Active-site copper and zinc ions modulate the quaternary structure of prokaryotic Cu,Zn superoxide dismutase

The influence of the constitutive metal ions on the equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase has been studied for the wild-type and for two mutant protein forms bearing a negative charge in the amino acid clusters at the dimer association interface. Depletion of copper and zinc dissociates the two mutant proteins into monomers, which reassemble toward the dimeric state upon addition of stoichiometric amounts of zinc. Pressure-dependent dissociation is observed for the copper-depleted wild-type and mutated enzymes, as monitored by the fluorescence shift of a unique tryptophan residue located at the subunit association interface. The spectral shift occurs slowly, reaching a plateau after 15-20 minutes, and is fully reversible. The recovery of the original fluorescence properties, after decompression, is fast (less than four minutes), suggesting that the isolated subunit has a relatively stable structure, and excluding the presence of stable intermediates during the dimer-monomer transition. The dimer dissociation process is still incomplete at 6.5 kbar for the copper-depleted wild-type and mutated enzymes, at variance with what is generally observed for oligomeric proteins that dissociate below 3 kbar. Measurement of the degree of dissociation, at two different protein concentrations, allows us to calculate the standard volume variation upon association, Delta V, and the dissociation constant K(d0), at atmospheric pressure, (25 ml/mol and 3 x 10(-7)M, respectively). The holoprotein is fully dimeric even at 6.5 kbar, which allows us to evaluate a lower Delta G degrees limit of 11.5 kcal/mol, corresponding to a dissociation constant K(d0)<10(-9)M.     

Pressure dissociation and conformational drift of the beta dimer of tryptophan synthase

Micromolar solutions of tryptophan synthase beta 2 dimer dissociate into monomers in the pressure range of 800-1600 bars as shown by studies of the spectral shift of the intrinsic fluorescence and of the fluorescence polarization of dansyl conjugates. At 25 degrees C the standard change in volume on dissociation (dV0) of the holoprotein was -162 mL mol-1, and the dissociation constant at 1 bar was K0 = 3.7 10(-10) M. Pyridoxal-reduced holoprotein and apoprotein had, within 10%, the same dV0, but K0 was decreased in the reduced protein (6 X 10(-11) M) and increased in the apoprotein (3.6 X 10(-9) M). At 4 degrees C the free energy of association of the holoprotein was reduced by 1.4 kcal mol-1, but dV0 was unchanged. In all the protein forms the decompression curves differed from the respective compression curves, indicating the loss of some free energy of association following separation of the monomers. This hysteretic behavior was largest in the apoprotein and amounted to a loss of 2.6 kcal mol-1 in the free energy of association. When the pressure was rapidly raised to 2.2 kbars, half-dissociation of the reduced pyridoxal beta 2 dimer took approximately 12 min. Upon return to atmospheric pressure reassociation was complete in 2-3 min and half of the enzyme activity was regained in 10 min; pyridoxal fluorescence recovered more slowly with a biphasic course. The independent return of these properties and the hysteretic behavior indicate that subunit separation is followed by a conformational drift like that observed in lactate dehydrogenase dissociated by either pressure or temperature or in enolase dissociated by dilution.     

Thermodynamic analysis of unfolding and dissociation in lactose repressor protein.

Lactose repressor protein, regulator of lac enzyme expression in Escherichia coli, maintains its structure and function at extremely low protein concentrations (<10(-)12 M). To examine the unfolding and dissociation of this tetrameric protein, structural transitions in the presence of varying concentrations of urea were monitored by fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation, and functional activities. The spectroscopic data demonstrated a single cooperative transition with no evidence of folded dimeric or monomeric species of this protein. These spectroscopic transitions were reversible provided a long incubation step was employed in the refolding reaction at approximately 3 M urea. The refolded repressor protein possessed the same functional and structural properties as wild-type repressor protein. The absence of concentration dependence expected for tetramer dissociation to unfolded monomer (M4 <--> 4U) in the spectral transitions indicates that the disruption of the monomer-monomer interface and monomer unfolding are a concerted reaction (M4 <--> U4) that may occur prior to the dissociation of the dimer-dimer interface. Thus, we propose that the unfolded monomers remain associated at the C-terminus by the 4-helical coiled-coil structure that forms the dimer-dimer interface and that this intermediate is the end point detected in the spectral transitions. Efforts to confirm the existence of this species by ultracentrifugation were inhibited by the aggregation of this intermediate. Based upon these observations, the wild-type fluorescence and CD data were fit to a model, M4 <--> U4, which resulted in an overall DeltaG degrees for unfolding of 40 kcal/mol. Using a mutant protein, K84L, in which the monomer-monomer interface is stabilized, sedimentation equilibrium results demonstrated that the dimer-dimer interface of lac repressor could persist at higher levels of urea than the monomer-monomer interface. The tetramer-dimer transition monitored using this mutant repressor yields a DeltaG degrees of 20.4 kcal/mol. Using this free energy value for the dissociation process of U4 <--> 4U, an overall free energy change of approximately 60 kcal/mol was calculated for dissociation of all interfaces and unfolding of the tetrameric lac repressor, reflecting the exceptional stability of this protein.     

Denaturant-induced equilibrium unfolding of concanavalin A is expressed by a three-state mechanism and provides an estimate of its protein stability

The urea and guanidine hydrochloride (GdnHCl)-induced denaturation of tetrameric concanavalin A (ConA) at pH 7.2 has been studied by using intrinsic fluorescence, 8-anilino-1-naphthalenesulfonate (ANS) binding, far-UV circular dichroism (CD), and size-exclusion chromatography. The equilibrium denaturation pathway of ConA, as monitored by steady state fluorescence, exhibits a three-state mechanism involving an intermediate state, which has been characterized as a structured monomer of the protein by ANS binding, far-UV CD and gel filtration size analysis. The three-state equilibrium is analyzed in terms of two distinct and separate dissociation (native tetramer<-->structured monomer) and unfolding (structured monomer<-->unfolded monomer) reaction steps, with the apparent transition midpoints (C(m)), respectively, at 1.4 and 4.5 M in urea, and at 0.8 and 2.4 M in GdnHCl. The results show that the free energy of stabilization of structured monomer relative to the unfolded state (-DeltaG(unf, aq)), is 4.4-5.5 kcal mol(-1), and that of native tetramer relative to structured monomer (-DeltaG(dis, aq)) is 7.2-7.4 kcal mol(-1), giving an overall free energy of stabilization (-DeltaG(dis&unf, aq)) of 11.6-12.9 kcal mol(-1) (monomer mass) for the native protein. However, the free energy preference at the level of quaternary tetrameric structure is found to be far greater than that at the tertiary monomeric level, which reveals that the structural stability of ConA is maintained mostly by subunit association.     

Hysteresis and conformational drift of pressure-dissociated glyceraldehydephosphate dehydrogenase

Pressure dissociation of yeast glyceraldehydephosphate dehydrogenase (GAPDH) was studied by fluorescence spectroscopy. Observations in the range of -5 to 30 degrees C indicate that monomer association into the tetramer proceeds with an enthalpy change of -14 kcal mol-1 and a large increase in entropy which at 25 degrees C amounts to 18 kcal mol-1. The large conformational drift and the low-temperature stability of the tetramer recovered after decompression facilitated a comparison of its properties with those of the native tetramer. Significant differences in absorption and fluorescence-excitation polarization spectra, yield of tryptophan fluorescence, and binding of anilinonaphthalenesulfonate and NADH were observed. At 0 degree C the standard free energies of association of the monomers into the native and drifted tetramers were respectively -32 and -29 kcal mol-1. The volume change upon association measured from the pressure span of the compression curves was 200-230 mL mol-1 but four times as large when derived from the displacement of the compression curves with total protein concentration. This large discrepancy can be explained by the existence in the native tetramer population of a distribution of free energies of association with a dispersion from the mean of about 6 kcal mol-1. At 0 degree C and 1 bar ATP and ADP decreased the stability of the GAPDH tetramer by changes in free energy of association of +3.7 and +4.1 kcal mol-1, respectively. NAD and c-AMP stabilized it by -2.3 and -1.3 kcal mol-1. The variation in sign and magnitude of the ligand-induced changes in free energy of association observed in this case, and previously in hexokinase [Ruan, K., & Weber, G. (1988) Biochemistry 27, 3295], and the heterogeneity of the free energy of association of GAPDH, revealed as indicated above, lead to the conclusion that oligomeric aggregates exist in a variety of conformations that depend upon the protein concentration, temperature, pressure, and the presence of specific ligands. The multiplicity of species revealed by the energetics raises questions about the significance of the structures of oligomeric proteins determined by X-ray crystallography.     

The thermodynamic analysis of protein stabilization by sucrose and glycerol against pressure-induced unfolding.

We have studied the reaction native left arrow over right arrow denatured for the 33-kDa protein isolated from photosystem II. Sucrose and glycerol have profound effects on pressure-induced unfolding. The additives shift the equilibrium to the left; they also cause a significant decrease in the standard volume change (DeltaV). The change in DeltaV was related to the sucrose and glycerol concentrations. The decrease in DeltaV varied with the additive: sucrose caused the largest effect, glycerol the smallest. The theoretical shift of the half-unfolding pressure (P1/2) calculated from the net increase in free energy by addition of sucrose and glycerol was lower than that obtained from experimental mea- surements. This indicates that the free energy change caused by preferential hydration of the protein is not the unique factor involved in the protein stabilization. The reduction in DeltaV showed a large contribution to the theoretical P1/2 shift, suggesting that the DeltaV change, caused by the sucrose or glycerol was associated with the protein stabilization. The origin of the DeltaV change is discussed. The rate of pressure-induced unfolding in the presence of sucrose or glycerol was slower than the refolding rate although both were significantly slower than that observed without any stabilizers.     

Exploration of pressure-induced dissociation of pyruvate oxidase.

The dissociation of pyruvate oxidase (PO) caused by pressure up to 220 MPa at various conditions was explored by measuring the intrinsic fluorescence spectra and polarization. At 5 degrees C and pH 7.6 the standard volume change (deltaV0) and free energy upon dissociation of the enzyme is -220 ml/mol and 29.83 kCal/mol, respectively. It was found that FAD was irreversibly removed during the pressure-dissociation of the enzyme. A much smaller standard volume change (-153 ml/mol) and lower free energy (24.92 kCal/mol) of apo-pyruvate oxidase (apo-PO) compared with the native enzyme indicated that FAD played very important role in stabilizing the enzyme and significantly influenced the standard volume change. The substrate pyruvic acid can significantly stabilize the enzyme against pressure in spite the standard volume for the enzyme in this case has a big increase relative to the native enzyme. The comparison of the intrinsic fluorescence of the native and the activated enzyme obtained by limited proteolysis indicated that the physical separation of alpha-peptide from the enzyme only occurred when the subunits were dissociated from each other under pressure.     

Molecular Dynamics/Free Energy Perturbation Studies of the Thermostable V74I Mutant of Ribonuclease HI

Abstract

Recent site-directed mutagenesis and thermodynamic studies have shown that the V74I mutant of Escherichia coli ribonuclease HI (RNase HI) is more stable than the wild type protein [Ishikawa et al., Biochemistry 32, 6171 (1993)]. In order to clarify the stabilization mechanism of this mutant, we calculated the free energy change due to the mutation Val 74→Ile in both the native and denatured states by free energy perturbations based on molecular dynamics (MD) simulations. We carried out inclusive MD simulations for the protein in water; i.e., fully solvated, no artificial constraints applied, and all long-range Coulomb interactions included. We found that the free energy of the mutant increased slightly relative to the wild type, in the native state by 1.60 kcal/mol, and in the denatured state by 2.25 kcal/mol. The unfolding free energy increment of the mutant (0.66 ± 0.19 kcal/mol) was in good agreement with the experimental value (0.6 kcal/mol). The hysteresis error in the free energy calculations, i.e., forward and reverse perturbations, was only ±0.19 kcal/mol. These results show that the V74I mutant is stabilized relative to the wild type by the increased free energy of the denatured state and not by a decrease in the free energy of the native state as had been proposed earlier based on the mutant X-ray structure. It was found that the stabilization was caused by a loss of solvation energy in the mutant denatured state and not by improved packing interactions inside the native protein.     

Dissociation of the lactose repressor protein tetramer using high hydrostatic pressure

Dissociation of lac repressor tetramer by high hydrostatic pressures was monitored with intrinsic tryptophan fluorescence. With the assumption of complete dissociation to monomer, tryptophan polarization data gave delta V a approximately 170 mL/mol and the concentration for 50% tetramer dissociation, C1/2, was 3.8 X 10(-8) M. Upon addition of inducer, the calculated delta V a increased to approximately 220 mL/mol and the C1/2 decreased to approximately 1 X 10(-8) M, a free energy difference of approximately 0.7 kcal. These results indicate a modest stabilization of the tetramer by the presence of inducer. Monitoring the average energy of tryptophan emission demonstrated that tetramer dissociation takes place over the same range of pressures as evidenced by the polarization data and IPTG dissociation can be more or less superimposed upon tetramer dissociation depending upon the ligand concentration used. Although the two transitions cannot be separated entirely, the delta V a for the region of the pressure dependence dominated by ligand dissociation was 69 mL/mol, an unexpectedly large value. For tetramer modified with methyl methanethiosulfonate, subunit dissociation was shifted to much higher pressures and IPTG dissociation did not occur. The delta V a for subunit association was calculated as approximately 160 mL/mol, and the C1/2 was 3.5 X 10(-9) M. Interactions at the subunit interface of the modified protein are apparently stronger than in the unmodified protein. The absence of inducer dissociation from the MMTS-modified tetramer by the application of high hydrostatic pressure suggests that the volume change for inducer binding to the modified protein is much smaller than that observed for the unmodified repressor.     

Hydrostatic pressure and calcium-induced dissociation of calpains

The dissociation of mu- and m-calpains was studied by fluorescence spectroscopy under high hydrostatic pressure (up to 650 MPa). Increasing pressure induced a red shift of the tryptophan fluorescence of the calcium-free enzyme. The concentration dependence of the spectral transition was consistent with a pressure-induced dissociation of the subunits. Rising temperature increased the stability of calpain heterodimers and confirmed the predominance of hydrophobic interactions between monomers. At saturating calcium, the spectral transition was not observed for native or iodoacetamide-inactivated calpains, indicating that they were already dissociated by calcium. The reaction volume was about -150 ml mol-1 for both isoforms, and the dissociation constants at atmospheric pressure are approximately 10-12 M and 10-15 M for mu- and m-calpains, respectively. This result indicates a tighter interaction in the isoform that requires higher calcium concentration for activity.     

Optical detection of a red-shifted Michaelis complex in the reaction of HCN with ferromyeloperoxidase. Evidence for conformational stabilization of high spin iron (II).

The reaction of HCN with ferromyeloperoxidase involves the sequential formation of two monocyanide complexes. The first complex, which forms immediately on mixing, is characterized by a red shift in the Soret band of the ferroperoxidase, and a dissociation constant (measured as a Michaelis constant) of 0.67 mM. The second complex arises from the first via a first order process, whose maximal rate is 0.095 s-1 at 25 degrees C, pH 7.0. This more stable complex is characterized by a blue shift in the Soret and alpha bands and by an overall dissociation constant in the region of 4.5 microM. This gives a free energy difference between the two complexes of around 3.0 kcal mol-1 and a difference in optical absorption of 15 nm (Soret). The measured Arrhenius activation energy for the conversion of the high energy, long wavelength complex to the low energy, short wavelength complex is 16.3 kcal mol-1. A larger blue shift is observed on protein denaturation (34 nm), after which the two-step binding reaction is not observed. This, and the magnitude of the activation energy in the spontaneous complex interconversion process, shows that the latter is a conformational process. In addition, it can be concluded that the unknown structural feature of the heme site which is responsible for the anomalous red shift in the optical spectrum of native ferromyeloperoxidase, is also the link between the ligand state of the iron and the protein conformation.     

Computational methodology for estimating changes in free energies of biomolecular association upon mutation. The importance of bound water in dimer-tetramer assembly for beta 37 mutant hemoglobins

The computational modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field that includes hydrogen bonding, Coulombic, and hydrophobic terms, was used to model the free energy of dimer-tetramer association in a series of deoxy hemoglobin beta 37 double mutants. Five of the analyzed mutants (beta 37W --> Y, beta 37W --> A, beta 37W --> G, beta 37W --> E, and beta 37W --> R) have been solved crystallographically and characterized thermodynamically and subsequently made a good test set for the calibration of our method as a tool for free energy prediction. Initial free energy estimates for these mutants were conducted without the inclusion of crystallographically conserved water molecules and systematically underestimated the experimentally calculated loss in free energy observed for each mutant dimer-tetramer association. However, the inclusion of crystallographic waters, interacting at the dimer-dimer interface of each mutant, resulted in HINT free energy estimates that were more accurate with respect to experimental data. To evaluate the ability of our method to predict free energies for de novo protein models, the same beta 37 mutants were computationally generated from native deoxy hemoglobin and similarly analyzed. Our theoretical models were sufficiently robust to accurately predict free energy changes in a localized region around the mutated residue. However, our method did not possess the capacity to generate the long-range secondary structural effects observed in crystallographically solved mutant structures. Final method analysis involved the computational generation of structurally and/or thermodynamically uncharacterized beta 37 deoxy hemoglobin mutants. HINT analysis of these structures revealed that free energy predictions for dimer-tetramer association in these models agreed well with previously observed energy predictions for structurally and thermodynamically characterized beta 37 deoxy hemoglobin mutants.     

Temperature and pressure effects on C112S azurin: Volume,expansivity, and flexibility changes

The pressure‐induced unfolding of the mutant C112S azurin from Pseudomonas aeruginosa was monitored both under steady state and dynamic conditions. The unfolding profiles were obtained by recording the spectral shift of the fluorescence emission as well as by phosphorescence intensity measurements. We evaluated the difference in free energy, ΔG, as a function of pressure and temperature. The dependence of ΔG on temperature showed concave profile at all pressures studied. A positive heat capacity change of about 4.3 kJ mol^?1 deg^?1 fitted all the curves. The volume change of the reaction showed a moderate dependence on temperature when compared with other proteins previously studied. The kinetic activation parameters (ΔV*, ΔH*, ΔS*) were obtained from upward and downward pressure‐jump experiments and used to characterize the volumetric and energetic properties of the transition state between native and unfolded protein. Our findings suggest that the folding and unfolding reaction paths passed through different transition states. The change in the phosphorescence lifetime with pressure pointed out that pressure‐induced unfolding occurred within two steps: the first leading to an increased protein flexibility, presumably caused by water penetration into the protein. Major structural changes of the tryptophan environment occurred in a second step at higher pressures. Proteins 2014; 82:1787–1798. © 2014 Wiley Periodicals, Inc.     

Anomalous pressure dissociation of large protein aggregates. Lack of concentration dependence and irreversibility at extreme degrees of dissociation of extracellular hemoglobin

The effects of hydrostatic pressure on the extracellular hemoglobin of Glossoscolex paulistus were investigated by studies of light scattering, intrinsic protein fluorescence, filtration chromatography, and oxygen binding. Pressure promoted a large decrease of light scattering consistent with the dissociation of the hemoglobin. Pressures up to 1.7 kbar caused dissociation with reversibility of the light scattering and fluorescence properties after return to atmospheric pressure. Higher pressures provoked additional dissociation with increasing loss of reversibility. After complete dissociation by incubation at 2.5 kbar followed by decompression, the protein continued mostly dissociated. The dissociated forms were distributed in two populations as based on size exclusion chromatography, one corresponding to small dissociated units (average Mr = 33,000) and the other population corresponding to the one-twelfth subunit (260,000 Mr). The pressure dissociation curves showed no significant dependence on protein concentration suggesting that the native hemoglobin population exists in a distribution of free-energies of association. Both the decrease of concentration dependence and the loss of ability to reassemble seem to increase with the complexity and size of the protein aggregate. These findings permit the conclusion that increased heterogeneity of free-energies of association with the size of the aggregate may result in the molecular individuality of large protein complexes such as subcellular particles and viruses.     

Dissociation of yeast hexokinase by hydrostatic pressure

The pressure-induced dissociation of the isozymes P1 and P2 of hexokinase was investigated by studies of the spectral shift of the intrinsic protein fluorescence and by the fluorescence polarization of dansyl conjugates. The free energy of association of the monomers at atmospheric pressure, Katm, was -14.2 kcal mol-1 at 20 degrees C and -11.4 kcal mol-1 at 0 degrees C. The positive enthalpy indicates that the association of the monomers is entropy-driven, overcoming the negative enthalpy of hydration of the subunit interfaces. At 0 degrees C and 1 bar, glucose stabilizes the association by -1.1 kcal mol-1 and the binding of both adenosine 5'-(beta, gamma-methylenetriphosphate) (AMPPCP) and glucose by an even larger amount, -1.34 kcal mol-1. Paradoxically, adenosine 5'-triphosphate (ATP), or AMPPCP, in the absence of glucose destabilizes the association by +0.34 kcal mol-1, while adenosine 5'-diphosphate (ADP) stabilizes it by -0.6 kcal mol-1. Comparison of dV0, the apparent standard volume of association, at different pHs and temperatures indicates that its value (115-160 mL mol-1) is strongly dependent upon the ionization of a group at the subunit interface with a pK near neutrality. Under dissociating pressures, trypsin action results in permanent dissociation of the dimer, confirming earlier observations of Colowick by less direct methods. The P1 and P2 enzymes differ in Katm and dV0 and markedly so in the effects of salt upon the stability of the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dimeric procaspase-3 unfolds via a four-state equilibrium process.

We have examined the folding and assembly of a catalytically inactive mutant of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the second transition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).     

Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: trimer dissociation can be separated from unfolding

The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure.     

Comparison of high pressure-induced dissociation of single-stranded DNA-binding protein (SSB) from high pressure-sensitive and high pressure-adapted marine Shewanella species

The effects of hydrostatic pressure on protein quaternary structure were compared for recombinant single-stranded DNA-binding protein (SSB) derived from piezosensitive, piezotolerant, and obligately piezophilic ("pressure-loving") marine Shewanella strains. The pressure-induced dissociation of the oligomeric SSB proteins was investigated using fluorescence anisotropy. The SSBs all exhibited striking similarity in the pressure-dependent behavior of the fluorescence intensity and emission spectrum as well as in their dissociation constants at atmospheric pressure. The free energies of subunit association into tetramers for all SSBs were between -27 and -30 kcal mol(-1). However, SSB from the piezosensitive Shewanella strain S. hanedai was more sensitive to pressure than that of the SSB proteins from the piezotolerant or piezophilic bacteria. The volume change of association obtained from the pressure dependence of dissociation at a fixed protein concentration (Delta V(p)) for SSB from S. hanedai was 394-402 ml mol(-1). The Delta V(p) values for SSB from the deeper-living Shewanellas were smaller and ranged from 253 to 307 ml mol(-1). Differences between the primary structures of the SSB proteins that could correlate with differences in sensitivity to pressure-induced dissociation were examined.     

Protein Thermostability Calculations Using Alchemical Free Energy Simulations

Thermal stability of proteins is crucial for both biotechnological and therapeutic applications. Rational protein engineering therefore frequently aims at increasing thermal stability by introducing stabilizing mutations. The accurate prediction of the thermodynamic consequences caused by mutations, however, is highly challenging as thermal stability changes are caused by alterations in the free energy of folding. Growing computational power, however, increasingly allows us to use alchemical free energy simulations, such as free energy perturbation or thermodynamic integration, to calculate free energy differences with relatively high accuracy. In this article, we present an automated protocol for setting up alchemical free energy calculations for mutations of naturally occurring amino acids (except for proline) that allows an unprecedented, automated screening of large mutant libraries. To validate the developed protocol, we calculated thermodynamic stability differences for 109 mutations in the microbial Ribonuclease Barnase. The obtained quantitative agreement with experimental data illustrates the potential of the approach in protein engineering and design.