Evidence for the Preservation of Native Inter- and Intra-Molecular Hydrogen Bonds in the Desolvated FK-Binding Protein·FK506 Complex Produced by Electrospray Ionization
Abstract:It is now well established that electrospray ionization (ESI) is capable of introducing noncovalent protein assemblies into a desolvated environment, thereby allowing their analysis by mass spectrometry. The degree to which native interactions from the solution phase are preserved in this environment is less clear. Site-directed mutagenesis of FK506-binding protein (FKBP) has been employed to probe specific intra-and inter-molecular interactions within the complex between FKBP and its ligand FK506. Collisional… Show more
“…This is justified by the observation that frequently protein molecules retain their structures and functions when transferred in gas phase 45-48 . Because of that, their intrinsic flexibility and therefore dielectric properties in vacuum should remain the same as in water phase.…”
Traditional implicit methods for modeling electrostatics in biomolecules use a two-dielectric approach: a biomolecule is assigned low dielectric constant while the water phase is considered as a high dielectric constant medium. However, such an approach treats the biomolecule-water interface as a sharp dielectric border between two homogeneous dielectric media and does not account for inhomogeneous dielectric properties of the macromolecule as well. Recently we reported a new development, a smooth Gaussian-based dielectric function which treats the entire system, the solute and the water phase, as inhomogeneous dielectric medium (J Chem Theory Comput. 2013 Apr 9; 9(4): 2126-2136.). Here we examine various aspects of the modeling of polar solvation energy in such inhomogeneous systems in terms of the solute-water boundary and the inhomogeneity of the solute in the absence of water surrounding. The smooth Gaussian-based dielectric function is implemented in the DelPhi finite-difference program, and therefore the sensitivity of the results with respect to the grid parameters is investigated, and it is shown that the calculated polar solvation energy is almost grid independent. Furthermore, the results are compared with the standard two-media model and it is demonstrated that on average, the standard method overestimates the magnitude of the polar solvation energy by a factor 2.5. Lastly, the possibility of the solute to have local dielectric constant larger than of a bulk water is investigated in a benchmarking test against experimentally determined set of pKa's and it is speculated that side chain rearrangements could result in local dielectric constant larger than 80.
“…This is justified by the observation that frequently protein molecules retain their structures and functions when transferred in gas phase 45-48 . Because of that, their intrinsic flexibility and therefore dielectric properties in vacuum should remain the same as in water phase.…”
Traditional implicit methods for modeling electrostatics in biomolecules use a two-dielectric approach: a biomolecule is assigned low dielectric constant while the water phase is considered as a high dielectric constant medium. However, such an approach treats the biomolecule-water interface as a sharp dielectric border between two homogeneous dielectric media and does not account for inhomogeneous dielectric properties of the macromolecule as well. Recently we reported a new development, a smooth Gaussian-based dielectric function which treats the entire system, the solute and the water phase, as inhomogeneous dielectric medium (J Chem Theory Comput. 2013 Apr 9; 9(4): 2126-2136.). Here we examine various aspects of the modeling of polar solvation energy in such inhomogeneous systems in terms of the solute-water boundary and the inhomogeneity of the solute in the absence of water surrounding. The smooth Gaussian-based dielectric function is implemented in the DelPhi finite-difference program, and therefore the sensitivity of the results with respect to the grid parameters is investigated, and it is shown that the calculated polar solvation energy is almost grid independent. Furthermore, the results are compared with the standard two-media model and it is demonstrated that on average, the standard method overestimates the magnitude of the polar solvation energy by a factor 2.5. Lastly, the possibility of the solute to have local dielectric constant larger than of a bulk water is investigated in a benchmarking test against experimentally determined set of pKa's and it is speculated that side chain rearrangements could result in local dielectric constant larger than 80.
“…*Denaturation curves for these proteins were fitted using a constrained m D−N value (determined from 17 independent measurements) to improve fitting precision (26) ESI-IMS-MS allows the separation of gas phase ions by virtue of their rotationally averaged collision cross-section (CCS), which depends on molecular shape. This approach has been used to interrogate protein ligand interactions, gas phase stabilization of protein complexes, and conformation-dependent dissociation (41)(42)(43)(44)(45). Using ESI-IMS-MS, the internal energy of HBc ions was increased by collisions with an inert buffer gas.…”
Significance
Hepatitis B virus (HBV) is a major pathogen, yet no fully effective therapies exist. HBc is the multifunctional, capsid-forming protein essential for HBV replication. HBc structural plasticity is reportedly functionally important. We analyzed the folding mechanism of HBc using a multidisciplinary approach, including microscale thermophoresis and ion mobility spectrometry–mass spectrometry. HBc folds in a 3-state transition with a dimeric, helical intermediate. We found evidence of a strained native ensemble wherein the energy landscapes for folding, allostery, and capsid formation are linked. Mutations thermodynamically trapped HBc in conformations unable to form capsids, suggesting chemical chaperones could elicit similar, potentially antiviral, effects.
“…Thus, one powerful method is to explore time-dependent perturbations in protein structure (Henzler-Wildman and Kern, 2007 ; van dem Bedem and Fraser, 2015 ). For example, pioneering efforts in nanoflow electrospray ionization (nESI) have revolutionized the study of large protein complexes with its increased sensitivity and preservation of weak non-covalent interactions (Hopper et al, 2012 ). Because detection occurs in the gas phase, this technique effectively captures a “snapshot” of a binding equilibrium that exists in solution.…”
Section: Dissecting Protein Network
In Vitro
and mentioning
Networks of protein–protein interactions (PPIs) link all aspects of cellular biology. Dysfunction in the assembly or dynamics of PPI networks is a hallmark of human disease, and as such, there is growing interest in the discovery of small molecules that either promote or inhibit PPIs. PPIs were once considered undruggable because of their relatively large buried surface areas and difficult topologies. Despite these challenges, recent advances in chemical screening methodologies, combined with improvements in structural and computational biology have made some of these targets more tractable. In this review, we highlight developments that have opened the door to potent chemical modulators. We focus on how allostery is being used to produce surprisingly robust changes in PPIs, even for the most challenging targets. We also discuss how interfering with one PPI can propagate changes through the broader web of interactions. Through this analysis, it is becoming clear that a combination of direct and propagated effects on PPI networks is ultimately how small molecules re-shape biology.
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