro Techniques; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D009010:Monte Carlo Method; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D064211:Connectin; D005353:Fibronectins; D007136:Immunoglobulins; D009124:Muscle Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C577285:TTN protein, human; D011494:Protein Kinases