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

Abstract

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 ...

Backbone additivity in the transfer model of protein solvation

“…42,43 Recent experimental measurements have indicated hydrogen bonding of urea with the NH group of a dialanine peptide, yet urea hydrogen bonding with the carbonyl group of the peptide was not as well resolved. 39 However, those authors expected to have significantly more hydrogen bonds between the peptide carbonyl group as opposed to the peptide NH groups, 39 a trend that we see in our simulations (Table I). In the literature, we find a scatter of results from different models.…”

“…15,39,40 By calculating the number of osmolyte and water-hydrogen bonds to the peptide backbone models, and separating them into whether the solution species act as a donor or acceptor, helps to describe the nature in which each species interacts with the peptide backbone model (Table I). As expected, TMAO rarely forms hydrogen bonds with the peptide backbone, whereas urea, on average, forms between one and two hydrogen bonds with the peptide backbone for all backbone conformations.…”

“…We see the denaturing osmolyte acting as a hydrogen bond donor with far more frequency than as a hydrogen bond acceptor. 22,39 Methods Each peptide backbone was created in its extended conformation using the CHARMM-27 all atom parameter set 41 and subjected to energy minimization using the steepest descent method. The osmolyte solutions consisted of 66 osmolyte molecules and 1614 TIP3P 54 water molecules in a cubic simulation box with lengths of 3.8 nm, resulting in 2M osmolyte concentration.…”

Incorporation of57Fe-isotopically enriched in apoferritin: formation and characterization of isotopically enriched Fe nanoparticles for metabolic studies

“…In contrast, guanidinium is barely more efficient than urea if stabilization is mainly due to salt bridges. 28 However, no other signicant species seem to be detected in the chromatogram aer ultraltration. The guanidinum treated samples were stored for over a month and during this time no apparent modication of the chromatographic prole was obtained (see ESI †).…”

Native state dynamics drive the unfolding of the SH3 domain of PI3 kinase at high denaturant concentration

“…Even less is known about how the thermal fluctuations that lead to protein unfolding are perturbed by denaturants such as guanidine hydrochloride (GdnHCl) and urea. Protein denaturants may act indirectly by disrupting the structure of water, thereby making hydrophobic groups more readily solvated (9,10), or directly by interacting more strongly than water with the protein backbone and side chains (11)(12)(13). To understand how denaturants act, it is necessary to determine whether the mechanism of unfolding of a protein, as well as the thermal fluctuations that enable unfolding, are the same in the absence of a chemical denaturant and in the presence of a high concentration of the denaturant.…”