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Knowledge of protein stability principles provides a means to increase protein stability in a rational way. Here we explore the feasibility of stabilizing proteins by replacing solvent-exposed hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or GLN: The rationale behind this is a previous observation that, in some cases, neutral hydrogen bonds may be more stable that charged ones. We identified, in the apoflavodoxin from Anabaena PCC 7119, three surface-exposed aspartate or glutamate residues involved in hydrogen bonding with a single partner and we mutated them to asparagine or glutamine, respectively. The effect of the mutations on apoflavodoxin stability was measured by both urea and temperature denaturation. We observed that the three mutant proteins are more stable than wild-type (on average 0.43 kcal/mol from urea denaturation and 2.8 degrees C from a two-state analysis of fluorescence thermal unfolding data). At high ionic strength, where potential electrostatic repulsions in the acidic apoflavodoxin should be masked, the three mutants are similarly more stable (on average 0.46 kcal/mol). To rule out further that the stabilization observed is due to removal of electrostatic repulsions in apoflavodoxin upon mutation, we analysed three control mutants and showed that, when the charged residue mutated to a neutral one is not hydrogen bonded, there is no general stabilizing effect. Replacing hydrogen-bonded charged Asp or Glu residues by Asn or Gln, respectively, could be a straightforward strategy to increase protein stability.

Electrostatic contributions to the conformational stability of apoflavodoxin were studied by measurement of the proton and salt-linked stability of this highly acidic protein with urea and temperature denaturation. Structure-based calculations of electrostatic Gibbs free energy were performed in parallel over a range of pH values and salt concentrations with an empirical continuum method. The stability of apoflavodoxin was higher near the isoelectric point (pH 4) than at neutral pH. This behavior was captured quantitatively by the structure-based calculations. In addition, the calculations showed that increasing salt concentration in the range of 0 to 500 mM stabilized the protein, which was confirmed experimentally. The effects of salts on stability were strongly dependent on cationic species: K + , Na + , Ca 2+ , and Mg 2+ exerted similar effects, much different from the effect measured in the presence of the bulky choline cation. Thus cations bind weakly to the negatively charged surface of apoflavodoxin. The similar magnitude of the effects exerted by different cations indicates that their hydration shells are not disrupted significantly by interactions with the protein.Site-directed mutagenesis of selected residues and the analysis of truncation variants indicate that cation binding is not site-specific and that the cation-binding regions are located in the central region of the protein sequence. Three-state analysis of the thermal denaturation indicates that the equilibrium intermediate populated during thermal unfolding is competent to bind cations. The unusual increase in the stability of apoflavodoxin at neutral pH affected by salts is likely to be a common property among highly acidic proteins.

Proteins perform many useful molecular tasks, and their biotechnological use continues to increase. As protein activity requires a stable native conformation, protein stabilisation is a major scientific and practical issue. Towards that end, many successful protein stabilisation strategies have been devised in recent years. In most cases, model proteins with a two-state folding equilibrium have been used to study and demonstrate protein stabilisation. Many proteins, however, display more complex folding equilibria where stable intermediates accumulate. Stabilising these proteins requires specifically stabilising the native state relative to the intermediates, as these are expected to lack activity. Here we discuss how to investigate the 'relevant' stability of proteins with equilibrium intermediates and propose a way to dissect the contribution of side chain interactions to the overall stability into the 'relevant' and 'nonrelevant' terms. Examples of this analysis performed on apoflavodoxin and in a single-chain mini antibody are presented. STABILISATION OF PROTEINS WITH A TWO-STATE EQUILIBRIUMThe conformational stability of a protein is the free energy difference of the native/denatured equilibrium.Where no intermediates complicate this equilibrium, the stability can be easily measured from thermal or chemical denaturation [1]. Fuelled by the interest in protein stability, small model proteins have been used to investigate both the principles and practical strategies of protein stabilisation [2,3]. Although some basic questions regarding what stabilises proteins may not be settled [4,5] there are now various ways to attempt, with a reasonable probability of success, the increase of protein stability from a judicious analysis of protein structure [6]. The question is, Are these strategies similarly useful to stabilise proteins with more complex equilibria? THE 'RELEVANT' CONFORMATIONAL STABILITY OF PROTEINS WITH COMPLEX EQUILIBRIA: THE THREE-STATE CASELet us consider a simple three-state folding equilibrium with a single intermediate conformation appearing at mildly denaturing conditions (e.g., moderate urea concentration or moderately high temperatures) before the full denaturation takes place.For proteins of this kind, the conformational stability is made of two terms that, respectively, represent the stability of the native conformation relative to the intermediate (∆G NI ) and that of the intermediate relative to the denatured state (∆G ID ). However, provided the intermediate is no longer active (and the odds are it will not be), the 'relevant' conformational stability is given by just the first term, ∆G NI . The point is that the energetics of NI equilibria are so poorly understood that it is not clear whether the strategies found to stabilise two-state proteins will work well for proteins with equilibrium intermediates. If the energetics of protein intermediates are close to those of denatured states, there is no problem; but if, energetically, intermediates are not very different from the native state ...

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The active form of many proteins is a non-covalent complex between a polypeptide (the apoprotein) and a cofactor. Most flavoproteins are in this category (1). Among them, flavodoxins have been widely used to quantitate the energetics of apoprotein-cofactor complexes (2-7) because they can be conveniently overexpressed, deprived from the FMN cofactor, and reconstituted. Studies carried out on several flavodoxins indicate that a large part of the binding energy derives from interactions of the phosphate moiety of FMN with hydrogen donors situated at the N terminus of the first ␣-helix (Fig.

The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small.

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