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Macromolecular Ion-Solvent Interactions in Charged Droplets

Final Report Summary - MISICD (Macromolecular Ion-Solvent Interactions in Charged Droplets)

Charged liquid droplets are common in both nature and technology; aerosols, thunder clouds and electrospray are just a few prominent examples. The composition of the droplets varies with the context, but typically a droplet is composed of solvent and charge carriers such as simple ions or macroions.
The macroions may be proteins or nucleic acids. Since the discovery in the late 1960s of the electrospray ionization (ESI) method there has been a widespread development of experimental techniques that utilise aerosols composed of highly charged droplets. A few notable examples are in the use of ESI in deposition of materials, sample preparation in synchrotron radiation and transfer of analytes from bulk solution into the gas phase for mass spectrometry (MS) analysis. A recent application of charged droplets that is investigated in the last four years is their usage as vessels for accelerating chemical reactions. The outcome of these applications is quite diverse. For instance, protein mass-spectrometry aims toward the understanding of the
interactions among biological molecules. Other applications include the efficient analysis of chemicals for increasing the security in transportation
by the detection of explosives in the airports. One should mention efficient chemical analysis of medical samples and application in industrial production as well.

My research aims at understanding the chemical and physical properties of the charged droplets. The project objectives focused on (i) protein-ion interactions in droplets; (ii) the stability of complexes of macromolecules in droplets and (iii) the physical origin of the ``star'' morphologies of droplets when they contain a non-fissile charge. Typical star morphologies are shown in Figure 1 (bottom-left and top-right panels). In our studies we use molecular simulations and theoretical approaches in describing the late stages of droplet evolution that are intrinsically difficult to capture in the experiments due to their short life-time. One of the main outcomes of our research is that we transform the manner in which scientists in the field of electrospray mass spectrometry have perceived until recently the stability of charged droplets and its interactions with the analytes.

The chemical and physical processes that take place within a droplet are complex, comparing to vapour/solvent phase chemistry, and determine the outcome of the experimental measurements. The major reason for that is that the droplet environment is dynamic. Droplets that are generated by
ESI undergo evaporation, fragmentation and develop charge induced instabilities. The charged induced instabilities are shown in the top and bottom-left panels of Figure 1. These factors affect the charge state of a macromolecule and the stability of a macromolecular complex (such as nucleic acids, proteins), which is the outcome of electrospray mass spectrometry.

Quantitatively the stability of a complex of proteins or nucleic acids is described by its binding affinity. A promising experimental method for the detection of the equilibrium constant of non-covalently bound complexes is electrospray ionization mass spectrometry (ESI-MS). ESI-MS is based on the
generation of charged droplets that carry the complexes from the bulk solution into the gaseous state.

Poorly understood processes that affect the stability of weak non-covalent protein complexes in the intervening droplet environment is a significant factor that precludes ESI-MS to become a high-throughput robust experimental method for the detection of protein-protein and protein-ligand equilibrium
constants. Moreover, the principles that govern the stability of protein complexes in droplets are still unknown. From another perspective, the study of the complex dissociation rate in a droplet is closely related to current research where electrosprayed droplets are used as vessels to perform chemical reactions. The factors that may affect the stability of the non-covalent complexes are: the droplet evaporation rate, the increase in the concentration of ions in evaporating droplets and the distinct droplet morphologies arising from the charge-induced instabilities. We have found the role of these factors in the stability of the nucleic acids and protein complexes by using molecular modelling. In the studies of protein complexes we presented evidence that a weak protein complex changes conformation and may dissociate in shrinking droplets relative to its stability in bulk solution. A typical complex dissociation and droplet fragmentation event is shown in Figure 2. Then the droplets containing these dissociated proteins divide. Our findings suggest that in some cases ESI-MS does not measure the correct association constants. The study is expected to stimulate research for systematic development of experimental protocols that stabilize weakly bound protein interfaces in droplets. In the study of the DNA complexes in droplets we found how the conformation of the desolvated DNA is affected by the presence of the salts in the droplet environment. In the presence of non-fissile highly charged ions, droplets attain distinct structures that are characterized by the formation of spikes. We characterized the spikes with respect to their
structure, dynamics and physical origin. The "star" droplet structures call for new experiments and exploitation possibly in the fields of material science and catalysis.

There is a multitude of long-term goals of this research. Electrospray ionisation mass spectrometry has the potential to become the method of choice in pharmaceutical and biochemical research for the analysis of protein-protein and protein-ligand interactions. However, many of these interactions are non-covalent and consequently, they may break-down during the desolvation of droplets. Molecular insight into the mechanisms of droplet desolvation, obtained by computational means, may improve the efficiency and quality of measurements, which in turn may lead to societal benefits, which may ultimately include for instance lower costs of drugs or improved security at airports because of more efficient chemical analysis.
From another perspective, an understanding of the physical chemistry of charged droplets also underpins advances in the field of atmospheric aerosols.
The molecular physics of such aerosols plays a major role in determining climates and environmental conditions. Hence, computational studies of charged droplets are an area of fundamental research that lies on a path towards informing major political decisions with profound societal impact.

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