The microsolvated
state of a molecule, represented by its interactions
with only a small number of solvent molecules, can play a key role
in determining the observable bulk properties of the molecule. This
is especially true in cases where strong local hydrogen bonding exists
between the molecule and the solvent. One method that can probe the
microsolvated states of charged molecules is differential mobility
spectrometry (DMS), which rapidly interrogates an ion’s transitions
between a solvated and desolvated state in the gas phase (i.e., few
solvent molecules present). However, can the results of DMS analyses
of a class of molecules reveal information about the bulk physicochemical
properties of those species? Our findings presented here show that
DMS behaviors correlate strongly with the measured solution phase
pKa and pKb values, and cell permeabilities of a set of structurally related
drug molecules, even yielding high-resolution discrimination between
isomeric forms of these drugs. This is due to DMS’s ability to separate species based upon only subtle (yet
predictable) changes in structure: the same subtle changes that can
influence isomers’ different bulk properties. Using 2-methylquinolin-8-ol
as the core structure, we demonstrate how DMS shows promise for rapidly
and sensitively probing the physicochemical properties of molecules,
with particular attention paid to drug candidates at the early stage
of drug development. This study serves as a foundation upon which
future drug molecules of different structural classes could be examined.
Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.
Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high-resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native-like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation-prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.
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