Direct characterization of the folded, unfolded and urea-denatured states of the C-terminal domain of the ribosomal protein L9

The stability of the isolated C-terminal domain of the ribosomal protein L9 (CTL9) is strongly dependent upon pH. Below pH 4.2, the folded and unfolded states are both populated significantly. Their interconversion is slow on the NMR chemical shift time-scale and separate, well-resolved resonances from each state are observed. This allows the hydrodynamic properties of both states to be studied under identical conditions by using pulse field gradient NMR experiments. Hydrodynamic radii of the folded, unfolded and urea denatured protein molecules at pD 3.8 have been derived. The acid-denatured protein has a significantly smaller hydrodynamic radius, 28.2A, compared to that of the urea-denatured protein, which is 33.6A at pD 3.8. Far-UV CD spectra show that there is more residual secondary structure retained in the acid-denatured ensemble than in the urea-denatured one. ANS binding experiments and analysis of the CD data show that this acid-denatured species is not a molten globule state. Diffusion measurements of CTL9 were conducted over the pD range from 2.1 to 7.0. The hydrodynamic radii of both the folded and the acid-unfolded protein start to increase below pD 4, with the radius of hydration of the acid-unfolded state increasing from 25.1A at pD 4.2 to 33.5A at pD 2.1. The hydrodynamic radius of the urea-denatured protein is much less sensitive to pH. The unfolded protein at pD 2.1, no urea, has almost the same hydrodynamic radius as the urea-denatured protein at pD 3.8. The CD spectra, however, show significant differences in residual secondary structure, and the acid-denatured state contains more structure.     

Specificity of the initial collapse in the folding of the cold shock protein

The two-state folding reaction of the cold shock protein from Bacillus caldolyticus (Bc-Csp) is preceded by a rapid chain collapse. A fast shortening of intra-protein distances was revealed by F?rster resonance energy transfer (FRET) measurements with protein variants that carried individual pairs of donor and acceptor chromophores at various positions along the polypeptide chain. Here we investigated the specificity of this rapid compaction. Energy transfer experiments that probed the stretching of strand beta2 and the close approach between the strands beta1 and beta2 revealed that the beta1-beta2 hairpin is barely formed in the collapsed form, although it is native-like in the folding transition state of Bc-Csp. The time course of the collapse could not be resolved by pressure or temperature jump experiments, indicating that the collapsed and extended forms are not separated by an energy barrier. The co-solute (NH4)2SO4 stabilizes both native Bc-Csp and the collapsed form, which suggests that the large hydrated SO4(2-) ions are excluded from the surface of the collapsed form in a similar fashion as they are excluded from folded Bc-Csp. Ethylene glycol increases the stability of proteins because it is excluded preferentially from the backbone, which is accessible in the unfolded state. The collapsed form of Bc-Csp resembles the unfolded form in its interaction with ethylene glycol, suggesting that in the collapsed form the backbone is still accessible to water and small molecules. Our results thus rule out that the collapsed form is a folding intermediate with native-like chain topology. It is better described as a mixture of compact conformations that belong to the unfolded state ensemble. However, some of its structural elements are reminiscent of the native protein.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 °C) and low (20 °C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T_m (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 °C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T_m was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 °C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T_m by 10.5 °C. In mineral media at 20 °C the corresponding changes of T_m were almost negligible. After a temperature shift from 40 to 20 °C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

Key role of coulombic interactions for the folding transition state of the cold shock protein

The cold shock protein CspB shows a five-stranded beta-sheet structure, and it folds rapidly via a native-like transition state. A previous Phi value analysis showed that most of the residues with Phi values close to one reside in strand beta1, and two of them, Lys5 and Lys7 are partially exposed charged residues. To elucidate how coulombic interactions of these two residues contribute to the energetic organisation of the folding transition state we performed comparative folding experiments in the presence of an ionic denaturant (guanidinium chloride) and a non-ionic denaturant (urea) and a double-mutant analysis. Lys5 contributes 6.6 kJ mol(-1) to the stability of the transition state, and half of it originates from screenable coulombic interactions. Lys7 contributes 5.3 kJ mol(-1), and 3.4 kJ mol(-1) of it are screened by salt. In the folded protein Lys7 interacts with Asp25, and the screenable coulombic interaction between these two residues is fully formed in the transition state. This suggests that long-range coulombic interactions such as those originating from Lys5 and Lys7 of CspB can be important for organizing and stabilizing native-like structure early in protein folding.     

Increased folding stability of TEM-1 beta-lactamase by in vitro selection

In vitro selections of stabilized proteins lead to more robust enzymes and, at the same time, yield novel insights into the principles of protein stability. We employed Proside, a method of in vitro selection, to find stabilized variants of TEM-1 β-lactamase from Escherichia coli. Proside links the increased protease resistance of stabilized proteins to the infectivity of a filamentous phage. Several libraries of TEM-1 β-lactamase variants were generated by error-prone PCR, and variants with increased protease resistance were obtained by raising temperature or guanidinium chloride concentration during proteolytic selections. Despite the small size of phage libraries, several strongly stabilizing mutations could be obtained, and a manual combination of the best shifted the profiles for thermal unfolding and temperature-dependent inactivation of β-lactamase by almost 20 °C to a higher temperature. The wild-type protein unfolds in two stages: from the native state via an intermediate of the molten-globule type to the unfolded form. In the course of the selections, the native protein was stabilized by 27 kJ mol^− 1 relative to the intermediate and the cooperativity of unfolding was strongly increased. Three of our stabilizing replacements (M182T, A224V, and R275L) had been identified independently in naturally occurring β-lactamase variants with extended substrate spectrum. In these variants, they acted as global suppressors of destabilizations caused by the mutations in the active site. The comparison between the crystal structure of our best variant and the crystal structure of the wild-type protein indicates that most of the selected mutations optimize helices and their packing. The stabilization by the E147G substitution is remarkable. It removes steric strain that originates from an overly tight packing of two helices in the wild-type protein. Such unfavorable van der Waals repulsions are not easily identified in crystal structures or by computational approaches, but they strongly reduce the conformational stability of a protein.     

The high-resolution NMR structure of the early folding intermediate of the Thermus thermophilus ribonuclease H

Elucidation of the high-resolution structures of folding intermediates is a necessary but difficult step toward the ultimate understanding of the mechanism of protein folding. Here, using hydrogen-exchange-directed protein engineering, we populated the folding intermediate of the Thermus thermophilus ribonuclease H, which forms before the rate-limiting transition state, by removing the unfolded regions of the intermediate, including an α-helix and two β-strands (51 folded residues). Using multidimensional NMR, we solved the structure of this intermediate mimic to an atomic resolution (backbone rmsd, 0.51 Å). It has a native-like backbone topology and shows some local deviations from the native structure, revealing that the structure of the folded region of an early folding intermediate can be as well defined as the native structure. The topological parameters calculated from the structures of the intermediate mimic and the native state predict that the intermediate should fold on a millisecond time scale or less and form much faster than the native state. Other factors that may lead to the slow folding of the native state and the accumulation of the intermediate before the rate-limiting transition state are also discussed.     

Oligomerization of the SPP1 scaffolding protein

Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His₆) and purified. The protein is an α-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His₆ is structurally a dimer of dimers. Incubation at temperatures above 37 °C correlated with a reduction of its α-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 °C. Refolding of gp11-His₆ thermally denatured at 65 °C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (∼ 90%) and a smaller fraction of 2.4 S dimers (∼ 10%). This population of gp11-His₆ was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His₆ showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His₆.     

The denatured state of N-PGK is compact and predominantly disordered

The organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I_b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I_k) and denatured (D)]. The transition to I_b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I_b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I_b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics of I_b indicate that it forms from the fully denatured state within 100 μs, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I_b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology.     

Temperature-dependent Hammond behavior in a protein-folding reaction: analysis of transition-state movement and ground-state effects

Characterization of the transition-state ensemble and the nature of the free-energy barrier for protein folding are areas of intense activity and some controversy. A key issue that has emerged in recent years is the width of the free-energy barrier and the susceptibility of the transition state to movement. Here we report denaturant-induced and temperature-dependent folding studies of a small mixed α-β protein, the N-terminal domain of L9 (NTL9). The folding of NTL9 was determined using fluorescence-detected stopped-flow fluorescence measurements conducted at seven different temperatures between 11 and 40 °C. Plots of the log of the observed first-order rate constant versus denaturant concentration, “chevron plots,” displayed the characteristic V shape expected for two-state folding. There was no hint of deviation from linearity even at the lowest denaturant concentrations. The relative position of the transition state, as judged by the Tanford β parameter, β_T, shifts towards the native state as the temperature is increased. Analysis of the temperature dependence of the kinetic and equilibrium m values indicates that the effect is due to significant movement of the transition state and also includes a contribution from temperature-dependent ground-state effects. Analysis of the Leffler plots, plots of ΔG^‡versus ΔG°, and their cross-interaction parameters confirms the transition-state movement. Since the protein is destabilized at high temperature, the shift represents a temperature-dependent Hammond effect. This provides independent confirmation of a recent theoretical prediction. The magnitude of the temperature-denaturant cross-interaction parameter is larger for NTL9 than has been reported for the few other cases studied. The implications for temperature-dependent studies of protein folding are discussed.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Desolvation and Development of Specific Hydrophobic Core Packing during Im7 Folding

Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val→Thr or Phe→Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Φ-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (> 2.5 kJ mol^− 1) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.     

Studies of the unfolding of an unstable mutant of staphylococcal nuclease: Evidence for low temperature unfolding and compactness of the high temperature unfolded state

Fluorescence and circular dichroism data as a function of temperature were obtained to characterize the unfolding of nuclease A and two of its less stable mutants. These spectroscopic data were obtained with a modified instrument that enables the nearly simultaneous detection of both fluorescence and CD data on the same sample. A global analysis of these multiple datasets yielded an excellent fit of a model that includes a change in the heat capacity change, ΔC_p, between the unfolded and native states. This analysis gives a ΔC_p of 2.2 kcal/mol/·K for thermal unfolding of the WT protein and 1.3 and 1.8 kcal/mol/K for the two mutants. These ΔC_p values are consistent with significant population of the cold unfolded state at ∼0°C. Independent evidence for the existence of a cold unfolded state is the observation of a separately migrating peak in size exclusion chromatography. The new chromatographic peak is seen near 0°C, has a partition coefficient corresponding to a larger hydrodynamic radius, and shows a red-shifted fluorescence spectrum, as compared to the native protein. Data also indicate that the high-temperature unfolded form of mutant nuclease is relatively compact. Size exclusion chromatography shows the high temperature unfolded form to have a hydrodynamic radius that is larger than that for the native form, but smaller than that for the urea or pH-induced unfolded forms. Addition of chemical denaturants to the high-temperature unfolded form causes a further unfolding of the protein, as indicated by an increase in the apparent hydrodynamic radius and a decrease in the rotational correlation time for Trp140 (as determined by fluorescence anisotropy decay measurements). Proteins 28:227–240, 1997 © 1997 Wiley-Liss Inc.     

Kinetic folding of Haloferax volcanii and Escherichia coli dihydrofolate reductases: haloadaptation by unfolded state destabilization at high ionic strength

Salts affect protein stability by multiple mechanisms (e.g., the Hofmeister effect, preferential hydration, electrostatic effects and weak ion binding). These mechanisms can affect the stability of both the native state and the unfolded state. Previous equilibrium stability studies demonstrated that KCl stabilizes dihydrofolate reductases (DHFRs) from Escherichia coli (ecDHFR, E. coli DHFR) and Haloferax volcanii (hvDHFR1, H. volcanii DHFR encoded by the hdrA gene) with similar efficacies, despite adaptation to disparate physiological ionic strengths (0.2 M versus 2 M). Kinetic studies can provide insights on whether equilibrium effects reflect native state stabilization or unfolded state destabilization. Similar kinetic mechanisms describe the folding of urea-denatured ecDHFR and hvDHFR1: a 5-ms stopped-flow burst-phase species that folds to the native state through two sequential intermediates with relaxation times of 0.1-3 s and 25-100 s. The latter kinetic step is very similar to that observed for the refolding of hvDHFR1 from low ionic strength. The unfolding of hvDHFR1 at low ionic strength is relatively slow, suggesting kinetic stabilization as observed for some thermophilic enzymes. Increased KCl concentrations slow the urea-induced unfolding of ecDHFR and hvDHFR1, but much less than expected from equilibrium studies. Unfolding rates extrapolated to 0 M denaturant, k_unf(H₂O), are relatively independent of ionic strength, demonstrating that the KCl-induced stabilization of ecDHFR and hvDHFR1 results predominantly from destabilization of the unfolded state. This supports the hypothesis from previous equilibrium studies that haloadaptation harnesses the effects of elevated salt concentrations on the properties of the aqueous solvent to enhance protein stability.     

Coulomb forces control the density of the collapsed unfolded state of barstar

Although it has been recently shown that unfolded polypeptide chains undergo a collapse on transfer from denaturing to native conditions, the forces determining the dynamics and the size of the collapsed form have not yet been understood. Here, we use single-molecule fluorescence resonance energy transfer experiments on the small protein barstar to characterize the unfolded chain in guanidinium chloride (GdmCl) and urea. The unfolded protein collapses on decreasing the concentration of denaturants. Below the critical concentration of 3.5 M denaturant, the collapse in GdmCl leads to a more dense state than in urea. Since it is known that GdmCl suppresses electrostatic interactions, we infer that Coulomb forces are the dominant forces acting in the unfolded barstar under native conditions. This hypothesis is clearly buttressed by the finding of a compaction of the unfolded barstar by addition of KCl at low urea concentrations.     

Downhill versus Barrier-Limited Folding of BBL 1: Energetic and Structural Perturbation Effects upon Protonation of a Histidine of Unusually Low pKa

A dispersion of melting temperatures at pH 5.3 for individual residues of the BBL protein domain has been adduced as evidence for barrier-free downhill folding. Other members of the peripheral subunit domain family fold cooperatively at pH 7. To search for possible causes of anomalies in BBL's denaturation behavior, we measured the pH titration of individual residues by heteronuclear NMR. At 298 K, the pK_a of His142 was close to that of free histidine at 6.47 ± 0.04, while that of the more buried His166 was highly perturbed at 5.39 ± 0.02. Protonation of His166 is thus energetically unfavorable and destabilizes the protein by ∼ 1.5 kcal/mol. Changes in C^α secondary shifts at pH 5.3 showed a decrease in helicity of the C-terminus of helix 2, where His166 is located, which was accompanied by a measured decrease of 1.1 ± 0.2 kcal/mol in stability from pH 7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only ∼ 1% of the structurally perturbed state will be present at the biologically relevant pH 7.6. Experiments at pH 5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pK_a induced by changes in temperature will have near-maximal effects on pH-dependent conformational equilibria and on propagation of experimental error. Accordingly, conventional barrier-limited folding predicts some dispersion of measured thermal unfolding curves of individual residues at pH 5.3.     

Thermodynamic and Kinetic Characterization of a Germ Line Human λ6 Light-Chain Protein: The Relation between Unfolding and Fibrillogenesis

Proteins encoded by the gene segment 6a of the λ variable light-chain repertoire are strongly associated with amyloid deposition. 6aJL2 is a model protein constructed with the predicted sequences encoded by the 6a and JL2 germ line genes. In this work, we characterized the urea- and temperature-induced unfolding of 6aJL2. In the short time scale, spectroscopic, hydrodynamic and calorimetric experiments were compatible with a two-state transition. Furthermore, ΔG, m and the midpoint urea concentration obtained from equilibrium experiments were compatible with those obtained from kinetic experiments. Since fibril formation is a slow process, samples were also incubated for longer times. After incubation for several hours at 37 °C, spectroscopic, hydrodynamic and calorimetric experiments revealed the presence of a partially unfolded off-pathway intermediate around the midpoint urea concentration (1.5-3.0 M urea). In vitro fibrillogenesis assays show that the maximum growth rate for fibril formation and the minimum lag time were obtained at urea concentrations where the partially unfolded state was populated (2.5 M urea at 37 °C). This indicates that this partially unfolded state is critical for in vitro fibril formation. Concentration-dependent kinetics and hydrodynamic properties of the intermediate were consistent with a soluble oligomeric state. The intermediate is formed around the midpoint urea concentration, where the native and unfolded states are equally populated and their rate of interconversion is the slowest. This situation may promote the slow accumulation of an intermediate state that is prone to aggregate.     

Apomyoglobin stability as dependent on urea concentration and temperature at two pH values

Equilibrium unfolding of apomyoglobin (ApoMb) in the presence of urea was studied as dependent on the temperature (5–2°C) at two pH values (5.7 and 6.2). Thermodynamic parameters of ApoMb transition from the native to the unfolded state were estimated under various conditions. Conformational changes in ApoMb were detected by tryptophan fluorescence and far-UV circular dichroism. The ApoMb stability and the cooperativity of its unfolding at 5°C were considerably lower than at other temperatures at both pH values, where ApoMb is in the native conformation.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 330–335.Original Russian Text Copyright © 2005 by Baryshnikova, Sharapov, Kashparov, Ilyina, Bychkova.     

Thermodynamically stable aggregation-resistant antibody domains through directed evolution

Protein aggregates are usually formed by interactions between unfolded or partially unfolded species, and often occur when a protein is denatured by, for example, heat or low pH. In earlier work, we used a Darwinian selection strategy to create human antibody variable domains that resisted heat aggregation. The repertoires of domains were displayed on filamentous phage and denatured (at 80 °C in pH 7.4), and folded domains were selected by binding to a generic ligand after cooling. This process appeared to select for domains with denatured states that resisted aggregation, but the domains only had low free energies of folding (ΔG_N-D^o = 15-20 kJ/mol at 25 °C in pH 7.4). Here, using the same phage repertoire, we have extended the method to the selection of domains resistant to acid aggregation. In this case, however, the thermodynamic stabilities of selected domains were higher than those selected by thermal denaturation (under both neutral and acidic conditions; ΔG_N-D^o = 26-47 kJ/mol at 25 °C in pH 7.4, or ΔG_N-D^o = 27-34 kJ/mol in pH 3.2). Furthermore, we identified a key determinant (Arg28) that increased the aggregation resistance of the denatured states of the domains at low pH without compromising their thermodynamic stabilities. Thus, the selection process yielded domains that combined thermodynamic stability and aggregation-resistant unfolded states. We suggest that changes to these properties are controlled by the extent to which the folding equilibrium is displaced during the process of selection.     

Structural investigation of cold activity and regulation of aspartate carbamoyltransferase from the extreme psychrophilic bacterium Moritella profunda

Aspartate carbamoyltransferase (EC 2.1.3.2) is extensively studied as a model for cooperativity and allosteric regulation. The structure of the Escherichia coli enzyme has been thoroughly analyzed by X-ray crystallography, and recently the crystal structures of two hyperthermophilic ATCases of the same structural class have been characterized. We here report the detailed functional and structural investigation of the ATCase from the psychrophilic deep sea bacterium Moritella profunda. Our analysis indicates that the enzyme conforms to the E. coli model in that two allosteric states exist that are influenced by similar homotropic interactions. The heterotropic properties differ in that CTP and UTP inhibit the holoenzyme, but ATP seems to exhibit a dual regulatory pattern, activating the enzyme at low concentrations and inhibiting it in the mM range. The crystal structure of the unliganded M. profunda ATCase shows resemblance to a more extreme T state reported previously for an E. coli ATCase mutant. A detailed molecular analysis reveals potential features of adaptation to cold activity and cold regulation. Moreover, M. profunda ATCase presents similarities with certain mutants of E. coli ATCase altered in their kinetic properties or temperature relationships. Finally, structural and functional comparison of ATCases across the full physiological temperature range agrees with an important, but fundamentally different role for electrostatics in protein adaptation at both extremes, i.e. an increased stability through the formation of ion pairs and ion pair networks at high physiological temperatures, and an increased flexibility through enhanced protein solvation at low temperatures.