Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering.
The mechanical unfolding of an immunoglobulin domain from the human muscle protein titin (TI I27) has been shown to proceed via a metastable intermediate in which the A-strand is detached. The structure and properties of this intermediate are characterised in this study. A conservative destabilising mutation in the A-strand has no effect on the unfolding force, nor the dependence of the unfolding force on the pulling speed, indicating that the unfolding forces measured in an AFM experiment are those required for the unfolding of the intermediate and not the native state. A mutant of TI I27 with the A-strand deleted (TI I27-A) is studied by NMR and standard biophysical techniques, combined with protein engineering. Molecular dynamics simulations show TI I27-A to be a good model for the intermediate. It has a structure very similar to the native state, and is surprisingly stable. Comparison with a Phi-value analysis of the unfolding pathway clearly shows that the protein unfolds by a different pathway under an applied force than on addition of denaturant.
Fowler
Susan B
SB
University of Cambridge, Department of Chemistry, MRC Centre for Protein Engineering, UK.
Best
Robert B
RB
Toca Herrera
José L
JL
Rutherford
Trevor J
TJ
Steward
Annette
A
Paci
Emanuele
E
Karplus
Martin
M
Clarke
Jane
J
D003198:Computer Simulation; D064211:Connectin; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry* / Q000235:genetics; D018625:Microscopy, Atomic Force / Q000379:methods; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary
D064211:Connectin; D007136:Immunoglobulins; D009124:Muscle Proteins; C577285:TTN protein, human; D011494:Protein Kinases