Conformation and orientation of a protein folding intermediate trapped by adsorption

Abstract

Although adsorption-induced conformational changes of proteins play an essential role during protein adsorption on interfaces, detailed information about these changes is lacking. To further the current understanding of protein adsorption, in this study, the orientation, conformation, and local stability of bovine ␣-lactalbumin (BLA) adsorbed on polystyrene nanospheres is characterized at the residue level by hydrogen͞deuterium exchange and 2D NMR spectroscopy. Most of the adsorbed BLA molecules have conformational properties similar to BLA molecules in the acid-induced molten globule state (A state). A folding intermediate of BLA is thus induced and trapped by adsorption of the protein on the hydrophobic interface. Several residues, clustered on one side of the adsorbed folding intermediate of BLA, have altered amide proton exchange protection factors compared to those of the A state of BLA. This side preferentially interacts with the interface and includes residues in helix C, the calcium binding site, and part of the ␤-domain. Local unfolding of this interacting part of the adsorbed protein seems to initiate the adsorption-induced unfolding of BLA. Adsorption-induced protein unfolding apparently resembles more the mechanical unfolding of a protein than the global unfolding of a protein as induced by denaturant, pH, or pressure. 2D macromolecular crowding prevented the minority of adsorbed BLA molecules, which arrived late at the interface, to unfold to the A state. Protein adsorption is a novel and challenging approach to probe features of the free energy landscapes accessible to unfolding proteins.bovine ␣-lactalbumin ͉ hydrogen͞deuterium exchange ͉ NMR A dsorption of protein molecules on solid interfaces is important in fields like biomedical materials engineering (1), chromatography (2), and nanotechnology (3, 4). To understand protein adsorption, knowledge of adsorption-induced conformational changes of proteins is essential (5, 6). Unfortunately, because of considerable experimental difficulties, detailed information at the submolecular level about these conformational changes is sparse (7-12), which hampers the further development of the theory of protein adsorption. To stimulate this development, we characterize here at the residue level, the orientation, conformation, and local stability of bovine ␣-lactalbumin (BLA) adsorbed on polystyrene nanospheres by hydrogen͞deuterium (H͞D) exchange and 2D NMR spectroscopy. BLA, a 14-kDa protein from milk, is chosen to study protein adsorption because its structure, stability, and folding behavior have been thoroughly investigated. In addition, the adsorption of BLA on a variety of interfaces has been studied.Information about the stability and dynamics of a protein at the level of single amino acids can be obtained by investigating its H͞D exchange characteristics (13). In this study, BLA molecules in the native state are added to solid polystyrene nanospheres in 2 H 2 O. All added protein molecules adsorb spontaneously and rapidly [i.e., within 15 ms (14)] on ...

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

“…In the interrupted H/D exchange methodology (13,35,36), an unstable protein species of interest is kept in D 2 O for a variable time period, t, during which exchange of amide protons for solvent deuterons occurs. After this period, conditions are changed such that the protein arrives into circumstances in which no, or very slow, hydrogen exchange occurs and hence exchange is effectively quenched.…”

“…Now it is possible to characterize an off-pathway molten globule in the absence of denaturant. We use the interrupted H/D exchange methodology (13,35,36) to reveal the stable core of the molten globule of Tyr 44 -flavodoxin and discuss why this species needs to unfold to produce native flavodoxin.…”

The effect of nanoscale surface curvature on the oligomerization of surface-bound proteins

“…protein-NP conjugates. Although NMR [62,63] and optical spectroscopy [26][27][28][29][30][31][32][33][34][35][36] have been used to characterize the ensemble average behaviour of suspensions of protein-NP conjugates, our use of SMFS provides the first investigation of the properties of proteins adsorbed to NP surfaces at the single-molecule level. Moreover, previous studies of bulk suspensions of protein-NP conjugates have suffered from the presence of impurities because suspensions of NPs are typically stabilized by charge or surfactants before the introduction of proteins.…”

“…Nuclear magnetic resonance (NMR) has been used to investigate a-LA when adsorbed onto the surface of polystyrene (PS) NPs [62] and the structure has been identified as a molten globule unfolding intermediate [63]. CD and fluorescence have also been used to detect a loss of tertiary structure of a-LA upon binding to PS NPs [64].…”

Experimental characterization of adsorbed protein orientation, conformation, and bioactivity

“…38,45,46 In addition to quantifying the secondary structural elements, the shifts in the near UV CD spectral range (260-320 nm) have been used to qualitatively determine the "molten globule" states in many adsorbed proteins, which reflect the protein's tertiary structure. 40,42,46,47 Similarly, in many proteins containing cofactors as an important functional part of the bioactive site, such as metal ions, shifts in the spectral features in the visible light related to the cofactor's position have also been used as indicators of the integrity of the binding site. 48,49 However, since the shape and magnitude of near-UV CD spectra are influenced by the type of protein, and strategies to quantify the tertiary structural shifts using CD techniques are lacking, CD is primarily used for secondary structural determination.…”