Dissecting the Dynamic Conformations of the Metamorphic Protein Lymphotactin

Abstract

A mass spectrometer provides an ideal laboratory to probe the structure and stability of isolated protein ions. Interrogation of each discrete mass/charge-separated species enables the determination of the intrinsic stability of a protein fold, gaining snapshots of unfolding pathways. In solution, the metamorphic protein lymphotactin (Ltn) exists in equilibrium between two distinct conformations, a monomeric (Ltn10) and a dimeric (Ltn40) fold. Here, we use electron capture dissociation (ECD) and drift tube ion mobility-mass spectrometry (DT IM-MS) to analyze both forms and use molecular dynamics (MD) to consider how the solution fold alters in a solvent-free environment. DT IM-MS reveals significant conformational flexibility for the monomer, while the dimer appears more conformationally restricted. These findings are supported by MD calculations, which reveal how salt bridges stabilize the conformers in vacuo. Following ECD experiments, a distinctive fragmentation pattern is obtained for both the monomer and dimer. Monomer fragmentation becomes more pronounced with increasing charge state especially in the disordered regions and C-terminal α-helix in the solution fold. Lower levels of fragmentation are seen in the β-sheet regions and in regions that contain salt bridges, identified by MD simulations. The lowest charge state of the dimer for which we obtain ECD data ([D+9H](9+)) exhibits extensive fragmentation with no relationship to the solution fold and has a smaller collision cross section (CCS) than charge states 10-13+, suggesting a "collapsed" encounter complex. Other charge states of the dimer, as for the monomer, are resistant to fragmentation in regions of β-sheets in the solution fold. This study provides evidence for preservation and loss of global fold and secondary structural elements, providing a tantalizing glimpse into the power of the emerging field of native top-down mass spectrometry.

Compaction of Duplex Nucleic Acids upon Native Electrospray Mass Spectrometry

“…45 Briefly optimized structures often have CCS values matching well with the experiments. 42,[46][47][48] Fabris and co-workers have recently underlined the difficulties in transposing to DNA the MD and CCS calculation protocols traditionally used for proteins. 43 As a way out they proposed to calibrate all traveling wave IMS data using short MD simulation results, but our study shows why this approach would lead to a misrepresentation of nucleic acid structures in the gas phase.…”

Linking molecular models with ion mobility experiments. Illustration with a rigid nucleic acid structure

“…Modelling the ESI process to generate solvent-free polyatomic ions and the subsequent ion structural changes in the gas phase however encounters three major challenges to be addressed in the future as follows: (1) realistic description of solvent evaporation effects on the species conformation, (2) partial charges location for a given charge state, and (3) the time scale (milliseconds for an IMS experiment). The first two issues have not yet been clarified for all biological molecules, although several attempts been made in this direction, [55,72,74,75,[105][106][107] whereas the latter point would require a rather nowadays unaffordable computer time, above all for large systems like biopolymers (to the best of our knowledge, the sub-milliseconds time scale in a MD simulation in gas phase has been achieved only for a 7-mer oligonucleotide). [77] Finally, although there is still room for improvement in CCS calculation approaches, algorithms and parameterization (especially for gases other than helium), we were pleased to find out that several of the currently available methods, although developed and parameterized for totally different molecules, transpose well to the 24-nucleotide nucleic acid structure studied here.…”

“…MD using classical methods are preferred for biomolecules, such as proteins, protein/protein complexes and large oligonucleotides, where a satisfactory conformational sampling is more desirable than accounting for electronic structure effects. [41,55,56] MD simulations can however be hampered by multiple local energy minima, where the biomolecule can be trapped. Several enhanced sampling techniques allow the species to overcome energetic barriers and explore other conformational 5À on the Synapt G1 and (C) Reconstructed collision cross section distributions for the ion [(dTG 4 T) basins, for example simulated annealing molecular dynamics, [57][58][59][60] replica-exchange molecular dynamics [61][62][63] and adaptively biased molecular dynamics.…”

“…Therefore, many ways of distributing the charges among these titrable groups are compatible with a specific net charge shown in the ESI spectra. To tackle this issue, different methodologies have been developed, which either take into account how the acidic strength (the pK a ) of the (conjugated) acid (e.g.ÀCOOH andÀNH 3 + ), varies depending on the surrounding environment, going from residues deeply buried in the protein (very low pK a ) up to residues surrounded nearly by only water (with a pK a practically equal to that of the lone residue in bulk water), [55,72] or consider energy states resulting from different protonation schemes. [73,74] In the case of nucleic acids, assuming that all the phosphates can be equally titrable, a different strategy must be used to obtain the net charge state, and two main methods are available in literature: the distributed charge (DC) and the localized charge (LC) method.…”

Interplay of Electrostatics and Hydrophobic Effects in the Metamorphic Protein Human Lymphotactin

“…The fact that hLtn40 shows a smaller number of clusters compared to hLtn10 at the same threshold is qualitatively consistent with the expectation that the dimeric structure is better stabilized than the monomer by the fairly extensive dimer interface and it features less disordered regions. 52 Electrostatic Effects: Salt Ion Distribution and Network of Charged Residues. Salt Ion Distribution around the Protein.…”