Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

Abstract

The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real "snap-shot" of the situation in solution can be obtained. (J Am Soc Mass Spectrom 2010, 21, 286 -289) © 2010 American Society for Mass Spectrometry E lectrospray is an exceptionally soft ionization technique, which has allowed the observation of numerous noncovalent complexes, including protein-protein, protein-small molecule, and protein-DNA complexes [1,2]. A major question is still whether the information obtained from gas-phase results is representative of the species present in solution. The term "noncovalent bonding" in biochemistry generally summarizes three types of intermolecular forces: electrostatic interactions (e.g., salt bridges), dipolar interactions (e.g., hydrogen bonds), and van der Waals interactions (e.g., hydrophobic interactions) [3]. In addition, protein-water interactions often play a major role in complex stabilization. In solution, hydrophobic interactions are very weak compared with the hydrogen bonds between water molecules. Hence, it is not the hydrophobic forces that are the actual reason for the interaction of nonpolar binding partners, but rather the surrounding water molecules that "force" hydrophobic binding partners to aggregate. This solvent-driven process is called the hydrophobic effect, and plays a major role in protein folding, adhesion of lipid bilayers, partitioning effects of drugs and metabolites, and many other aggregation phenomena in chemistry and biology [4][5][6]. The hydrophobic effect is defined by the thermodynamics of mixing hydrophobic compounds with water, and is dominated by large, entropic factors [4,7]. Obviously, in the absence of a hydr...

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

“…Even when carefully controlling the instrument parameters, complexes that are predominatly stabilized by nonpolar interactions are prone to dissociation in the gas phase [25]. This so-called in-source dissociation can lead to an artificially low binding constants based on the reduced abundance of the complex ions [21].…”

“…In some studies it was shown that the gas-phase stability reflects the binding properties in solution [37][38][39]. More frequently, however, a correlation between the gas-phase stability and the solution-phase stability is absent [25,26,28,[40][41][42][43][44][45][46], for example for leucine-zippers and acyl-CoA binding protein (ACBP) and a series of acyl CoA derivatives [28]. If binding properties in solution correlate with gas-phase stability it has to be assumed that the dominant interactions are very similar in solution and in the gas phase, and that solvent mediation play only a minor role (see Daniel at al.…”

Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

“…To date, there have been few reports of the application of ESI-MS to directly characterize hydrophobic protein-ligand interactions [23,[83][84][85][86]. In fact, it has been suggested that the direct ESI-MS assay does not give results that accurately reflect the solution equilibria for protein-ligand interactions that are dominated by hydrophobic bonding [87]. The underlying argument is that because hydrophobic bonding requires the presence of water, the dehydrated complexes are unstable in the gas phase [49,88].…”

Does chemical cross-linking with NHS esters reflect the chemical equilibrium of protein-protein noncovalent interactions in solution?

“…In another study dealing with the domain interplay of uPAR, the detected amount of cross-linked uPAR-ATF complex exceeded the level predicted from the equilibrium binding situation after 15 min cross-linking reaction at pH 7.4 [26]. In contrast, Bich et al detected complex levels of about 90% of the predicted values on average after chemical cross-linking with the amine reactive homobifunctional cross-linker 1,1=-(suberoyldioxy)bisazabenzotriazole (SBAT) and MALDI-MS detection [27]. The question arises whether the chemical stabilization of the noncovalent complex can disturb the solution phase equilibrium and, if so, under which conditions a shift could be expected.…”