Search

Flavodoxins are classified in two groups according to the presence or absence of a approximately 20-residue loop of unknown function. In the accompanying paper (36), we have shown that the differentiating loop from the long-chain Anabaena PCC 7119 flavodoxin is a peripheral structural element that can be removed without preventing the proper folding of the apoprotein. Here we investigate the role played by the loop in the stability and folding mechanism of flavodoxin by comparing the equilibrium and kinetic behavior of the full-length protein with that of loop-lacking, shortened variants. We show that, when the loop is removed, the three-state equilibrium thermal unfolding of apoflavodoxin becomes two-state. Thus, the loop is responsible for the complexity shown by long-chain apoflavodoxins toward thermal denaturation. As for the folding reaction, both shortened and wild type apoflavodoxins display three-state behavior but their folding mechanisms clearly differ. Whereas the full-length protein populates an essentially off-pathway transient intermediate, the additional state observed in the folding of the shortened variant analyzed seems to be simply an alternative native conformation. This finding suggests that the long loop may also be responsible for the accumulation of the kinetic intermediate observed in the full-length protein. Most revealing, however, is that the influence of the loop on the overall conformational stability of apoflavodoxin is quite low and the natively folded shortened variant Delta(120-139) is almost as stable as the wild type protein. The fact that the loop, which is not required for a proper folding of the polypeptide, does not even play a significant role in increasing the conformational stability of the protein supports our proposal (36) that the differentiating loop of long-chain flavodoxins may be related to a recognition function, rather than serving a structural purpose.

Flavodoxins are well known one-domain α/β electron-transfer proteins that, according to the presence or absence of a ∼20-residue loop splitting the fifth β-strand of the central β-sheet, have been classified in two groups: long and short-chain flavodoxins, respectively. Although the flavodoxins have been extensively used as models to study electron transfer, ligand binding, protein stability and folding issues, the role of the loop has not been investigated. We have constructed two shortened versions of the long-chain Anabaena flavodoxin in which the split β-strand has been spliced to remove the original loop. The two variants have been carefully analyzed using various spectroscopic and hydrodynamic criteria, and one of them is clearly well folded, indicating that the long loop is a peripheral element of the structure of long flavodoxins. However, the removal of the loop (which is not in contact with the cofactor in the native structure) markedly decreases the affinity of the apoflavodoxin-FMN complex. This seems related to the fact that, in long flavodoxins, the adjacent tyrosine-bearing FMN binding loop (which is longer and thus more flexible than in short flavodoxins) is stabilized in its competent conformation by interactions with the excised loop. The modest role played by the long loop of long flavodoxins in the structure of these proteins (and in its conformational stability, see Lopez-Llano, J., Maldonado, S., Jain, S., Lostao, A., Godoy-Ruiz, R., Sanchez-Ruiz, Cortijo, M., Fernandez-Recio, J., and Sancho, J. (2004) J. Biol. Chem. 279, 47184–47191) opens the possibility that its conservation in so many species is related to a functional role yet to be discovered. In this respect, we discuss the possibility that the long loop is involved in the recognition of some flavodoxin partners. In addition, we report on a structural feature of flavodoxins that could indicate that the short flavodoxins derive from the long ones.

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.

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal α/β protein organized in three layers: a central β-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (kex < 10−1 min−1). According to their distribution in the structure, protein stability is highest on the β-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other α/β (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central β-strands and in helix 5 is common to the cores. Proteins 2001;43:476–488. © 2001 Wiley-Liss, Inc.

Flavodoxins are well known one-domain alpha/beta electron-transfer proteins that, according to the presence or absence of a approximately 20-residue loop splitting the fifth beta-strand of the central beta-sheet, have been classified in two groups: long and short-chain flavodoxins, respectively. Although the flavodoxins have been extensively used as models to study electron transfer, ligand binding, protein stability and folding issues, the role of the loop has not been investigated. We have constructed two shortened versions of the long-chain Anabaena flavodoxin in which the split beta-strand has been spliced to remove the original loop. The two variants have been carefully analyzed using various spectroscopic and hydrodynamic criteria, and one of them is clearly well folded, indicating that the long loop is a peripheral element of the structure of long flavodoxins. However, the removal of the loop (which is not in contact with the cofactor in the native structure) markedly decreases the affinity of the apoflavodoxin-FMN complex. This seems related to the fact that, in long flavodoxins, the adjacent tyrosine-bearing FMN binding loop (which is longer and thus more flexible than in short flavodoxins) is stabilized in its competent conformation by interactions with the excised loop. The modest role played by the long loop of long flavodoxins in the structure of these proteins (and in its conformational stability, see Lopez-Llano, J., Maldonado, S., Jain, S., Lostao, A., Godoy-Ruiz, R., Sanchez-Ruiz, Cortijo, M., Fernandez-Recio, J., and Sancho, J. (2004) J. Biol. Chem. 279, 47184-47191) opens the possibility that its conservation in so many species is related to a functional role yet to be discovered. In this respect, we discuss the possibility that the long loop is involved in the recognition of some flavodoxin partners. In addition, we report on a structural feature of flavodoxins that could indicate that the short flavodoxins derive from the long ones.

Flavodoxins are classified in two groups according to the presence or absence of a approximately 20-residue loop of unknown function. In the accompanying paper (36), we have shown that the differentiating loop from the long-chain Anabaena PCC 7119 flavodoxin is a peripheral structural element that can be removed without preventing the proper folding of the apoprotein. Here we investigate the role played by the loop in the stability and folding mechanism of flavodoxin by comparing the equilibrium and kinetic behavior of the full-length protein with that of loop-lacking, shortened variants. We show that, when the loop is removed, the three-state equilibrium thermal unfolding of apoflavodoxin becomes two-state. Thus, the loop is responsible for the complexity shown by long-chain apoflavodoxins toward thermal denaturation. As for the folding reaction, both shortened and wild type apoflavodoxins display three-state behavior but their folding mechanisms clearly differ. Whereas the full-length protein populates an essentially off-pathway transient intermediate, the additional state observed in the folding of the shortened variant analyzed seems to be simply an alternative native conformation. This finding suggests that the long loop may also be responsible for the accumulation of the kinetic intermediate observed in the full-length protein. Most revealing, however, is that the influence of the loop on the overall conformational stability of apoflavodoxin is quite low and the natively folded shortened variant Delta(120-139) is almost as stable as the wild type protein. The fact that the loop, which is not required for a proper folding of the polypeptide, does not even play a significant role in increasing the conformational stability of the protein supports our proposal (36) that the differentiating loop of long-chain flavodoxins may be related to a recognition function, rather than serving a structural purpose.

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.

Flavodoxins are one domain α β electron transfer proteins that participate in photosynthetic reactions. All flavodoxins carry a molecule of flavin mononucleotide (FMN), non-covalently bound, that confers redox properties to the protein. There are two structurally distinct flavodoxins, short ones and long flavodoxins; the latter contain an extra loop with unknown function. We have undertaken the study of the stability and folding of the apoflavodoxin from Anabaena (a long flavodoxin) and the analysis of the interaction between the apoflavodoxin and FMN. Our studies indicate that apoflavodoxin folds in a few seconds to a form that is competent in FMN binding. The stability of this apoflavodoxin is low and its urea denaturation can be described by a two-state mechanism. The role of the different parts of the apoflavodoxin is the stability and structure of the whole protein is being investigated using mutagenesis and specific cleavage to generate apoflavodoxin fragments. The X-ray structure of apoflavodoxin is very similar to that of its complex with FMN, the main difference being the conformation of the two aromatic residues that sandwich FMN in the complex. In apoflavodoxin these groups interact with each other so closing the FMN binding site. Despite this fact, apoflavodoxin binds FMN tightly and rapidly, and the resulting holoflavodoxin displays a high conformational stability. We have found that one role of the aromatic residues that interact with FMN is to help to retain bound the reduced form of the cofactor whose complex with apoflavodoxin is otherwise too weak.

We have destabilized apoflavodoxin by site-specific excision of its C-terminal helix. The resulting flavodoxin fragment (Fld1-149) is compact and monomeric at pH 7.0, with spectroscopic properties of a molten globule and a low conformational stability. To study if Fld1-149 is cooperatively stabilized, we have measured the equilibrium urea unfolding by fluorescence, circular dichroism, and size-exclusion chromatography. The three techniques produced coincident unfolding curves. Furthermore, the thermal unfolding seems also to be cooperative as the same temperature of half-denaturation is obtained using fluorescence and circular dichroism. Fld1-149 displays cold denaturation. The equilibrium properties of Fld1-149 demonstrate that molten globules lacking well-defined tertiary interactions can still be cooperatively stabilized and that cooperatively may appear in protein conformations of very low stability. This suggests that protein folding intermediates, can, in principle, be cooperatively stabilized.