Constant Enthalpy Change Value during Pyrophosphate Hydrolysis within the Physiological Limits of NaCl

A decrease in water activity was thought to result in smaller enthalpy change values during PPi hydrolysis, indicating the importance of solvation for the reaction. However, the physiological significance of this phenomenon is unknown. Here, we combined biochemistry and calorimetry to solve this problem using NaCl, a physiologically occurring water activity-reducing reagent. The pyrophosphatase activities of extremely halophilic Haloarcula japonica, which can grow at ∼4 m NaCl, and non-halophilic Escherichia coli and Saccharomyces cerevisiae were maximal at 2.0 and 0.1 m NaCl, respectively. Thus, halophilic and non-halophilic pyrophosphatases exhibit distinct maximal activities at different NaCl concentration ranges. Upon calorimetry, the same exothermic enthalpy change of −35 kJ/mol was obtained for the halophile and non-halophiles at 1.5–4.0 and 0.1–2.0 m NaCl, respectively. These results show that solvation changes caused by up to 4.0 m NaCl (water activity of ∼0.84) do not affect the enthalpy change in PPi hydrolysis. It has been postulated that PPi is an ATP analog, having a so-called high energy phosphate bond, and that the hydrolysis of both compounds is enthalpically driven. Therefore, our results indicate that the hydrolysis of high energy phosphate compounds, which are responsible for biological energy conversion, is enthalpically driven within the physiological limits of NaCl.This work was supported by Grant-in-aid for Scientific Research on Innovative Areas 20118005 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan

Single-point amino acid substitutions at the 119th residue of thermolysin and their pressure-induced activation

AbstractThe effect of amino acid substitution at the 119th site of thermolysin (TLN) on the pressure activation behavior of this enzyme was studied for four mutants at pressures <300 MPa. For Q119Q, Q119N and Q119R, the highest activation was observed to be over 30 times that at atmospheric pressure and the activation volumes (ΔV‡) were about −75 ml/mol. However, we obtained only 10 times higher activation for Q119E and Q119D (ΔV‡∼−60 ml/mol). The intrinsic fluorescence of TLN changed at pressures >300 MPa, and the latter two mutants showed a smaller ΔGapp and ΔVapp of transition than the wild type. These results are discussed with respect to the hydration change in the enzyme protein around the substituted region

Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N &harr; In&nbsp;&harr; D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we &ldquo;reverse engineered&rdquo; PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis

Reversible and Fast Association Equilibria of a Molecular Chaperone, gp57A, of Bacteriophage T4

The association of a molecular chaperone, gp57A, of bacteriophage T4, which facilitates formation of the long and short tail fibers, was investigated by analytical ultracentrifugation, differential scanning microcalorimetry, and stopped-flow circular dichroism (CD) to establish the association scheme of the protein. Gp57A is an oligomeric α-helix protein with 79 amino acids. Analysis of the sedimentation velocity data by direct boundary modeling with Lamm equation solutions together with a more detailed boundary analysis incorporating association schemes led us to conclude that at least three oligomeric species of gp57A are in reversible and fast association equilibria and that a 3(mer)-6(mer)-12(mer) model described the data best. On the other hand, differential scanning microcalorimetry revealed a highly reversible two-step transition of dissociation/denaturation, both of which accompanied decrease in CD at 222 nm. The melting curve analysis revealed that it is consistent with a 6(mer)-3(mer)-1(mer) model. The refolding/association kinetics of gp57A measured by stopped-flow CD was consistent with the interpretation that the bimolecular reaction from trimer to hexamer was preceded by a fast α-helix formation in the dead-time. Trimer or hexamer is likely the functional oligomeric state of gp57A

Analysis and Control of Protein Crystallization Using Short Peptide Tags That Change Solubility without Affecting Structure, Thermal Stability, and Function

Short peptide tags attached to recombinant proteins are emerging as an important tool for biochemical research. Here, we report the effects of 10 Solubilization Controlling Peptide (SCP) tags on crystallization behavior of a bovine pancreatic trypsin inhibitor (BPTI) variant. The tags did not affect structure, thermodynamics, and activities of BPTI. Moreover, eight of the tagged variants crystallized under the same condition, and six of them diffracted at high resolution. All variants with long-term solubility (<i>LS</i>) between 1 and 6 mg/mL produced crystals that diffracted well, while variants with <i>LS</i> < 1 and >6 mg/mL did not crystallize, produced poorly diffracting crystals, or crystallized under a different condition. The only exception was a glutamine tagged variant, which had an <i>LS</i> of 5 mg/mL, but fast aggregation kinetics, and produced mere needles unsuitable for further analysis. Crystal structures indicated that most tags were largely invisible, indicating high flexibility, without having interactions with nearby residues. Therefore, short peptides, introducing a mere 5–7 residue elongation, could provide a useful technology for tuning protein solubility without affecting its other properties and hence for overcoming problems associated with excessively low or high solubility, such as in crystallization