Mechanical Unfolding of a Titin Ig Domain: Structure of Transition State Revealed by Combining Atomic Force Microscopy, Protein Engineering and Molecular Dynamics Simulations

• Published in: Journal of molecular biology Vol. 330; no. 4; pp. 867 - 877
• Main Authors: Best, Robert B., Fowler, Susan B., Toca Herrera, José L., Steward, Annette, Paci, Emanuele, Clarke, Jane
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 18.07.2003
• Subjects: AFM; Computer Simulation; Connectin; immunoglobulin; Immunoglobulins - chemistry; Microscopy, Atomic Force; Models, Molecular; muscle; Muscle Proteins - chemistry; Muscles - metabolism; Mutation; Protein Denaturation; Protein Folding; Protein Kinases - chemistry; Protein Structure, Tertiary; Proteins - chemistry; Thermodynamics; titin; titin; MD, molecular dynamics; immunoglobulin; Ig, immunoglobulin; TI I27, the 27th Ig-like domain from the I band of human cardiac titin; muscle; AFM; protein folding; AFM, atomic force microscopy; f and ‡ o, the transition states investigated by force and denaturant unfolding, respectively
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/S0022-2836(03)00618-1

Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering Φ-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations in the core do not affect the force required to unfold the protein. As has been shown before, mechanical strength lies in the region of the A′ and G-strands but, contrary to previous suggestions, the results indicate clearly that side-chain interactions play a significant role in maintaining mechanical stability. Since Φ-values calculated from molecular dynamics simulations are the same as those determined experimentally, we can, with confidence, use the molecular dynamics simulations to analyse the structure of the transition state in detail, and are able to show loss of interactions between the A′ and G-strands with associated A–B and E–F loops in the transition state. The key event is not a simple case of loss of hydrogen bonding interactions between the A′ and G-strands alone. Comparison with Φ-values from traditional folding studies shows differences between the force and “no-force” transition states but, nevertheless, the region important for kinetic stability is the same in both cases. This explains the correspondence between hierarchy of kinetic stability (measured in stopped-flow denaturant studies) and mechanical strength in these titin domains.