Reversible Oligomerization and Reverse Hydrophobic Effect Induced by Isoleucine Tags Attached at the C-Terminus of a Simplified BPTI Variant.

Protein amorphous aggregation has become the focus of great attention, as it can impair the ability of cells to function properly. Here, we evaluated the effects of three peptide tags, consisting of one, three, and five consecutive isoleucines attached at the C-terminus end of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, BPTI-19A, on the thermal stability and oligomerization by circular dichroism spectrometry and differential scanning calorimetry in detail. All of the BPTI-19A variants exhibited a reversible and apparently two-state thermal transition like BPTI-19A at pH 4.7. The thermal transition of the five-isoleucine-tagged variant showed clear protein-concentration dependence, where the apparent denaturation temperature decreased as the protein concentration increased. Quantitative analysis indicated that this phenomenon originated from the presence of reversibly oligomerized (RO) states at high temperatures. The results also illustrated that the thermodynamic stability difference between the native and the monomeric denatured state in all the proteins was destabilized by the hydrophobic tags and was well explained by the reverse hydrophobic effect due to the tags. The existence of the RO states was confirmed by both analytical ultracentrifugation and dynamic light scattering. This indicated that the five-isoleucine hydrophobic tag is strong enough to induce intermolecular hydrophobic contact among the denatured molecules leading to oligomerization, and even one- or three-isoleucine tags are effective enough to generate intramolecular hydrophobic contact, thus provoking denaturation through the reverse hydrophobic effect.

Protocols of IATC, DSC, and PPC: The Multistate Structural Transition of Cytochrome c.

The recent development of high-precision calorimeters allows us to monitor the structural transition of biomolecules by calorimetry and thereby characterize the thermodynamic property changes accompanying three-dimensional structure changes. We developed isothermal acid-titration calorimetry (IATC) to evaluate the pH dependence of protein enthalpy. Using the double deconvolution method with precise differential scanning calorimetry (DSC), we revealed that the MG state is an equilibrium intermediate state of the reversible thermal three-state transition of the protein, and we successfully determined its volumetric properties by pressure perturbation calorimetry (PPC). Our findings underscore the importance of a precise calorimetry and analysis model for protein research.

Thermodynamics of the Thermal Denaturation of Acid Molten Globule State of Cytochrome c Indicate a Reversible High-Temperature Oligomerization Process.

In this study, we performed differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) analysis of the thermal transition of cytochrome c from an acidic molten globule (MG) state with the protein concentrations of 0.5-18.2 mg/mL. DSC profiles were highly reversible and showed clear protein-concentration dependence, indicating that reversible oligomerization occurred accompanying the thermal transition from the MG state. The DSC and PPC data required at least a six-state model (MG1 ⇄ MG2 ⇄ D ⇄ 1/2 I2 ⇄ 1/3 I3 ⇄ 1/4 I4) including three new oligomeric states: dimer (I2), trimer (I3), and tetramer (I4) in addition to the three monomeric states previously characterized. Dynamic light scattering confirmed the oligomerization during the thermal transition. The partial specific volumes of these oligomeric states were found to be smaller than those of the monomeric states, MG2 and D, indicating dehydration of hydrophobic surface or hydration of released anions may occur with the reversible oligomerization.

Enzymatic and thermodynamic profiles of a heterotetramer lactate dehydrogenase isozyme in swine.

Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the final step of glycolysis and produces NAD+. In somatic cells, LDH forms homotetramers and heterotetramers that are encoded by two different genes: LDHA (skeletal muscle type, M) and LDHB (heart type, H). Analysis of LDH isozymes is important for understanding the physiological role of homotetramers and heterotetramers and for optimizing inhibition of their enzymatic activity as it may result in distinct effects. Previously, we reported that hydroxychloroquine (HCQ) inhibited LDH activity, but we did not examine isozyme specificity. In the present study, we isolated heterotetrameric LDH (H2M2) from swine brain, determined its kinetic and thermodynamic properties, and examined the effect of HCQ on its activity compared to homotetrameric LDH isozymes. We show that: (1) the Km values for H2M2-mediated catalysis of pyruvate or lactate were intermediate compared to those for the homotetrameric isozymes, M4 and H4 whereas the Vmax values were similar; (2) the Km and Vmax values for H2M2-mediated catalysis of NADH were not significantly different among LDH isozymes; (3) the values for activation energy and van't Hoff enthalpy changes for pyruvate reduction of H2M2 were intermediate compared to those for the homotetrameric isozymes; (4) the temperature for half residual activity of H2M2 was closer to that for M4 than for H4. We also show that HCQ had different affinities for various LDH isozymes.

Unusual Reversible Oligomerization of Unfolded Dengue Envelope Protein Domain 3 at High Temperatures and Its Abolition by a Point Mutation.

We report differential scanning calorimetry (DSC) experiments between 10 and 120 °C of Dengue 4 envelope protein domain 3 (DEN4 ED3), a small 107-residue monomeric globular protein domain. The thermal unfolding of DEN4 ED3 was fully reversible and exhibited two peculiar endothermic peaks. AUC (analytical ultracentrifugation) experiments at 25 °C indicated that DEN4 ED3 was monomeric. Detailed thermodynamic analysis indicated that the two endothermic peaks separated with an increasing protein concentration, and global fitting of the DSC curves strongly suggested the presence of unfolded tetramers at temperatures around 80-90 °C, which dissociated to unfolded monomers at even higher temperatures. To further characterize this rare thermal unfolding process, we designed and constructed a DEN4 ED3 variant that would unfold according to a two-state model, typical of globular proteins. We thus substituted Val 380, the most buried residue at the dimeric interface in the protein crystal, with less hydrophobic amino acids (Ala, Ser, Thr, Asn, and Lys). All variants showed a single heat absorption peak, typical of small globular proteins. In particular, the DSC thermogram of DEN4 V380K indicated a two-state reversible thermal unfolding independent of protein concentration, indicating that the high-temperature oligomeric state was successfully abolished by a single mutation. These observations confirmed the standard view that small monomeric globular proteins undergo a two-state unfolding. However, the reversible formation of unfolded oligomers at high temperatures is a truly new phenomenon, which was fully inhibited by an accurately designed single mutation.

Conserved and unique thermodynamic properties of lactate dehydrogenases in an ectothermic organism, the teleost Microstomus achne, and an endothermic organism, bovine.

It is widely believed that enzymatic activities in ectothermic organisms adapt to environmental temperatures. However, to date, no study has thoroughly compared multiple thermodynamic enzymatic characteristics across species living in dramatically different environments. To start to address this gap, we compared the characteristics of lactate dehydrogenase (LDH) purified from the muscles from slime flounder Microstomus achne white muscle and bovine skeletal muscle (bM4) and heart. The K m and V max for pyruvate reduction were about three times higher for M. achne LDH than bM4 Surprisingly, maximum LDH activity was observed at ∼30 °C and ∼50 °C for M. achne and bovine LDHs, respectively, suggesting that the maximum enzymatic activity of LDH is set at a temperature ∼20 °C higher than environmental or body temperature across species. Although K m and V max values of these LDHs increased with temperature, the V max/K m ratio for M. achne LDH and bM4 was independent. Differential scanning calorimetry and enthalpy change measurements confirmed that M. achne and bovine muscle-specific LDHs shared similar properties. Based on the present findings and previous reports, we hypothesize that the function and thermodynamic properties of muscle LDH are highly conserved between a teleost adapted to cold, M. achne, and bovine.

Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum.

This work aims to improve thermostability of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM) by random and site-directed mutagenesis. From error prone PCR, a mutated CgAM with higher thermostability at 50 °C compared to the wild-type was selected and sequenced. The result showed that the mutant contains a single mutation of A406V. Site-directed mutagenesis was then performed to construct A406V and A406L. Both mutated CgAMs showed higher intermolecular transglucosylation activity with an upward shift in the optimum temperature and a slight increase in the optimum pH for disproportionation and cyclization reactions. Thermostability of both mutated CgAMs at 35-40 °C was significantly increased with a higher peak temperature from DSC spectra when compared to the wild-type. A406V had a greater effect on activity and thermostability than A406L. The catalytic efficiency values kcat/Km of A406V- and A406L-CgAMs were 2.9 and 1.4 times higher than that of the wild-type, respectively, mainly due to a significant increase in kcat. LR-CD product analysis demonstrated that A406V gave higher product yield, especially at longer incubation time and higher temperature, in comparison to the wild-type enzyme.

IATC, DSC, and PPC Analysis of Reversible and Multistate Structural Transition of Cytochrome c.

Development of precise calorimeters has enabled us to monitor the structural transition of biomolecules by calorimetry to characterize the thermodynamic property changes accompanying three-dimensional structure change. We developed isothermal acid-titration calorimetry to evaluate the pH dependence of protein enthalpy, and demonstrated the thermodynamic transition between the native and molten globule (MG) states of cytochrome c with very small enthalpy change (~20 kJ/mol) by this method. The double deconvolution method with precise differential scanning calorimetry has revealed the MG state as an equilibrium intermediate state of the reversible thermal transition of the protein, and pressure perturbation calorimetry has succeeded in determining its volumetric properties. These examples strongly indicate the importance of a precise calorimetry and analysis model in the field of protein research.

Unique structural changes in calcium-bound calmodulin upon interaction with protein 4.1R FERM domain: novel insights into the calcium-dependent regulation of 4.1R FERM domain binding to membrane proteins by calmodulin.

Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca(2+) but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca(2+)-dependent manner. We have previously mapped a Ca(2+)-independent CaM-binding site, pep11 (A(264)KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca(2+) (Ca(2+)/CaM), interacts simultaneously with pep11 and with Ser(185) in A(181)KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca(2+)/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca(2+)/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca(2+). Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca(2+). Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca(2+)/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca(2+)/CaM-dependent regulation of R30 binding to membrane proteins.

Volumetric properties of the molten globule state of cytochrome c in the thermal three-state transition evaluated by pressure perturbation calorimetry.

Thermodynamic properties, including volumetric properties, provide important information on protein stability and folding. Pressure perturbation calorimetry (PPC) is an effective technique for evaluating the temperature dependence of the thermal expansion coefficient, α(p), of biomaterials. Recently, the thermal N-to-D transition of cytochrome c at nearly pH 4 was found to be a three-state transition including a molten globulelike intermediate state. In this study, the thermal N-to-D transition of cytochrome c was examined by PPC measurements with a three-state model. As far as we know, this is the first report of a three-state analysis of PPC, since two-state analyses are traditionally applied. The V(p) of the MG1-like intermediate state in the N-to-D transition at pH 4 was found to be the value between the partial volumes of the N and D states, suggesting an increase of the hydrophobic hydration in this intermediate state.

Structural stabilization of protein 4.1R FERM domain upon binding to apo-calmodulin: novel insights into the biological significance of the calcium-independent binding of calmodulin to protein 4.1R.

In erythrocytes, 4.1R80 (80 kDa isoform of protein 4.1R) binds to the cytoplasmic tail of the transmembrane proteins band 3 and GPC (glycophorin C), and to the membrane-associated protein p55 through the N- (N-terminal), α- (α-helix-rich) and C- (C-terminal) lobes of R30 [N-terminal 30 kDa FERM (4.1/ezrin/radixin/moesin) domain of protein 4.1R] respectively. We have shown previously that R30 binds to CaM (calmodulin) in a Ca2+-independent manner, the equilibrium dissociation constant (Kd) for R30-CaM binding being very similar (in the submicromolar range) in the presence or absence of Ca2+. In the present study, we investigated the consequences of CaM binding on R30's structural stability using resonant mirror detection and FTIR (Fourier-transform IR) spectroscopy. After a 30 min incubation above 40° C, R30 could no longer bind to band 3 or to GPC. In contrast, R30 binding to p55, which could be detected at a temperature as low as 34° C, was maintained up to 44° C in the presence of apo-CaM. Dynamic light scattering measurements indicated that R30, either alone or complexed with apo-CaM, did not aggregate up to 40° C. FTIR spectroscopy revealed that the dramatic variations in the structure of the ß-sheet structure of R30 observed at various temperatures were minimized in the presence of apo-CaM. On the basis of Kd values calculated at various temperatures, ΔCp and ΔG° for R30 binding to apo-CaM were determined as -10 kJ · K(-1) · mol-1 and ~ -38 kJ · mol(-1) at 37° C (310.15 K) respectively. These data support the notion that apo-CaM stabilizes R30 through interaction with its ß-strand-rich C-lobe and provide a novel function for CaM, i.e. structural stabilization of 4.1R80.

Thermodynamic and structural properties of the acid molten globule state of horse cytochrome C.

To understand the stabilization, folding, and functional mechanisms of proteins, it is very important to understand the structural and thermodynamic properties of the molten globule state. In this study, the global structure of the acid molten globule state, which we call MG1, of horse cytochrome c at low pH and high salt concentrations was evaluated by solution X-ray scattering (SXS), dynamic light scattering, and circular dichroism measurements. MG1 was globular and slightly (3%) larger than the native state, N. Calorimetric methods, such as differential scanning calorimetry and isothermal acid-titration calorimetry, were used to evaluate the thermodynamic parameters in the transitions of N to MG1 and MG1 to denatured state D of horse cytochrome c. The heat capacity change, ΔC(p), in the N-to-MG1 transition was determined to be 2.56 kJ K(-1) mol(-1), indicating the increase in the level of hydration in the MG1 state. Moreover, the intermediate state on the thermal N-to-D transition of horse cytochrome c at pH 4 under low-salt conditions showed the same structural and thermodynamic properties of the MG1 state in both SXS and calorimetric measurements. The Gibbs free energy changes (ΔG) for the N-to-MG1 and N-to-D transitions at 15 °C were 10.9 and 42.2 kJ mol(-1), respectively.

Thermodynamic properties of BPTI variants with highly simplified amino acid sequences.

We report the first detailed thermodynamic analysis of simplified proteins by differential scanning calorimetry (DSC). The experiments were carried out with five simplified BPTI variants, whose structures and activities have been reported, in which several residues not essential for specifying the tertiary structure were replaced by alanine. In most aspects, the thermodynamics of simplified proteins were very similar to, if not essentially identical with, those of natural proteins. In particular, they undergo a highly cooperative two-state thermal unfolding process with a large enthalpy change, which is a thermodynamic hallmark of the native state of natural globular proteins. Furthermore, the specific enthalpy and entropy changes upon unfolding at 110 degrees C were close to values invariably observed for small natural globular proteins (55 J g(-1) and ~16 J K(-1) g(-1), respectively). On the other hand, two simplified BPTI variants, BPTI-21 and BPTI-22 (containing 21 and 22 alanine residues), were enthalpically stabilized while entropically destabilized with respect to the reference BPTI-[5,55] molecule. This peculiar type of entropy-enthalpy compensation is in sharp contrast to the usual enthalpy destabilization/entropy stabilization observed in mutational studies of natural proteins. Overall, we conclude that a thermodynamic native state can be achieved by proteins encoded with extensively simplified sequences.

A molten globule-like intermediate state detected in the thermal transition of cytochrome c under low salt concentration.

To understand the stabilization mechanism of the transient intermediate state in protein folding, it is very important to understand the structure and stability of the molten globule state under a native condition, in which the native state exists stably. The thermal transitions of horse cytochrome c were thermodynamically evaluated by highly precise differential scanning calorimetry (DSC) at pH 3.8-5.0. The heat capacity functions were analyzed using double deconvolution and the nonlinear least-squares method. An intermediate (I) state is clearly confirmed in the thermal native (N)-to-denatured (D) transition of horse cytochrome c. The mole fraction of the intermediate state shows the largest value, 0.4, at nearly 70 degrees C at pH 4.1. This intermediate state was also detected by the circular dichroism (CD) method and was found to have the properties of the molten globule-like structure by three-state analysis of the CD data. The Gibbs free-energy change between N and I, DeltaG(NI), and that between N and D, DeltaG(ND), were evaluated to be 9-22 kJ mol(-1) and 41-45 kJ mol(-1), respectively at 15( ) degrees C and pH 4.1.

Direct observation of the enthalpy change accompanying the native to molten-globule transition of cytochrome c by using isothermal acid-titration calorimetry.

The enthalpy change accompanying the reversible acid-induced transition from the native (N) to the molten-globule (MG) state of bovine cytochrome c was directly evaluated by isothermal acid-titration calorimetry (IATC), a new method for evaluating the pH dependence of protein enthalpy. The enthalpy change was 30 kJ/mol at 30 degrees C, pH 3.54, with 500 mM KCl. The results of the global analysis of the temperature dependence of the excess enthalpy from 20 to 35 degrees C demonstrated that the N to MG transition is a two-state transition with a small heat capacity change of 1.1 kJ K(-1) mol(-1). The present findings were also indicative of the pH dependence of the enthalpy and the heat capacity of the MG state, -13 kJ mol(-1) pH(-1) and -1.0 kJ K(-1) mol(-1) pH(-1), respectively, at 30 degrees C within a pH range from 2 to 3.

Isothermal acid-titration calorimetry for evaluating the pH dependence of protein stability.

A new method, which can be called as isothermal acid-titration calorimetry (IATC), was proposed for evaluating the enthalpy of protein molecules as a function of pH using isothermal titration calorimetry (ITC). This measurement was used to analyze the acid-denaturation of bovine ribonuclease A. The enthalpy change by acid-denaturation of this protein was estimated as 310 kJ/mol at pH 2.8 and 40 degrees C. This value agreed well with the enthalpy change obtained by differential scanning calorimetry. The midpoint pH and proton binding-number difference observed by IATC agreed well with those of the acid transition of the three-dimensional structure monitored by circular dichroism spectrometry. The van't Hoff enthalpy of the transition was derived from the temperature dependence of the midpoint pH and the proton binding-number difference. It agreed well with the calorimetric enthalpy change directly observed by IATC, strongly indicating that there was no stable intermediate state during the acid transition of this protein.