Monitoring Unfolding of Titin I27 Single and Bi Domain with High-Pressure NMR Spectroscopy

Abstract

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the folding energy landscape. Simulations, when corroborated by experimental data yielding global information on the folding process, can provide this level of insight. Molecular dynamics (MD) has often been combined with force spectroscopy experiments to decipher the unfolding mechanism of titin immunoglobulin-like single or multidomain, the giant multimodular protein from sarcomeres, yielding information on the sequential events during titin unfolding under stretching. Here, we used high-pressure NMR to monitor the unfolding of titin I27 Ig-like single domain and tandem. Because this method brings residue-specific information on the folding process, it can provide quasiatomic details on this process without the help of MD simulations. Globally, the results of our high-pressure analysis are in agreement with previous results obtained by the combination of experimental measurements and MD simulation and/or protein engineering, although the intermediate folding state caused by the early detachment of the AB β-sheet, often reported in previous works based on MD or force spectroscopy, cannot be detected. On the other hand, the A'G parallel β-sheet of the β-sandwich has been confirmed as the Achilles heel of the three-dimensional scaffold: its disruption yields complete unfolding with very similar characteristics (free energy, unfolding volume, kinetics rate constants) for the two constructs.

Folding of the Ig-Like Domain of the Dengue Virus Envelope Protein Analyzed by High-Hydrostatic-Pressure NMR at a Residue-Level Resolution

“…From the analysis of the residue-specific denaturation curves, the first event in DEN4-ED3 unfolding was found to be the rupture of the junction between the N-and C-terminal ß-strands 1 and 9, on one edge of the ß-sandwich. Interestingly, this behavior is rather similar to what has been recently shown for the I27 domain of the multimodular giant protein Titin from the striated muscle cell, which also adopts an Ig-like fold, but with a significantly different sequence (sequence identity <7%) [27]. In Titin I27, the N-and C-termini are involved in ß-sheet structures: a small, distorted antiparallel (AB) ß-sheet, and a longer parallel (A'G) ß-sheet (where the capital letters stand for the ß-strands numbering, following the usual convention for Ig-like modules).…”

“…Besides, our P-jump experiments yield a value of k f 0 = 1.14 (± 0.08) × 10 −3 s −1 for the rate constant for folding of DEN4-ED3, more than one order of magnitude lower than the value previously measured for Titin I27 (k f 0 = 2.24 (± 0.11) × 10 −2 s −1 ) [27]. This seems to corroborate the observation made by Clarke et al [32], who found a positive linear correlation between the logarithm of the rate constant for folding and the stability of Ig-like protein.…”

“…DEN4-ED3 presents the characteristic core organization described by Clarke et al [32] for Ig-like domains; the buried hydrophobic residues borne by strands 2 (B, in the usual Ig-fold convention) and 8 (F) are closely entangled and form the "spinal column" of the core, whereas hydrophobic residues from strands 4, 7, and 9 (C,E, and G) pack onto this central scaffold (Figure 8). Even if a similar hydrophobic core has been reported for Titin I27 [27], closer inspection reveals some differences: strand C contributes also to the spinal column of the hydrophobic core, with the indole ring of Trp34 being inserted between the buried hydrophobic residues from strands B and F. The remaining hydrophobic residues from strands E and G make a dense network of hydrophobic interactions around this compact core, mainly organized around Trp34 (Figure 8). It is tempting to speculate that the more concerted unfolding transition for Titin I27 is a consequence of the more integrated packing patterns of its core.…”

“…In the present study, we used high-pressure NMR spectroscopy to monitor the unfolding of ED3 from dengue-4 virus (DEN4-ED3) and shed insight into the structural details of its folding pathway. In particular, we found that the folding pathway of DEN4-ED3 shares similarities with that of the I27 module from the giant multimodular protein Titin found in the striated muscle cells [27], which also adopts an Ig-like fold. In both proteins, unfolding starts with the disruption of the N-and C-terminal strands on the edge of the ß-sandwich, but the Transition State Ensemble (TSE) of DEN4-ED3 was found to be significantly more hydrated, and hence less structured, than that of Titin I27.…”

“…Nevertheless, the NMR measurement of global folding kinetic parameters remains possible. For this, we recorded series of 1D proton NMR spectra after P-jumps [25,27]. The proton resonance at 0.96 ppm, corresponding to unfolded-state methyl proton resonances, was used as a probe to measure the global relaxation times τ ( Figure 7A).…”