Activity and stability of a thermostable alpha-amylase compared to its mesophilic homologue: mechanisms of thermal adaptation

To elucidate how enzymes adapt to extreme environmental conditions, a comparative study with a thermostable alpha-amylase from Bacillus licheniformis (BLA) and its mesophilic homologue from Bacillus amyloliquefaciens (BAA) was performed. We measured conformational stability, catalytic activity, and conformational fluctuations on the picosecond time scale for both enzymes as a function of temperature. The objective of this study is to analyze how these properties are related to each other. BLA shows its maximal catalytic activity at about 90-95 degrees C and a strongly reduced activity (only 20% of the maximum) at room temperature. Although B. licheniformis itself is a mesophilic organism, BLA shows an activity profile typical for a thermophilic enzyme. In contrast to this, BAA exhibits its maximal activity at about 80 degrees C but with a level of about 60% activity at room temperature. In both cases the unfolding temperatures T(m) are only 6 degrees C (BAA, T(m) = 86 degrees C) and 10 degrees C (BLA, T(m) = 103 degrees C), respectively, higher than the temperatures for maximal activity. In contrast to many previous studies on other thermophilic-mesophilic pairs, in this study a higher structural flexibility of the thermostable BLA was measured as compared to the mesophilic BAA. The findings of this study neither indicate a proportionality between the observed dynamics and the catalytic activity nor support the idea of more "rigid" thermostable proteins, as often proposed in the concept of "corresponding states".     

Chemical modification of lysine residues in Bacillus alpha-amylases: effect on activity and stability

Chemical modification of lysine residues in two bacterial alpha-amylases, a mesophilic enzyme from Bacillus amyloliquefaciens (BAA) and a thermophilic enzyme from Bacillus licheniformis (BLA) was carried out using citraconic anhydride. 13 +/- 1 residues in BAA and 10 +/- 1 residues in BLA were found modified under defined experimental conditions. Modification brought about dramatic enhancement of thermal stability of BAA and catalytic activity of BLA. Such alterations were found dependent on the temperature and pH. Results obtained on Tm, the extent of deamidation, changes in the circular dichroism (CD) spectra and kinetic parameters before and after modification are discussed in terms of their contributions to the mechanism of irreversible thermoinactivation and activity enhancement.     

Comparative studies on trifluoroethanol (TFE) state of a thermophilic alpha-amylase and its mesophilic counterpart: limited proteolysis, conformational analysis, aggregation and reactivation of the enzymes

Detailed circular dichroism (CD), scattering and quenching studies, 1-anilinonaphthalene-8-sulfonate (ANS) binding, irreversible thermoinactivation, activity measurements and proteolytic digestion of bacterial alpha-amylases have been carried out to elucidate the effect of trifluoroethanol (TFE) on the structure of these enzymes. Under high concentrations of TFE both of the alpha-amylases, a thermostable alpha-amylase from Bacillus licheniformis (BLA) and its mesophilic counterpart from Bacillus amyloliquefaciens (BAA), acquire partially folded state characterized by an enhanced content of the secondary structure (helix) and reduced tertiary structures. According to ANS binding studies, we suggest that the TFE states induced by TFE/water mixture are not the molten globule state in the alpha-amylase folding pathway. In addition, data shows significant reversible aggregation of both enzymes in TFE/water mixtures with concentration between 10 and 60% (v/v). However, reversibility is more in case of BAA. As expected, in the absence of TFE, the thermophilic enzyme compared to mesophilic enzyme, shows a greater resistance to digestion by thermolysin. With respect to fluorescence quenching by acrylamide and potassium iodide, the thermophilic enzyme, BLA, is characterized by higher structural flexibility as compared to the BAA. On the other hand, in the presence of TFE, the enzymes are digested by protease to produce large protein fragments. It is proposed that highly helical secondary structures, acquired by BAA and BLA when dissolved in aqueous TFE, prevent binding and adaptation of the protein substrate at the active site of the protease.     

Nucleotide sequence, structural investigation and homology modeling studies of a Ca2+-independent alpha-amylase with acidic pH-profile

The novel alpha-amylase purified from locally isolated strain, Bacillus sp. KR-8104, (KRA) (Enzyme Microb Technol; 2005; 36: 666-671) is active in a wide range of pH. The enzyme maximum activity is at pH 4.0 and it retains 90% of activity at pH 3.5. The irreversible thermoinactivation patterns of KRA and the enzyme activity are not changed in the presence and absence of Ca(2+) and EDTA. Therefore, KRA acts as a Ca(2+)-independent enzyme. Based on circular dichroism (CD) data from thermal unfolding of the enzyme recorded at 222 nm, addition of Ca(2+) and EDTA similar to its irreversible thermoinactivation, does not influence the thermal denaturation of the enzyme and its T(m). The amino acid sequence of KRA was obtained from the nucleotide sequencing of PCR products of encoding gene. The deduced amino acid sequence of the enzyme revealed a very high sequence homology to Bacillus amyloliquefaciens (BAA) (85% identity, 90% similarity) and Bacillus licheniformis alpha-amylases (BLA) (81% identity, 88% similarity). To elucidate and understand these characteristics of the alpha-amylase, a model of 3D structure of KRA was constructed using the crystal structure of the mutant of BLA as the platform and refined with a molecular dynamics (MD) simulation program. Interestingly enough, there is only one amino acid substitution for KRA in comparison with BLA and BAA in the region involved in the calcium-binding sites. On the other hand, there are many amino acid differences between BLA and KRA at the interface of A and B domains and around the metal triad and active site area. These alterations could have a role in stabilizing the native structure of the loop in the active site cleft and maintenance and stabilization of the putative metal triad-binding site. The amino acid differences at the active site cleft and around the catalytic residues might affect their pKa values and consequently shift its pH profile. In addition, the intrinsic fluorescence intensity of the enzyme at 350 nm does not show considerable change at pH 3.5-7.0.     

Comparison of the molten globule states of thermophilic and mesophilic alpha-amylases

In recent years great interest has been generated in the process of protein folding, and the formation of intermediates during the folding process has been proven with new experimental strategies. In the present work, we have examined the molten globule state of Bacillus licheniformis alpha-amylase (BLA) by intrinsic fluorescence and circular dichroism spectra, 1-anilino naphthalene-8-sulfonate (ANS) binding and proteolytic digestion by pepsin, for comparison to its mesophilic counterpart, Bacillus amyloliquefaciens alpha-amylase (BAA). At pH 4.0, both enzymes acquire partially folded state which show characteristics of molten globule state. They unfold in such a way that their hydrophobic surfaces are exposed to a greater extent compared to the native forms. Chemical denaturation studies by guanidine hydrochloride and proteolytic digestion with pepsin show that molten globule state of BLA is more stable than from BAA. Results from gel filtration indicate that BAA has the same compactness at pH 4.0 and 7.5. However, molten globule state of BLA is less compact than its native state. The effects of polyols such as trehalose, sorbitol and glycerol on refolding of enzymes from molten globule to native state were also studied. These polyols are effective on refolding of mesophilic alpha-amylase but only slightly effect on BLA refolding. In addition, the folding pathway and stability of intermediate state of the thermophilic and the mesophilic alpha-amylases are discussed.     

Reversible and irreversible unfolding of multi-domain proteins

In contrast to single-domain proteins unfolding of larger multi-domain proteins is often irreversible. In a comparative case study on three different multi-domain proteins (phosphoglycerate kinase: PGK and two homologous alpha-amylases: TAKA and BLA) we investigated properties of unfolded states and their ability to fold back into the native state. For this purpose guanidine hydrochloride, alkaline pH, and thermal unfolded states were characterized. Structural alterations upon unfolding and refolding transitions were monitored using fluorescence and CD spectroscopy. Static and dynamic light scattering was employed to follow aggregation processes. Furthermore, proper refolding was also investigated by enzyme activity measurements. While for PGK at least partial reversible unfolding transitions were observed in most cases, we found reversible unfolding for TAKA in the case of alkaline pH and GndHCl induced unfolding. BLA exhibits reversible unfolding only under conditions with high concentrations of protecting osmolytes (glycerol), indicating that aggregation of the unfolded state is the main obstacle to achieve proper refolding for this protein. Structural properties, such as number and size of domains, secondary structure contents and compositions within domains, and domain topology were analyzed and considered in the interpretation of differences in refolding behavior of the investigated proteins.     

Thermostability of irreversible unfolding alpha-amylases analyzed by unfolding kinetics

For most multidomain proteins the thermal unfolding transitions are accompanied by an irreversible step, often related to aggregation at elevated temperatures. As a consequence the analysis of thermostabilities in terms of equilibrium thermodynamics is not applicable, at least not if the irreversible process is fast with respect the structural unfolding transition. In a comparative study we investigated aggregation effects and unfolding kinetics for five homologous alpha-amylases, all from mesophilic sources but with rather different thermostabilities. The results indicate that for all enzymes the irreversible process is fast and the precedent unfolding transition is the rate-limiting step. In this case the kinetic barrier toward unfolding, as measured by unfolding rates as function of temperature, is the key feature in thermostability. The investigated enzymes exhibit activation energies (E(a)) between 208 and 364 kJmol(-1) and pronounced differences in the corresponding unfolding rates. The most thermostable alpha-amylase from Bacillus licheniformis (apparent transition temperature, T(1/2) approximately 100 degrees C) shows an unfolding rate which is four orders of magnitude smaller as compared with the alpha-amylase from pig pancreas (T(1/2) approximately 65 degrees C). Even with respect to two other alpha-amylases from Bacillus species (T(1/2) approximately 86 degrees C) the difference in unfolding rates is still two orders of magnitude.     

Amino acid residues stabilizing a Bacillus alpha-amylase against irreversible thermoinactivation

The alpha-amylase of Bacillus licheniformis (BLA) is stable and active at high temperature. More than 80% of its activity is retained after heat treatment at 90 degrees C for 30 min, and the optimum temperature for its activity is 80-85 degrees C. In contrast, the alpha-amylase of Bacillus amyloliquefaciens (BAA), the amino acid sequence of which shows 80% homology with that of BLA, is rapidly inactivated at 90 degrees C. Various chimeric genes were constructed from the structural genes for the two enzymes, and their products were analyzed for stability as to irreversible thermoinactivation. Two regions in the amino acid sequence of BLA comprising Gln178 (region I) and the 255th-270th residues (region II), respectively, were shown to determine the thermostability of BLA. Region I plays a major role in determining the thermostability. By means of site-directed mutagenesis of the BAA gene, deletion of Arg176 and Gly177 in region I and substitutions of alanine for Lys269 and aspartic acid for Asn266 in region II were shown to be responsible for the enhancement of the thermostability. Mutant BAAs containing the above deletion and substitutions showed almost the same thermostability as BLA as to irreversible thermoinactivation. Nevertheless, the mutant BAAs showed a temperature optimum as low as that of BAA (65 degrees C), indicating that they are still susceptible to reversible inactivation at temperatures higher than 65 degrees C.     

The thermal denaturation of ribonuclease A in aqueous-methanol solvents.

Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below -10 degrees C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70 degrees C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn.HCl at 70 degrees C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by alpha-helix, with a midpoint at 38 degrees C, was at a much higher temperature than that monitored by beta-sheet, 26 degrees C, which also corresponded to that observed by delta A286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of beta-sheet structure is decoupled from that of alpha-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of alpha-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core beta-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.     

How aggregation and conformational scrambling of unfolded states govern fluorescence emission spectra

In a case study on five homologous alpha-amylases we analyzed the properties of unfolded states as obtained from treatments with GndHCl and with elevated temperatures. In particular the wavelength of the tryptophan fluorescence emission peak (lambda(max)) is a valuable parameter to characterize properties of the unfolded state. In all cases with a typical red shift of the emission spectrum occurring during structural unfolding we observed a larger magnitude of this shift for GndHCl-induced unfolding as compared to thermal unfolding. Although a quantitative relation between aggregation and reduction of the unfolding induced red shifts cannot be given, our data indicate that protein aggregation contributes significantly to smaller magnitudes of red shifts as observed during thermal unfolding. In addition, other properties of the unfolded states, most probable structural compactness or simply differences in the conformational scrambling, also affect the magnitude of red shifts. For the irreversible unfolding alpha-amylases studied here, transition temperatures and magnitudes of red shifts are strongly depending on heating rates. Lower protein concentrations and smaller heating rates lead to larger red shifts upon thermal unfolding, indicating that under these conditions the protein aggregation is less pronounced.     

Association of heat-induced conformational change with activity loss of Rubisco.

Circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) were used to investigate the thermal conformational change associated with the activity loss of spinach Rubisco. CD and intrinsic fluorescence demonstrated a three stage thermal unfolding of Rubisco. At 25-45 degrees C, the secondary structure did not change but the tertiary and/or quaternary structure changed obviously with increased temperature. In 45-60 degrees C, the secondary structure showed much change with increased temperature and the tertiary and/or quaternary structure changed much faster. Over 60 degrees C, whole conformation changed abruptly with increased temperature and finally unfolded completely. DSC, CD and activity assays after annealing showed that the conformational change and the activity loss of Rubisco were completely reversible if the heating temperature was below 45 degrees C, partly reversible between 45 and 60 degrees C, and irreversible beyond 60 degrees C.     

Osmolytes stabilize ribonuclease S by stabilizing its fragments S protein and S peptide to compact folding-competent states

Osmolytes stabilize proteins to thermal and chemical denaturation. We have studied the effects of the osmolytes sarcosine, betaine, trimethylamine-N-oxide, and taurine on the structure and stability of the protein.peptide complex RNase S using x-ray crystallography and titration calorimetry, respectively. The largest degree of stabilization is achieved with 6 m sarcosine, which increases the denaturation temperatures of RNase S and S pro by 24.6 and 17.4 degrees C, respectively, at pH 5 and protects both proteins against tryptic cleavage. Four crystal structures of RNase S in the presence of different osmolytes do not offer any evidence for osmolyte binding to the folded state of the protein or any perturbation in the water structure surrounding the protein. The degree of stabilization in 6 m sarcosine increases with temperature, ranging from -0.52 kcal mol(-1) at 20 degrees C to -5.4 kcal mol(-1) at 60 degrees C. The data support the thesis that osmolytes that stabilize proteins, do so by perturbing unfolded states, which change conformation to a compact, folding competent state in the presence of osmolyte. The increased stabilization thus results from a decrease in conformational entropy of the unfolded state.     

Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific DeltaGmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state.     

A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (DeltaG(unf) = DeltaH - TDeltaS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for alpha-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30 degrees C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.     

Mechanisms of irreversible thermal inactivation of Bacillus alpha-amylases

Molecular mechanisms of irreversible thermal inactivation of two bacterial alpha-amylases, from the mesophile Bacillus amyloliquefaciens and from the thermophile Bacillus stearothermophilus, have been elucidated in the pH range of relevance to enzymatic catalysis. At pH 5.0, 6.5, and 8.0, B. amyloliquefaciens alpha-amylase irreversibly inactivates due to a monomolecular conformational process, formation of incorrect (scrambled) structures which subsequently undergo aggregation. At the last pH, this process can be suppressed by the presence of the substrate starch and consequently a covalent process, deamidation of asparagine and/or glutamine residues, becomes the cause of loss of enzymatic activity at 90 degrees C. Monomolecular conformational scrambling is the predominant cause of irreversible inactivation of B. stearothermophilus alpha-amylase at 90 degrees C at pH 5.0, 6.5, and 8.0. At pH 6.5 another contributing inactivation mechanism is the deamidation of amide residues, and at pH 8.0, O2 oxidation of the enzyme's cysteine residue.     

Why is one Bacillus alpha-amylase more resistant against irreversible thermoinactivation than another?

Half-lives of Bacillus alpha-amylases at 90 degrees C and pH 6.5 greatly increase in the series from Bacillus amyloliquefaciens to Bacillus stearothermophilus to Bacillus licheniformis, e.g. the difference in thermostability between the first and the third enzymes exceeds 2 orders of magnitude. This stabilization is achieved by lowering the rate constant of monomolecular conformational scrambling, which is the cause of irreversible thermoinactivation of B. amyloliquefaciens and B. stearothermophilus alpha-amylases, so that for B. licheniformis alpha-amylase, another process, deamidation of Asn/Gln residues, emerges as the cause of inactivation. The extra thermostability of the thermophilic enzyme was found to be mainly due to additional salt bridges involving a few specific lysine residues (Lys-385 and Lys-88 and/or Lys-253). These stabilizing electrostatic interactions reduce the extent of unfolding of the enzyme molecule at high temperatures, consequently making it less prone to forming incorrect (scrambled) structures and thus decreasing the overall rate of irreversible thermoinactivation. The implications of these findings for protein engineering are discussed.     

Thermodynamics and mechanism of cutinase stabilization by trehalose

Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2 degrees . Despite this stabilizing effect, trehalose also favors pathways that lead to irreversible denaturation. Stopped-flow kinetics of cutinase folding and unfolding was measured and temperature was introduced as experimental variable to assess the mechanism and thermodynamics of protein stabilization by trehalose. The main stabilizing effect of trehalose was to delay the rate constant of the unfolding of an intermediate. A full thermodynamic analysis of this step has revealed that trehalose induces the phenomenon of entropy-enthalpy compensation, but the enthalpic contribution increases more significantly leading to a net stabilizing effect that slows down unfolding of the intermediate. Regarding the molecular mechanism of stabilization, trehalose increases the compactness of the unfolded state. The conformational space accessible to the unfolded state decreases in the presence of trehalose when the unfolded state acquires residual native interactions that channel the folding of the protein. This residual structure results into less hydrophobic groups being newly exposed upon unfolding, as less water molecules are immobilized upon unfolding.     

Effect of hydrostatic pressure on conformational changes of canine milk lysozyme between the native, molten globule, and unfolded states

The effect of pressure on the unfolding of the native (N) and molten globule (MG) state of canine milk lysozyme (CML) was examined using ultraviolet (UV) spectroscopy at pH 4.5 and 2.0, respectively. It appeared that the thermally induced unfolding was promoted by the increase of pressure from atmospheric to 100 MPa, which indicates that both the N and MG states of CML unfolded with the decrease of the partial molar volume change (DeltaV). The volume changes needed for unfolding were estimated from the free energy change vs. pressure plots, and these volume changes became less negative from 20 to 60 degrees C. The DeltaV values at 25 degrees C were obtained for the N-MG (-46 cm3/mol) and MG-unfolded-state (U) transition (-40 cm3/mol). With regards to the MG-U transition, this value is contrastive to that of bovine alpha-lactalbumin (BLA) (0.9 cm3/mol), which is homologous to CML. Previous studies revealed that the MG state of CML was significantly more stable, and closer to the N state in structure, than that of BLA. In contrast to the swollen hydrophobic core of the MG state of BLA, our results suggest that the MG state of CML possesses a tightly packed hydrophobic core into which water molecules cannot penetrate.     

Linkage of proton binding to the thermal unfolding of Sso7d from the hyperthermophilic archaebacterium Sulfolobus solfataricus.

In this study the pH dependence of the thermal stability of Sso7d from Sulfolobus solfataricus is analyzed. This small globular protein of 63 residues shows a very marked dependence of thermal stability on pH: the denaturation temperature passes from 65.2 degrees C at pH 2.5 to 97.9 degrees C at pH 4.5. Analysis of the data points out that the binding of at least two protons is coupled to the thermal unfolding. By linking the proton binding to the conformational unfolding equilibrium, a thermodynamic model, which is able to describe the dependence upon the solution pH of both the excess heat capacity function and the denaturation Gibbs energy change for Sso7d, is developed. The decreased stability in very acid conditions is due to the binding of two protons on identical and noninteracting sites of the unfolded state. Actually, such sites are two carboxyl groups possessing very low pKa values in the native structure, probably involved in salt-bridges on the protein surface.     

Thermal unfolding of Apo and Holo Desulfovibrio desulfuricans flavodoxin: cofactor stabilizes folded and intermediate states

We here compare thermal unfolding of the apo and holo forms of Desulfovibrio desulfuricans flavodoxin, which noncovalently binds a flavin mononucleotide (FMN) cofactor. In the case of the apo form, fluorescence and far-UV circular dichroism (CD) detected transitions are reversible but do not overlap (T(m) of 50 and 60 degrees C, respectively, pH 7). The thermal transitions for the holo form follow the same pattern but occur at higher temperatures (T(m) of 60 and 67 degrees C for fluorescence and CD transitions, respectively, pH 7). The holoprotein transitions are also reversible and exhibit no protein concentration dependence (above 10 microM), indicating that the FMN remains bound to the polypeptide throughout. Global analysis shows that the thermal reactions for both apo and holo forms proceed via an equilibrium intermediate that has approximately 90% nativelike secondary structure and significant enthalpic stabilization relative to the unfolded states. Incubation of unfolded holoflavodoxin at high temperatures results in FMN dissociation. Rebinding of FMN at these conditions is nominal, and therefore, cooling of holoprotein heated to 95 degrees C follows the refolding pathway of the apo form. However, FMN readily rebinds to the apoprotein at lower temperatures. We conclude that (1) a three-state thermal unfolding behavior appears to be conserved among long- and short-chain, as well as apo and holo forms of, flavodoxins and (2) flavodoxin's thermal stability (in both native and intermediate states) is augmented by the presence of the FMN cofactor.