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- Materials and Methods
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Mannosylglycerate is a compatible solute typical of thermophilic marine microorganisms that has a remarkable ability to protect proteins from thermal denaturation. This ionic solute appears to be a universal stabilizing agent, but the extent of protection depends on the specific protein examined. To understand how mannosylglycerate confers protection, we have been studying its influence on the internal motions of a hyperstable staphylococcal nuclease (SNase). Previously, we found a correlation between the magnitude of protein stabilization and the restriction of fast backbone motions. We now report the effect of mannosylglycerate on the fast motions of side-chains and on the slower unfolding motions of the protein. Side-chain motions were assessed by 13CH3 relaxation measurements and model-free analysis while slower unfolding motions were probed by H/D exchange measurements at increasing concentrations of urea. Side-chain motions were little affected by the presence of different concentrations of mannosylglycerate or even by the presence of urea (0.25M), and show no correlation with changes in the thermodynamic stability of SNase. Native hydrogen exchange experiments showed that, contrary to reports on other stabilizing solutes, mannosylglycerate restricts local motions in addition to the global motions of the protein. The protein unfolding/folding pathway remained undisturbed in the presence of mannosylglycerate but the solute showed a specific effect on the local motions of β-sheet residues. This work reinforces the link between solute-induced stabilization and restriction of protein motions at different timescales, and shows that the solute preferentially affects specific structural elements of SNase.
- Top of page
- Materials and Methods
- Supporting Information
Cell membranes are permeable to water; hence, the ability to cope with changes in water activity is a prerequisite for cell survival. Most cells are prepared to counteract an increase in the external osmotic pressure by accumulating small molecules designated osmolytes. Organisms adapted to hypersaline environments accumulate high levels of osmolytes such as glycerol or KCl. Even at molar concentrations, these compounds do not disturb the physiological functions of macromolecules, thereby earning the name of “compatible solutes”.1, 2 Interestingly, the role of compatible solutes goes beyond that of balancing the osmotic gradient across the cell membrane, and some of these compounds are involved in the response to other types of stress, such as heat, oxidative, and acid stress.3 Conversely, common osmolytes, such as trehalose or ectoine, are able to confer increased stability on proteins under a range of environmental insults.
Organic osmolytes, and other compatible solutes, fall into few categories of chemical compounds, namely amino acids and derivatives, sugars and derivatives, polyols, and betaines. Trehalose, glycerol, glycine-betaine and ectoine are solutes typical of mesophiles, that is, organisms that grow optimally at moderate temperatures. However, marine organisms that thrive at high temperatures accumulate specific solutes which usually bear a negative charge at physiological pH and are rarely found as part of stress adaptation in mesophilic organisms. Appropriately, these compounds appear to be better than uncharged osmolytes at stabilizing proteins against thermal denaturation.4
For several years, we have studied compatible solutes of organisms adapted to hot environments (hyper/thermophiles). In particular, we are interested in the mechanisms underlying protein stabilization by mannosylglycerate (MG), a solute widespread in hyperthermophiles that is remarkably efficient in the stabilization of protein structures. For example, the melting temperature of SNase has an increment of 8°C in the presence of 0.5M mannosylglycerate.5
Understanding the molecular mechanisms that govern protein stabilization by osmolytes and other compatible solutes became a subject of great interest, not only because of the intrinsic biotechnological significance of increasing the performance of enzymes and other proteins under operational conditions, but also because of the medical relevance of finding effective suppressors of protein misfolding and aggregation. Since the pioneering studies by Nozaki and Tanford,6, 7 many researchers have made important contributions.8–11 There is now a much better understanding of the main thermodynamic features associated with protein folding/unfolding in the presence of uncharged osmolytes, but the effects on the structure and dynamics of the native and the denatured forms remain largely unexplored. In particular, further studies are necessary to clarify the relationship between protein dynamics and stabilization by compatible solutes. Previously, the effect of different solutes on a wide range of backbone motions was monitored by using several NMR methods. Small torsional fluctuations (ps-ns time scale) were studied by spin-relaxation measurements, chemical exchange on the millisecond scale was assessed by magnetization transfer experiments, and events in the second-to-minute time frame were probed with H/D exchange experiments.12 A strong correlation was established between the subnanosecond backbone motions (described by the generalized order parameter) and changes in the melting temperature (Tm) of the protein induced by different solutes. The effect of MG was also evident on the slowest time scale, providing site-specific information about the thermodynamic stability of the protein. However, motions on the millisecond time scale were little affected. Thus, there was strong evidence for a link between restriction of fast protein backbone motions and protein stabilization.
In the light of these results, we consider it important to address the following questions. Is the dynamic behavior of protein side-chains on the ns-ps timescale also important for the mechanism of protein stabilization by compatible solutes? Is the effect of stabilizing solutes on the slow timescale motions specific for the global motions of the protein, as previously suggested?13 And do stabilizing solutes affect the protein folding/unfolding pathway?
To study the effect of solutes on the dynamics of protein side-chains, we measured 13C relaxation rates of the methyl groups of a staphylococcal nuclease (SNase) variant (P117G/H124L/S128A), providing 46 probes of motion on the subnanosecond time scale. Although they are typically located in the close packed interior of proteins, methyl bearing side-chains usually display significant motions in addition to the rotation of the methyl group. Analysis of 13C relaxation data using the model-free formalism14, 15 is a common procedure to extract information about the motion of the carbon–carbon bond that connects the methyl group to the side-chain, and the rotation of the CH vectors about the symmetry axis. The methyl groups are expected to report on the effects of different solutes on the dynamics of the protein hydrophobic core in the subnanosecond time scale.
The thermodynamic equilibrium of proteins implies unfolding and refolding of the molecules, even under conditions that favor the folded form. This creates transient non-native states that represent a minute fraction of the protein population under normal conditions and allows the protein folding/unfolding pathway to be studied using NMR H/D exchange experiments. Most backbone NHs exchange with the solvent only when the protein molecule is in one of the non-native states. In the so-called EX2 exchange regime, proton exchange rates depend on the equilibrium constant between the native and non-native states.16 The motions involved in visiting these states may be local motions that expose little new surface area, or nearly global motions that expose large patches of normally buried peptide segments.16 Stabilizing solutes reduce the exchange rates of NHs,12, 13, 17 which implies a shift in the equilibrium of the protein population towards the native state. The observation of a greater effect on the NHs that exchange more slowly has been explained as a consequence of solutes opposing large increases in protein surface area, as the slower exchanging protons are expected to exchange only through large scale unfolding motions. However, not all slower-exchanging NHs require large protein motions; hence, it is not easy to establish a relationship between the effect of stabilizing solutes and exchange rates. Investigating the scale of the motions associated with NH exchange may clarify the matter and deal with our second query: is the effect of stabilizing solutes really specific for the global motions of the protein?
The dependence of individual H/D exchange rates on denaturant (urea) concentration can be used to differentiate between local and global backbone motions. The slope of this dependence, called the m-value, relates to the increase in surface exposure that occurs when the protein visits the higher energy exchange-competent state while remaining under largely native conditions (folded population around 99%).18, 19 Values close to zero are characteristic of local motions while large m-values indicate that NH exchange requires nearly global motions.20, 21 Englander and coworkers found that protein assembly proceeds via cooperative folding of protein segments which they called foldons,20 with H/D exchange measurements used to show which amides are involved in each foldon. Experiments of this type in the absence and presence of MG should address our last question regarding the possible effect of stabilizing solutes on the cooperative unfolding motions of the protein.
By addressing these questions, this work shows that stabilization of a variant of SNase by MG has little effect on side-chain dynamics but restricts local fluctuations of NHs as well as global opening motions. Moreover, there is an intriguing insight into the mechanism of stabilization in the observation of specific effects of MG on the local motions of the β-sheets in SNase.