Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography

Abstract

Hydrogen exchange has been a useful technique for studying the conformational state of proteins, both in bulk solution and at interfaces, for several decades. Here, we propose a physically-based model of simultaneous protein adsorption, unfolding and hydrogen exchange in HIC. An accompanying experimental protocol, utilizing mass spectrometry to quantify deuterium labeling, enables the determination of both the equilibrium partitioning between conformational states and pseudo-first order rate constants for folding and unfolding of adsorbed protein. Unlike chromatographic techniques, which rely on the interpretation of bulk phase behavior, this methodology utilizes the measurement of a molecular property (solvent exposure) and provides insight into the nature of the unfolded conformation in the adsorbed phase. Three model proteins of varying conformational stability, α-chymotrypsinogen A, β-lactoglobulin B, and holo α-lactalbumin, are studied on Sepharose ™ HIC resins possessing assorted ligand chemistries and densities. α-Chymotrypsinogen, the most conformationally stable protein in the set, exhibits no change in solvent exposure at all of the conditions studied, even when isocratic pulse-response chromatography suggests nearly irreversible adsorption. Apparent unfolding energies of adsorbed β-lactoglobulin B and holo α-lactalbumin range from −4 to 3 kJ/mol and are dependent on resin properties and salt concentration. Characteristic pseudo-first order rate constants for surfaceinduced unfolding are 0.2 to 0.9 min −1 . While poor protein recovery in HIC is often associated with irreversible unfolding, this study documents that non-eluting behavior can occur when surface unfolding is reversible or does not occur at all. Further, this hydrogen exchange technique can be used to assess the conformation of adsorbed protein under conditions where the protein is non-eluting and chromatographic methods are not applicable.

Methods for characterization of biochromatography media

“…Proteins may change their native conformation upon binding to HIC surfaces . This phenomenon has been studied for modern HIC media by use of deuterium exchange, circular dichroism, spectroscopic techniques, and isocratic elution techniques . McCue et al.…”

Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces

“…Among the goals of predictive methods is identifying the conditions where target proteins would be sufficiently stable during HIC. While there is evidence that resins of higher hydrophobicity destabilize proteins more than those of lesser hydrophobicity [13][14][15], no generalized empirical or theoretical framework exists to identify the resins and the mobile phase conditions that can destabilize a protein.…”

Direct Observation of the Orientation Dynamics of Single Protein‐Coated Nanoparticles at Liquid/Solid Interfaces

“…It is known that (NH 4 ) 2 SO 4 binds water strongly; raising the concentration of the salt decreases charge shielding, promotes exposure of the hydrophobic patches both in the molecules and on the stationary phase, results in increased hydrophobic affinity between the molecules and the stationary phase, and promotes the unfolding of proteins from their native state. [11] A recent study on the structure of BSA molecules absorbed on AuNRs with synchrotron radiation X-ray absorption spectroscopy and molecular dynamics simulations showed that the proteins undergo only partial spreading after AuNR binding, and that their structural flexibility is preserved, thus implying that they can still respond to the local environment change. [12] Hence, although the protein-coated AuNRs are quite large, their interfacial behavior is similar to that of proteins alone, so they can be treated as large artificial semielastic molecules for the purpose of protein-surface interaction studies.…”