The hydration structure of guanidinium and thiocyanate ions: Implications for protein stability in aqueous solution

Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).protein unfolding | dry molten globule | steady-state FRET D enaturants such as guanidinium chloride (GdmCl) and urea are classic perturbants used to probe the thermodynamics and kinetics of protein conformational changes, although their mechanism of action is poorly understood (1-15). In some of these studies, a dry molten globular (DMG) state has been observed on the native side of the free-energy barrier (16-18). The DMG is an expanded form of the native protein in which at least some of the side-chain packing interactions are disrupted without solvation of the hydrophobic core; it was originally postulated to explain heat-induced unfolding of proteins (19,20). Recently, unfolding intermediates resembling the DMG have also been observed in the absence of denaturants, and it has been suggested that the DMG exists in rapid equilibrium with the native state (21-23). The fact that the DMG can be observed by the addition of denaturants is, however, counterintuitive, because traditional models of chemical denaturation rely on changes in solvent-accessible surface area (SASA) (3,24,25) and there is no notable change in SASA during the formation of the DMG.Recent computational studies have suggested an alternative model of chemical denaturation in which denaturants first interact directly with the protein surface, causing the protein to swell, and then penetrate the core (the so-called "direct interaction" mechanism) (9,11,12,26). One of the consequences of this model is that denaturants unfold proteins in two steps (9, 12). In the first step, denaturant molecules displace water molecules within the fir...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride

Jha

Marqusee

2014

“…On the other hand, the absence of guanidinium to peptide H-bonding is consistent with the behavior of guanidinium in solution. It can, in principle, form H-bonds with water (39) but neutron diffraction experiments find that it has no recognizable hydration shell, presumably because of geometric constraints (18). Rather than forming an extensive H-bonding network, guanidinium tends to engage in transient stacking interactions with itself (39) and with other planar groups (10,36,39).…”

Section: Discussionmentioning

confidence: 99%

“…The mechanism of osmolyte action continues to be controversial. Opposing positions favor either a direct interaction between protein and osmolyte (8-11), such as hydrogen bonding, or an indirect effect mediated by the alteration of water structure (12, 13), or a mixture of both (14)(15)(16)(17)(18)(19). It has been difficult to distinguish between these direct and indirect models because the osmolyte-protein interaction is so weak.…”

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confidence: 99%

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Lim

Rösgen

Englander

2009

354

370

The mechanism by which urea and guanidinium destabilize protein structure is controversial. We tested the possibility that these denaturants form hydrogen bonds with peptide groups by measuring their ability to block acid-and base-catalyzed peptide hydrogen exchange. The peptide hydrogen bonding found appears sufficient to explain the thermodynamic denaturing effect of urea. Results for guanidinium, however, are contrary to the expectation that it might H-bond. Evidently, urea and guanidinium, although structurally similar, denature proteins by different mechanisms.denaturation ͉ hydrogen exchange ͉ osmolyte ͉ solvation B ecause of its fundamental importance, the study of protein molecular stability has engaged the attention of protein chemists for the last 100 years (1, 2). Experimental and theoretical investigations of protein stability have often focused on small-molecule osmolytes that can be used to modulate stability in vitro (3-6) and are used by virtually all organisms to counter biochemical stress in vivo (7). The mechanism of osmolyte action continues to be controversial. Opposing positions favor either a direct interaction between protein and osmolyte (8-11), such as hydrogen bonding, or an indirect effect mediated by the alteration of water structure (12, 13), or a mixture of both (14)(15)(16)(17)(18)(19). It has been difficult to distinguish between these direct and indirect models because the osmolyte-protein interaction is so weak.It is a thermodynamic truism (20-22) that the concentration of destabilizing osmolytes must be enriched relative to water molecules in the vicinity of the protein surface that becomes newly exposed upon unfolding (preferential interaction). In a thermodynamic sense, denaturing osmolytes ''bind'' selectively to the increased surface of unfolded proteins and thus bias the folded/ unfolded equilibrium toward denaturation. Similarly, stabilizing osmolytes must be preferentially excluded from the protein surface. This is true independently of the physical mechanism of the ''binding'' or ''antibinding'' interaction. Thermodynamically based studies directed at the binding/antibinding problem have been designed to measure the transfer free energy of the various component groups of proteins between water and osmolyte solutions. Results show that the main-chain peptide group makes the largest contribution to the energetics, accounting for Ϸ80% of the effect of urea and of strong stabilizers such as trimethyl amine N-oxide (TMAO) (23). Similar determinations for guanidinium are not yet available, but it seems clear that guanidinium also favorably interacts with the peptide group (8,11,24).To search for the mechanistic basis of thermodynamic destabilization due to urea and guanidinium, we tested the possibility that they exert their denaturing effect through peptide group hydrogen bonding. This can be done experimentally by the straightforward measurement of acid-and base-catalyzed hydrogen exchange (HX) in a small-molecule peptide model. Osmolyte that is H-bonded to the peptide NH ...

“…Consequently, its mechanism of action has been subjected to extensive studies (7)(8)(9)(10)(11)(12)(13)(14). Although different interpretations have been put forth (15-17), the generally accepted notion is that Gdm + denatures a protein by preferentially interacting with its peptide groups (7,11,18), including certain side chains (11,(19)(20)(21)(22). In particular, it has been hypothesized that Gdm + , which exists in aqueous solution as a rigid, flat object (12,18,19), can engage in stacking interactions with amino acids consisting of planar side chains, such as arginine (Arg) (19), asparagine (Asn) (11,20), glutamine (Gln) (11,20), and aromatic residues (20)(21)(22).…”

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confidence: 99%

Do guanidinium and tetrapropylammonium ions specifically interact with aromatic amino acid side chains?

Ding

Mukherjee

Chen

et al. 2017

Many ions are known to affect the activity, stability, and structural integrity of proteins. Although this effect can be generally attributed to ion-induced changes in forces that govern protein folding, delineating the underlying mechanism of action still remains challenging because it requires assessment of all relevant interactions, such as ion-protein, ion-water, and ion-ion interactions. Herein, we use two unnatural aromatic amino acids and several spectroscopic techniques to examine whether guanidinium chloride, one of the most commonly used protein denaturants, and tetrapropylammonium chloride can specifically interact with aromatic side chains. Our results show that tetrapropylammonium, but not guanidinium, can preferentially accumulate around aromatic residues and that tetrapropylammonium undergoes a transition at ∼1.3 M to form aggregates. We find that similar to ionic micelles, on one hand, such aggregates can disrupt native hydrophobic interactions, and on the other hand, they can promote α-helix formation in certain peptides.T he stability of a protein, or more precisely, the free energy difference between its folded and unfolded states, can be modulated by various solution properties. For example, addition of another solute to a protein solution can result in either a decrease or an increase in this protein's stability (1-3). The most noticeable example in this regard is the Hofmeister series (4-6), a group of ions that are ranked based on their protein-denaturing abilities. Among this series, the guanidinium ion (Gdm + ) is the most widely used chemical agent in protein denaturation due to its strong destabilizing effect and high solubility in water. Consequently, its mechanism of action has been subjected to extensive studies (7)(8)(9)(10)(11)(12)(13)(14). Although different interpretations have been put forth (15-17), the generally accepted notion is that Gdm + denatures a protein by preferentially interacting with its peptide groups (7,11,18), including certain side chains (11,(19)(20)(21)(22). In particular, it has been hypothesized that Gdm + , which exists in aqueous solution as a rigid, flat object (12,18,19), can engage in stacking interactions with amino acids consisting of planar side chains, such as arginine (Arg) (19), asparagine (Asn) (11,20), glutamine (Gln) (11,20), and aromatic residues (20)(21)(22). However, to the best of our knowledge, the only experimental evidence that supports this hypothesis comes from crystallographic data (20)(21)(22), which show that the guanidinium group of Arg is more frequently found to be stacked against the side chain of tyrosine (Tyr) or tryptophan (Trp) in proteins. Therefore, additional experimental studies that can directly probe such stack interactions in solution are needed.Another ion in the Hofmeister series that has a similar proteindenaturing capability to Gdm + is tetrapropylammonium (TPA + ) (23). However, a recent study by Dempsey et al. (24) found that although TPA + , like Gdm + , can denature a tryptophan (Trp) zipper β-hairpin (i.e....