Reflections on Charge State Distributions Protein

Authors: Omar M Hamdy Ryan R Julian

Publish Date: 2011/11/11

Volume: 23, Issue: 1, Pages: 1-6

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Abstract

The connection between charge state distributions protein structure and mechanistic details of electrospray are discussed in relation to the emerging field of gas phase structural biology Comparisons are drawn with the established area of enzymatic catalysis in organic solvents which shares many similar challenges Charge solvation emerges as a dominant force in both systems that must be dealt with to enable kinetic trapping of native structures in foreign environments Potential methods for mediating unfavorable charge solvation effects are discussed and ironically do not include partial solvation by water The importance of timescale in relation to the evolution of protein structure during the process of electrospray ionization is discussed Finally several prospects for future endeavors are highlightedElectrospray ionization ESI is great It can be used to gently transfer just about anything except perhaps small animals into the gas phase in an ionized state 1 2 The full impact that ESI will have on chemistry biochemistry biology medicine and other areas has not been fully determined but will certainly be substantial The utility and importance of ESI cannot therefore be reasonably disputed however ESI is not completely or perhaps even well understood The basics can be inferred a voltage difference is used to create charged droplets these droplets fission evaporate and eventually yield ions Exactly how these events occur particularly at a molecular level is not known 3 Similarly the influence that the electrospray process has on the molecules being ionized is not fully understood Why not There are experimental challenges ie the droplets of interest are very small and exist only transiently in an awkward to access environment Furthermore methods for confident molecular level characterization of such droplets may not exist There are also philosophical barriers—we know everything that we need to in order to make ESI useful so why invest the time and money Then there is the issue of source diversity which is rarely addressed in relation to how ESI works Electrospray itself can be found in numerous variations all differing in some way with respect to liquid flow rates gas flow rates geometries voltages ion optics materials etc In addition there are numerous closely related methods such as nanospray paperspray electrosonic spray and desorption electrospray DESI just to mention a few 4 5 Do all of these variations operate under similar general principals or are there important differences The point is at the moment we do not knowOne of the interesting phenomena associated with ESI is that molecules are frequently observed in multiple charge states especially larger molecules such as proteins 6 7 The collection of charge states observed for a particular molecule under a given set of experimental conditions is referred to as the charge state distribution Charge state distributions can be influenced by a variety of factors For example the addition of organic solvent and acid to an aqueous protein solution will frequently lead to a dramatic broadening and shift of the charge state distribution to lower m/z higher charge states In contrast addition of buffer such as ammonium acetate to an aqueous solution frequently favors lower charge states and narrower distributions These observations are presumably linked to protein structure where unfolded proteins are able to accommodate more charge which leads to higher and broader charge state distributions while folded proteins typically exhibit lower and narrower charge state distributionsIt is clear from ion mobility experiments that in the gas phase higher charge states do correspond to more unfolded proteins while lower charge states represent more compact structures 8 9 It is therefore often inferred that such structures were present in the original solutions It is entirely possible that this is the case however it is also possible that proteins may unfold during the process of ESI itself This issue will be discussed further below but it is sufficient for the moment to say that our ignorance about the mechanistic process of ESI makes it dangerous to assume that structures present in solution are always or perhaps even can be directly transferred into the gas phase This issue is particularly important given the increasing interest in the area of gas phase structural biology where gas phase methods including structural characterization in vacuo are employed to examine protein structure Proponents for this field argue that proteins can be transferred with structural fidelity into the gas phase and meaningfully examined within that environment while others argue that the gas phase is a wildly unnatural medium that in no way mimics aqueous or cellular conditions and that examination of protein structure in the gas phase is a waste of timeLet us take a brief aside to examine this issue from another angle Oddly enough insight into the dilemma of protein structure in the gas phase can be acquired by examination of enzymatic catalysis in organic solvents which is actually a fairly established field and has been reviewed 10 11 Organic solvents more specifically anhydrous organic solvents—at least to the extent that is possible are not typically associated with biology or proteins but are more comparable to the gas phase than water in many respects Interestingly even though most proteins are not soluble in organic solvents they are frequently employed for catalytic transformations in organics despite this shortcoming In fact several applications in industry have been used to generate kilograms of product in this fashion 10 Instinctively one might assume that proteins would be denatured in organic solvents however experimental characterization though limited has suggested that this is not the case 12 13 Furthermore the ultimate test for structural fidelity with proteins is generally considered to be functionality which can obviously be retained and utilized in organic media It is therefore reasonable to conclude that sufficient core structure is retained in organic solvents to allow for the observed catalysisMany of these experiments are carried out by lyophilization of the protein which is then dispersed in an organic solvent by vigorous and constant agitation Importantly the conditions under which the protein is lyophilized can have significant impact on catalytic activity If the protein is denatured prior to or during lyophilization no catalysis is observed This further supports retention of protein structure as being important for successful catalysis In addition experiments have shown that the addition of salts or crown ethers to solution prior to lyophilization greatly enhances catalytic activity 14 15 There are multiple potential explanations for this observation For example it has been suggested that proteins become very rigid in organic solvents due to loss of the dynamic hydrogen bonding environment afforded by water If this rigidity concept is correct then salts or crown ethers may restore a degree of flexibility to the enzymes which is necessary for catalysis Another not mutually incompatible way of looking at this issue relates to charge solvation It is likely that counterions or crown ethers enhance solvation of charged groups on the protein surface during and after lyophilization which helps mitigate loss of structure due to Coulombic forces Interestingly solvation of the charge groups by retention of water itself is not a viable option Addition of water even in small quantities is not beneficial for catalysis and leads to rapid denaturation 10 When these observations are considered together it would appear that proteins retain structure in organic media due to kinetic trapping In other words although native structures are likely not the lowest energy states in organic solvents the barriers to rearrangement to the lowest energy structures are sufficiently large to prevent this from occurring The addition of water likely lowers the kinetic barriers enabling transitions between different structural statesTo sum up there are a few important takehome points One proteins can be kinetically trapped in their native state in nonaqueous environments and retain enzymatic activity under certain conditions The theoretical possibility for gas phase structural biology is therefore possible Two water actually facilitates protein unfolding in denaturing environments As a result many of the water/organic solution combinations frequently employed in ESI are likely to be highly denaturing ironically because of the combination not just the organic Three lack of charge solvation can interfere with kinetic trapping of native states The gas phase is even worse at charge solvation than organic solvents making this a primary obstacle which will interfere with retention of native or nativelike structures

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