Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
The "physiological" behaviour of DNA/RNA structural motifs in gas phase
In the last decade, molecular modelling methodologies anchored to mass spectrometry (MS) techniques gained their momentum in the field of structural biology. Native MS, where non-covalent interactions are preserved, is made possible by electrospray ionization (ESI), which desolvates the species gently. The ultimate goal is to preserve the salient structural features of the biomacromolecules passing from physiological solutions into gas phase. Furthermore, hyphenating ESI-MS with ion mobility spectrometry (IMS) allows one to separate the species also according to their shape, which can be visualised in atomistic detail with computer simulations (D'Atri et al., 2014). The physical property linking experiments to theory is the collision cross section (CCS), on the one hand measured with IMS and on the other hand calculated on gas-phase structures with different collision models.
The desolvation process and the subsequent milliseconds drift needed to reach the detector however still pose unanswered questions about which structural traits nucleic acids retain in vacuo. The help brought by computational tools is two-fold: exploration of the conformational space with molecular dynamics (MD) techniques and calculation of the CCS with an adapted method. Both computations have to reckon with accuracy of the results and reasonable speed to obtain them, therefore an efficient and reliable protocol is desirable.
Here we show how the work-flow developed in our laboratory is successfully applicable to the investigation of nucleic acids constructs softly electrosprayed from physiological solutions. After calibrating the procedure on a rigid and well conserved structure such as a DNA G-quadruplex, we could show that 12-mer DNA duplexes experience a considerable sequence-dependent compaction in gas phase.
Their experimental CCS is far smaller than the one that a B-helix would adopt, and also far smaller than the values reported by the Bowers group on more densely charged duplexes (Gidden et al., 2004; Baker et al., 2007). Multiple co-existing conformations are observed, in that the CCS distributions are much broader than the instrumental peak width. Theoretical CCSs evaluated on candidate structures obtained via diverse modelling approaches led us to propose that the duplexes, upon desolvation, do not adopt the distorted helices proposed by Orozco's (Rueda et al., 2003) or Bowers' groups. They either convert into unstructured globular dimers (but this would not explain the persistence of a memory of the sequence in MS/MS experiments), or undergo a compaction through a mechanism in which the helix collapses along the longitudinal axis. We termed this mechanism "major groove zipping" (the most consistent with all observations to date): hydrogen bonds forming between protonated (donor) and deprotonated (acceptor) phosphate groups of the major groove backbone are able to significantly contract the duplex.
D'Atri, V., Porrini, M., Rosu F. & Gabelica V. (2014). Special Feature: Tutorial. Linking MolecularModels with Ion Mobility Experiments. Illustration with a rigid nucleic acid structure. Submitted toJournal of Mass Spectrometry.
Gidden, J., Ferzoco A., Baker, E.S., & Bowers M.T. (2004). Duplex formation and the onset of helicityin poly d(CG)n oligonucleotides in a solvent-free environment. J. Am. Chem. Soc., 126, 15132-15140.Rueda, M., Kalko, S.G., Luque, F.J., & Orozco, M. (2003). The structure and dynamics of DNA in the gas phase. J. Am. Chem. Soc., 125, 8007-8014.
Massimiliano Porrini 1, 2
1Univ. Bordeaux, IECB