High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

Thermodynamic analysis of cavity creating mutations in an engineered leucine zipper and energetics of glycerol-induced coiled coil stabilization

Protein stability in vitro can be influenced either by introduction of mutations or by changes in the chemical composition of the solvent. Recently, we have characterized the thermodynamic stability and the rate of folding of the engineered dimeric leucine zipper A(2), which has a strengthened hydrophobic core [Dürr, E., Jelesarov, I., and Bosshard, H. R. (1999) Biochemistry 38, 870-880]. Here we report on the energetic consequences of a cavity introduced by Leu/Ala substitution at the tightly packed dimeric interface and how addition of 30% glycerol affects the folding thermodynamics of A(2) and the cavity mutants. Folding could be described by a two-state transition from two unfolded monomers to a coiled coil dimer. Removal of six methylene groups by Leu/Ala substitutions destabilized the dimeric coiled coil by 25 kJ mol(-1) at pH 3.5 and 25 degrees C in aqueous buffer. Destabilization was purely entropic at around room temperature and became increasingly enthalpic at elevated temperatures. Mutations were accompanied by a decrease of the unfolding heat capacity by 0.5 kJ K(-1) mol(-1). Addition of 30% glycerol increased the free energy of folding of A(2) and the cavity mutants by 5-10 kJ mol(-1) and lowered the unfolding heat capacity by 25% for A(2) and by 50% for the Leu/Ala mutants. The origin of the stabilizing effect of glycerol varied with temperature. Stabilization of the parent leucine zipper A(2) was enthalpic with an unfavorable entropic component between 0 and 100 degrees C. In the case of cavity mutants, glycerol induced enthalpic stabilization below 50 degrees C and entropic stabilization above 50 degrees C. The effect of glycerol could not be accounted for solely by the enthalpy and entropy of transfer or protein surface from water to glycerol/water mixture. We propose that in the presence of glycerol the folded coiled coil dimer is better packed and displays less intramolecular fluctuations, leading to enhanced enthalpic interactions and to an increase of the entropy of folding. This work demonstrates that mutational and solvent effects on protein stability can be thermodynamically complex and that it may not be sufficient to only analyze changes of enthalpy and entropy at the unfolding temperature (T(m)) to understand the mechanisms of protein stabilization.     

The pressure-temperature free energy-landscape of staphylococcal nuclease monitored by (1)H NMR

The thermodynamic stability of staphylococcal nuclease was studied against the variation of both temperature and pressure by utilizing (1)H NMR spectroscopy at 750 MHz in 20 mM Mes buffer containing 99.9 % (2)H(2)O, pH 5.3. Equilibrium fractions of folded and unfolded protein species were evaluated with the proton signals of two histidine residues as monitor in the pressure range of 30-3300 bar and in the temperature range of 1.5 degrees C-35 degrees C. From the multi-parameter fit of the experimental data to the Gibbs energy equation expressed as a simultaneous function of pressure and temperature, we determined the compressibility change (Deltabeta), the volume change at 1 bar (DeltaV degrees ) and the expansivity change (Deltaalpha) upon unfolding among other thermodynamic parameters: Deltabeta=0.02(+/-0.003) ml mol(-1) bar(-1); Deltaalpha=1.33(+/-0.2) ml mol(-1) K(-1); DeltaV degrees =-41.9(+/-6. 3) ml mol(-1) (at 24 degrees C); DeltaG degrees =13.18(+/-2) kJ mol(-1) (at 24 degrees C); DeltaC(p)=13.12(+/-2) kJ mol(-1) K(-1); DeltaS degrees =0.32(+/-0.05) kJ mol(-1) K(-1 )(at 24 degrees C). The result yields a three-dimensional free energy surface, i.e. the free energy-landscape of staphylococcal nuclease on the P-T plane. The significantly positive Deltabeta and Deltaalpha values suggest that, in the pressure-denatured state, staphylococcal nuclease forms a loosely packed and fluctuating structure. The slight but statistically significant difference between the unfolding transitions of the His8 and His124 environments is considered to reflect local fluctuations in the native state, leading to pre-melting of the His124 environment prior to the cooperative unfolding of the major part of the protein.     

Buried water molecules contribute to the conformational stability of a protein

This study sought to attain a better understanding of the contribution of buried water molecules to protein stability. The 3SS human lysozyme lacks one disulfide bond between Cys77 and Cys95 and is significantly destabilized compared with the wild-type human lysozyme (4SS). We examined the structure and stability of the I59A-3SS mutant human lysozyme, in which a cavity is created at the mutation site. The crystal structure of I59A-3SS indicated that there were ordered new water molecules in the cavity created. The stability of I59A-3SS is 5.5 kJ/mol less than that of 3SS. The decreased stability of I59A-3SS (5.5 kJ/mol) is similar to that of Ile to Ala mutants with newly introduced water molecules in other globular proteins (6.3 +/- 2.1 kJ/mol), but is less than that of Ile/Leu to Ala mutants with empty cavities (13.7 +/- 3.1 kJ/mol). This indicates that water molecules partially compensate for the destabilization by decreasing hydrophobic and van der Waals interactions. These results provide further evidence that buried water molecules contribute to protein stability.     

Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI.

The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein. The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves. Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C. Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins.     

Human serum albumin interaction with formononetin studied using fluorescence anisotropy, FT-IR spectroscopy, and molecular modeling methods

Interaction of formononetin with a model transport protein, human serum albumin (HSA), has been studied using fluorescence anisotropy, FT-IR spectroscopy, and molecular modeling methods. Upon binding with HSA, the fluorescence spectrum of formononetin exhibits appreciable hypsochromic shift along with an enhancement in the fluorescence intensity. Gradual addition of HSA led to a marked increase in fluorescence anisotropy (r). From the value of fluorescence anisotropy, it is argued that the drug is located in a restricted environment of protein. The binding constant (K approximately 1.6 x 10(5) M(-1)) and the standard free energy change (DeltaG(0) approximately -29.9 kJ/mol) of formononetin-HSA interaction have been calculated according to the relevant fluorescence data. Fourier transform infrared measurements have shown that the secondary structures of the protein have been changed by the interaction of formononetin with HSA. Computational mapping of the possible binding sites of formononetin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA.     

Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH

The Thermotoga neapolitana xylose isomerase (TNXI) is extremely thermostable and optimally active at 95 degrees C. Its derivative, TNXI Val185Thr (V185T), is the most active type II xylose isomerase reported, with a catalytic efficiency of 25.1 s(-1) mM(-1) toward glucose at 80 degrees C (pH 7.0). To further optimize TNXI's potential industrial utility, two rounds of random mutagenesis and low temperature/low pH activity screening were performed using the TNXI V185T-encoding gene as the template. Two highly active mutants were obtained, 3A2 (V185T/L282P) and 1F1 (V185T/L282P/F186S). 1F1 was more active than 3A2, which in turn was more active than TNXI V185T at all temperatures and pH values tested. 3A2 and 1F1's high activities at low temperatures were due to significantly lower activation energies (57 and 44 kJ/mol, respectively) than that of TNXI and V185T (87 kJ/mol). Mutation L282P introduced a kink in helix alpha7 of 3A2's (alpha/beta)8 barrel. Surprisingly, this mutation kinetically destabilized 3A2 only at pH 5.5. 1F1 displayed kinetic stability slightly above that of TNXI V185T. In 1F1, mutation F186S creates a cavity that disrupts a four-residue network of aromatic interactions. How the conformation of the neighboring residues is affected by this cavity and how these conformational changes increase 1F1's stability still remain unclear.     

Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.     

Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions

The bacterial cold shock proteins (Csp) are used by both experimentalists and theoreticians as model systems for analyzing the Coulombic contributions to protein stability. We employ Proside, a method of directed evolution, to identify stabilized variants of Bs-CspB from Bacillus subtilis. Proside links the increased protease resistance of stabilized protein variants to the infectivity of a filamentous phage. Here, three cspB libraries were used for in vitro selections to explore the stabilizing potential of charged amino acids in Bs-CspB. In the first library codons for nine selected surface residues were partially randomized, in the second one random mutations were introduced non-specifically by error-prone PCR, and in the third one the spontaneous mutation rate of the phage in Escherichia coli was used. Stabilizing mutations were found at the surface positions 1, 3, 46, 48, 65, and 66. The contributions of these mutations to stability were characterized by analyzing them individually and in combination. The best combination (M1R, E3K, K65I, and E66L) increased the midpoint of thermal unfolding of Bs-CspB from 53.8 to 85.0 degrees C. The effects of most mutations are strongly context dependent. A good example is provided by the E3R mutation. It is strongly stabilizing (DeltaDeltaGD=11.1kJ mol(-1)) in the wild-type protein, but destabilizing (DeltaDeltaGD=-4.0kJ mol(-1)) in the A46K/S48R/E66L variant. The stabilizations by charge mutations did not correlate well with the corresponding changes in the protein net charge, and they could not be ascribed to the formation of ion pairs. Previous theoretical analyses did not identify the stabilization caused by the mutations at positions 1, 46, and 48. Also, electrostatics calculations based on protein net charge or charge asymmetry did not predict well the stability changes that occur when charged residues in Bs-CspB are mutated. It remains a challenge to model the Coulombic interactions of charged residues in a protein and to determine their contributions to the Gibbs free energy of protein folding.     

A disease state mutation unfolds the parkin ubiquitin-like domain

E3 ubiquitin ligases are essential enzymes in the ubiquitination pathway responsible for the recognition of specific E2 conjugating enzymes and for transferring ubiquitin to a substrate targeted for degradation. In autosomal recessive juvenile Parkinson's disease, an early onset form of Parkinson's disease, point mutations in the E3 ligase parkin are one of the most commonly observed traits. Parkin is a multidomain E3 ligase that contains an N-terminal ubiquitin-like domain that interacts with, and effects the ubiquitination of, substrates such as cyclin E, p38 and synphilin. In this work we have examined the folding and structure of the parkin ubiquitin-like domain (Ubld) and of the protein with two causative disease mutations (K48A and R42P). Parallel experiments with the protein ubiquitin were done in order to determine if the same mutations were detrimental to the ubiquitin structure and stability. Despite similar folds between the parkin Ubld and ubiquitin, urea unfolding experiments show that the parkin Ubld is surprisingly approximately 10.6 kJ/mol less stable than ubiquitin. The K48A mutation had little effect on the stability of the parkin Ubld or ubiquitin indicating that this mutation contributes to defective protein-protein interactions. In contrast, the single point mutation R42P in parkin's Ubld caused poor expression and degradation of the protein. To avoid these problems, a GB1-Ubld fusion protein was characterized by NMR spectroscopy to show that the R42P mutation causes the complete unfolding of the parkin Ubld. This observation provides a rationale for the more rapid degradation of parkin carrying the R42P mutation in vivo, and its inability to interact with some substrate proteins. Our work provides the first structural and folding insight into the effects of causative mutations within the ubiquitin-like domain in autosomal recessive juvenile Parkinson's disease.     

Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity

Activity and stability of the proto-oncogene c-Myb are regulated by post-translational modifications, though the molecular mechanisms underlying such control are only partially understood. Here we describe the functional interaction of c-Myb with Pin1, an isomerase that binds to phosphorylated Ser/Thr-Pro motifs. We found that co-expression of c-Myb and Pin1 led to a net increase of c-Myb transactivation activity, both on reporter constructs as well as on an endogenous target gene. DNA-binding studies revealed that Pin1 did not increase the association of c-Myb with its response element in DNA. The increase of c-Myb transactivation activity was strictly dependent on the presence of an active catalytic center in Pin1. We provide evidence that c-Myb and Pin1 physically interacted, both upon ectopic expression of the proteins in HEK-293 cells as well as in the more physiological setting of HL60 cells, where c-Myb and Pin1 are resident proteins. By point mutating each individual Ser/Thr-Pro motif in c-Myb as well as by using deletion mutants we show that S528 in the EVES-motif was the docking site for Pin1. Mass spectrometry confirmed that S528 is phosphorylated in vivo. Finally, functional studies showed that mutation of S528 to alanine almost abolished the increase of transactivation activity by Pin1. This study reveals a new paradigm by which phosphorylation controls c-Myb function.     

Infrequent cavity-forming fluctuations in HPr from Staphylococcus carnosus revealed by pressure- and temperature-dependent tyrosine ring flips

Infrequent structural fluctuations of a globular protein is seldom detected and studied in detail. One tyrosine ring of HPr from Staphylococcus carnosus, an 88-residue phosphocarrier protein with no disulfide bonds, undergoes a very slow ring flip, the pressure and temperature dependence of which is studied in detail using the on-line cell high-pressure nuclear magnetic resonance technique in the pressure range from 3 MPa to 200 MPa and in the temperature range from 257 K to 313 K. The ring of Tyr6 is buried sandwiched between a beta-sheet and alpha-helices (the water-accessible area is less than 0.26 nm2), its hydroxyl proton being involved in an internal hydrogen bond. The ring flip rates 10(1)-10(5) s(-1) were determined from the line shape analysis of H(delta1, delta2) and H(epsilon1,epsilon2) of Tyr6, giving an activation volume DeltaV++ of 0.044 +/- 0.008 nm3 (27 mL mol(-1)), an activation enthalpy DeltaH++ of 89 +/- 10 kJ mol(-1), and an activation entropy DeltaS++ of 16 +/- 2 JK(-1) mol(-1). The DeltaV++) and DeltaH++ values for HPr found previously for Tyr and Phe ring flips of BPTI and cytochrome c fall within the range of DeltaV(double dagger) of 28 to 51 mL mol(-1) and DeltaH++ of 71 to 155 kJ mol(-1). The fairly common DeltaV++ and DeltaH++ values are considered to represent the extra space or cavity required for the ring flip and the extra energy required to create a cavity, respectively, in the core part of a globular protein. Nearly complete cold denaturation was found to take place at 200 MPa and 257 K independently from the ring reorientation process.     

Shape and energetics of a cavity in c-Myb probed by natural and non-natural amino acid mutations.

The shape and the energetics of a functional cavity in the R2 subdomain (90-141) of the c-Myb DNA-binding domain were investigated by spectroscopy and thermodynamic analysis. We focused on the valine 103 residue located in front of the cavity. Nine mutants, in which valine 103 was substituted with alanine, 2-aminobutyric acid, norvaline, norleucine, leucine, isoleucine, allo -isoleucine, cyclohexylglycine, and cyclohexylalanine, were chemically synthesized and analyzed. These mutants provided a wide distribution of sizes which ranged from forming additional cavity space to filling and overflowing the cavity space. Temperature-scanning circular dichroism measurements and differential scanning calorimetry revealed a linear relationship between the van't Hoff enthalpy and the thermal transition temperature for the cavity-filling mutations. On the other hand, the mutants with side-chains larger than the side-chain of leucine resulted in a relatively low transition enthalpy and temperature, most likely due to the exposure of the side-chain to solvent and the increase in the entropy of the folded states. Branching at the beta-carbon atom reduced the unfolding free energy due to the steric constraint in the cavity. In particular, the mutational elongation of the side-chain from beta-carbon to the trans -to-CO direction proved to be more hindered than that from beta-carbon to the trans -to-NH. The unfolding free energy versus side-chain volume formed a bell-shaped plot with a maximum free energy for the leucine mutant. The difference in the transition free energy for cavity-filling mutants with beta-unbranched side-chains were two to four times larger than the difference in the transfer energy from organic solvent to water. Therefore, the increase in unfolding free energy would most likely be attributed to van der Waals interactions in the cavity wall, which would be a origin of stabilization by the sliding of tryptophan 95 into the cavity upon DNA binding.     

Characterizing a partially folded intermediate of the villin headpiece domain under non-denaturing conditions: contribution of His41 to the pH-dependent stability of the N-terminal subdomain

The contribution of interactions involving the imidazole ring of His41 to the pH-dependent stability of the villin headpiece (HP67) N-terminal subdomain has been investigated by nuclear magnetic resonance (NMR) spin relaxation. NMR-derived backbone N-H order parameters (S2) for wild-type (WT) HP67 and H41Y HP67 indicate that reduced conformational flexibility of the N-terminal subdomain in WT HP67 is due to intramolecular interactions with the His41 imidazole ring. These interactions, together with desolvation effects, contribute to significantly depress the pKa of the buried imidazole ring in the native state. 15N R1rho relaxation dispersion data indicate that WT HP67 populates a partially folded intermediate state that is 10.9 kJ mol(-1) higher in free energy than the native state under non-denaturing conditions at neutral pH. The partially folded intermediate is characterized as having an unfolded N-terminal subdomain while the C-terminal subdomain retains a native-like fold. Although the majority of the residues in the N-terminal subdomain sample a random-coil distribution of conformations, deviations of backbone amide 1H and 15N chemical shifts from canonical random-coil values for residues within 5A of the His41 imidazole ring indicate that a significant degree of residual structure is maintained in the partially folded ensemble. The pH-dependence of exchange broadening is consistent with a linear three-state exchange model whereby unfolding of the N-terminal subdomain is coupled to titration of His41 in the partially folded intermediate with a pKa,I=5.69+/-0.07. Although maintenance of residual interactions with the imidazole ring in the unfolded N-terminal subdomain appears to reduce pKa,I compared to model histidine compounds, protonation of His41 disrupts these interactions and reduces the difference in free energy between the native state and partially folded intermediate under acidic conditions. In addition, chemical shift changes for residues Lys70-Phe76 in the C-terminal subdomain suggest that the HP67 actin binding site is disrupted upon unfolding of the N-terminal subdomain, providing a potential mechanism for regulating the villin-dependent bundling of actin filaments.     

Heme-pocket-hydration change during the inactivation of cytochrome P-450camphor by hydrostatic pressure.

Hydrostatic pressure has been used to convert cytochrome P-450camphor to cytochrome P-420. The latter is an inactivated but soluble and undenaturated form of cytochrome P-450camphor. Using camphor analogues as probes of the active site we show that the inactivation volume change is directly correlated to the initial degree of hydration of the heme pocket. The values range between -73 ml/mol and -197 ml/mol [Di Primo, C., Hui Bon Hoa, G., Douzou, P. & Sligar, S. G. (1990) Eur. J. Biochem. 193, 383-386] for a totally hydrated (substrate-free, low-spin, six coordinated heme iron) and a non-hydrated (camphor-bound, high-spin, five coordinated heme iron) heme pocket. These results suggest that the larger value, -197 ml/mol, for the inactivation volume change is due to a hydration change of the heme pocket resulting from the displacement of the substrate during the compression and the subsequent entrance of water molecules. Similarly, the stability of the protein against compression is correlated with water accessibility to the active site. Increase in substrate mobility by loss of specific interactions with both regions of well defined secondary structure of cytochrome P-450camphor results in an increase of water accessibility and decrease of stability. Thus for camphor and adamantanone which strongly interact with the protein and exclude water from the active site [Poulos, T. L., Finzel, B. C. & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700; Raag, R. & Poulos, T. L. (1989) Biochemistry 28, 917-922] the increase in stability compared to the free protein is roughly 30 kJ/mol at 20 degrees C. With smaller substrates such as norcamphor, which loosely fits into the active site and does not completely exclude water [Raag, R. & Poulos, T. L. (1989) Biochemistry 28, 917-922], the increase in stability is only 7 kJ/mol. Finally these results suggest that cytochrome P-420 induced by hydrostatic pressure is a unique form where the active site is hydrated and camphor is displaced from its binding site.