Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

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 ↔ In ↔ 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 "reverse engineered" 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.

Thermodynamic Analysis of Point Mutations Inhibiting High-Temperature Reversible Oligomerization of PDZ3.

Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation, exhibiting two endothermic peaks because of the formation of reversible oligomers at high temperature (N↔I6↔D). This contrasts sharply with the standard two-state denaturation model observed for small, globular proteins. We performed an alanine scanning analysis by individually mutating three hydrophobic residues at the crystallographic oligomeric interface (Phe340, Leu342, and Ile389) and one away from the interface (Leu349, as a control). DSC analysis indicated that PDZ3-F340A and PDZ3-L342A exhibited a single endothermic peak. Furthermore, PDZ3-L342A underwent a perfect two-state denaturation, as evidenced by the single endothermic peak and confirmed by detailed DSC analysis, including global fitting of data measured at different protein concentrations. Reversible oligomerization (RO) at high temperatures by small globular proteins is a rare event. Furthermore, our present study showing that a point mutation, L342A, designed based on the crystal structure inhibited RO is surprising because RO occurs at a high-temperature. Future studies will determine how and why mutations designed using crystal structures determined at ambient temperatures influence the formation of RO at high temperatures, and whether high-temperature ROs are related to the propensity of proteins to aggregate or precipitate at lower temperatures, which would provide a novel and unique way of controlling protein solubility and aggregation.

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.

Significance of H461 at subsite +1 in substrate binding and transglucosylation activity of amylomaltase from Corynebacterium glutamicum.

Amylomaltase (AM) catalyzes inter- and intra-molecular transglycosylation reactions of glucan to yield linear and cyclic oligosaccharide products. The functional roles of the conserved histidine at position 461 in the active site of AM from Corynebacterium glutamicum (CgAM) was investigated. H461 A/S/D/R/W were constructed, their catalytic properties were compared to the wild-type (WT). A significant decrease in transglucosylation activities was observed, especially in H461A mutant, while hydrolysis activity was barely affected. The transglucosylation factor of the H461A-CgAM was decreased by 8.6 folds. WT preferred maltotriose (G3) as substrate for disproportionation reaction, but all H461 mutants showed higher preference for maltose (G2). Using G3 substrate, kcat/Km values of H461 mutated CgAMs were 40-64 folds lower, while the Km values were twice higher than those of WT. All mutants could not produce large-ring cyclodextrin (LR-CD) product. The heat capacity profile indicated that WT had higher thermal stability than H461A. The X-ray structure of WT showed two H-bonds between H461 and heptasaccharide analog at subsite +1, while no such bonding was observed from the model structure of H461A. The importance of H461 on substrate binding with CgAM was evidenced. We are the first to mutate an active site histidine in AM to explore its function.

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.

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.

Roles of N287 in catalysis and product formation of amylomaltase from Corynebacterium glutamicum.

Amylomaltase catalyzes intermolecular and intramolecular transglucosylation reactions to form linear and cyclic oligosaccharides, respectively. The aim of this work is to investigate the structure-function relationship of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM). Site-directed mutagenesis was performed to substitute Tyr for Asn287 (N287Y) to determine its role in controlling amylomaltase activity and product formation. Expression of the wild-type (WT) and N287Y was achieved by cultivating recombinant cells in the medium containing lactose at 16 °C for 14 h. The purified mutated enzyme showed a significant decrease in all transglucosylation activities while hydrolysis activity was not changed. Optimum temperature and pH for disproportionation reaction were slightly changed upon mutation while those for cyclization reaction were not changed. Interestingly, N287Y showed a change in large-ring cyclodextrin (LR-CD) product profile in which the larger size was observed together with an increase in thermostability and substrate preference for G5 in addition to G3. The secondary structure of the mutated enzyme was slightly changed in related to the WT as evidenced from circular dichroism analysis. This work thus demonstrates that N287 is required for transglucosylation activities of CgAM. Having an aromatic residue in this position increased thermostability, changed product profile and substrate preference but demolished most enzyme activities.

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.

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.

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.

Blocking PSD95-PDZ3's amyloidogenesis through point mutations that inhibit high-temperature reversible oligomerization (RO).

The third PDZ domain of the postsynaptic density protein 95 (PSD95-PDZ3; 11 kDa, 103 residues) has a propensity to form amyloid fibrils at high temperatures. At neutral pH, PDZ3 is natively folded, but it exhibits a peculiar three-state thermal unfolding with a reversible oligomerization (RO) equilibrium at high temperatures, which is uncharacteristic in the unfolding of a small globular protein as PDZ3 is. Here, we examined the RO's role in PDZ3's amyloidogenesis at high-temperature using two variants (F340A and L342A) that suppress the high-temperature RO and five single-alanine-mutated variants, where we mutated surface-exposed hydrophobic residues to alanine. Circular Dichroism (CD), Analytical Ultracentrifuge (AUC), and other spectroscopic measurements confirmed the retention of the native structure at ambient temperature. Differential Scanning Calorimetry (DSC) was used to assess the presence or absence of the high-temperature RO, and the amyloidogenicity of the variants was measured by Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM). By comparing the fraction of RO and the ThT signal, we found that mutations that suppressed the high-temperature RO strongly inhibited amyloidogenesis. On the other hand, all variants forming RO also formed amyloids under the same conditions as the wild-type PDZ3.

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 characterization of OsGID1-gibberellin binding using calorimetry and docking simulations.

Gibberellins (GAs) are phytohormones regulating various developmental processes in plants. In rice, the initial GA-signaling events involve the binding of a GA to the soluble GA receptor protein, GID1. Although X-ray structures for certain GID1/GA complexes have recently been determined, an examination of the complexes does not fully clarify how GID1s discriminate among different GAs. Herein, we present a study aimed at defining the types of forces important to binding via a combination of isothermal titration calorimetry (ITC) and computational docking studies that employed rice GID1 (OsGID1), OsGID1 mutants, which were designed to have a decreased possible number of hydrogen bonds with bound GA, and GA variants. We find that, in general, GA binding is enthalpically driven and that a hydrogen bond between the phenolic hydroxyl of OsGID1 Tyr134 and the C-3 hydroxyl of a GA is a defining structural element. A hydrogen-bond network that involves the C-6 carboxyl of a GA that directly hydrogen bonds the hydroxyl of Ser198 and indirectly, via a two-water-molecule network, the phenolic hydroxyl of Tyr329 and the NH of the amide side-chain of Asn255 is also important for GA binding. The binding of OsGID1 by GA(1) is the most enthalpically driven association found for the biologically active GAs evaluated in this study. This observation might be a consequence of a hydrogen bond formed between the hydroxyl at the C-13 position of GA(1) and the main chain carbonyl of OsGID1 Phe245. Our results demonstrate that by combining ITC experiments and computational methods much can be learned about the thermodynamics of ligand/protein binding.

Syntheses of cellotriose and cellotetraose analogues as transition state mimics for mechanistic studies of cellulases.

Cellotriose and cellotetraose analogues carrying cyclohexene rings were developed as molecular probes which are expected to mimic the transition state conformation of hydrolysis by cellulases. The cyclohexene ring was placed at the pyranose ring being expected to locate the -1 subsite of the enzyme. In order to evaluate these probes, sulfur derivatives of cellotriose and cellotetraose were also synthesized as the enzyme tolerant analogues which mimic the stable conformations of the natural cellulose. The binding assays using differential scanning calorimetry revealed that introduction of the cyclohexene ring is effective to the complexation with an endoglucanase, NCE5 from Humicola insolens.

Thermodynamic and structural analysis of highly stabilized BPTIs by single and double mutations.

Enhancing protein conformational stability is an important aspect of protein engineering and biotechnology. However, protein stabilization is difficult to rationalize as it often results from the small cumulative and intertwined effects of multiple mutations. Here, we analyzed the mechanisms behind a remarkable 13 degrees stabilization produced by a single A14G and a double A14GA38V mutation in BPTI-[5,55], a natively folded bovine pancreatic trypsin inhibitor variant. Differential scanning calorimetry analysis of three BPTI-[5,55] variants (A14G, A38V, and A14GA38V) indicated that the A14G mutation stabilized the structure enthalpically, whereas the A38V stabilization was entropy driven. We also determined the structure of the A14GA38V mutant at 1.09 A resolution, whereas the A38V variant did not crystallize, and we previously reported the A14G variant's structure (2ZJX). The overall structures of the A14G and A14GA38V variants were very similar to that of wild-type BPTI, but small local structure perturbations around residues 14 and 38 strongly suggested potential factors contributing to the enthalpy stabilization. First, the A14G mutation displaced the local backbone structures around residues 14 and 38 by up to 0.7 A, presumably increasing local van der Waals interactions. Next, this displacement produced steric clashes between neighboring residue's side-chains in all but the variants containing the A14G mutation. Noteworthy, these clashes are not predicted from the wild type BPTI structure. These observations provide one of the first unambiguous analyses of how a subtle interplay between the sidechain and backbone structures can have a major effect on protein stability.

Characterization of a sHsp of Schizosaccharomyces pombe, SpHsp15.8, and the implication of its functional mechanism by comparison with another sHsp, SpHsp16.0.

There exist two small heat shock proteins (sHsps) in the fission yeast, Schizosaccharomyces pombe (S. pombe), whose expressions are highly induced by heat stress. We have previously expressed, purified, and characterized one of the sHsps, SpHsp16.0. In this study, we examined the other sHsp, SpHsp15.8. It suppressed the thermal aggregation of citrate synthase (CS) from porcine heart and dithiothreitol-induced aggregation of insulin from bovine pancreas with very high efficiency. Almost one SpHsp15.8 subunit was sufficient to protect one protein molecule from aggregation. Like SpHsp16.0, SpHsp15.8 dissociated into small oligomers and then interacted with denatured substrate proteins. SpHsp16.0 exhibited a clear enthalpy change for denaturation occurring over 60 degrees C in differential scanning calorimetry (DSC). However, we could not observe any significant enthalpy change in the DSC of SpHsp15.8. The difference is likely to be caused by the adhesive characteristics of SpHsp15.8. The oligomer dissociation of SpHsp15.8 and SpHsp16.0 and their interactions with denatured substrate proteins were studied by fluorescence polarization analysis (FPA). Both sHsps exhibited a temperature-dependent decrease of fluorescence polarization, which correlates with the dissociation of large oligomers to small oligomers. The dissociation of the SpHsp15.8 oligomer began at about 35 degrees C and proceeded gradually. On the contrary, the SpHsp16.0 oligomer was stable up to approximately 45 degrees C, but then dissociated into small oligomers abruptly at this temperature. Interestingly, SpHsp16.0 is likely to interact with denatured CS in the dissociated state, while SpHsp15.8 is likely to interact with CS in a large complex. These results suggest that S. pombe utilizes two sHsps that function in different manners, probably to cope with a wide range of temperatures and various denatured proteins.

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.

Hydrophobic surface residues can stabilize a protein through improved water-protein interactions.

Protein stabilization is difficult to rationalize, but the detailed thermodynamic and structural analysis of a series of carefully designed mutants may provide experimental insights into the mechanisms underlying stabilization. Here, we report a systematic structural and thermodynamic analysis of bovine pancreatic trypsin inhibitor (BPTI) variants that are significantly stabilized through a single amino acid substitution at residue 38, which is located in a loop mostly exposed on the protein surface. Differential scanning calorimetry indicated that the BPTI-[5,55]Gly14 variants with a single mutation at position 38 were stabilized in an enthalpy-driven manner and that the magnitude of the stabilization increased as the hydrophobicity of residue 38 increased. This increase in the thermal stability of BPTI was unexpected because a hydrophobic residue on a protein surface is usually destabilizing. To identify the structural determinants of this stabilization, we determined the crystal structures of six BPTI-[5,55]Gly14 variants (Gly14 Gly38 , Gly14 Ala38 , Gly14 Val38 , Gly14 Leu38 , Gly14 Ile38 , and Gly14 Lys38 ) at high resolutions and showed that they retain essentially the same structure as the wild-type BPTI. A more detailed examination of their structures indicated that the extent of thermal stabilization correlated with both improved local packing and increased hydration around the substitution sites. In particular, the number of water molecules near residue 38 increased upon mutation to a hydrophobic residue suggesting that improved hydration contributed to the enthalpy-driven stabilization. Increasing a protein's thermal stability by the placement of a hydrophobic amino acid on the protein surface is a novel and unexpected phenomenon, and its exact nature is worth further examination, as it may provide a generic method for stabilizing proteins in an enthalpy-driven manner. DATABASE: The coordinates and structure factors of Gly14 Gly38 , Gly14 Ile38 , Gly14 Leu38 , and Gly14 Lys38 variants of BPTI-[5,55] are deposited in the Protein Data Bank under the PDB entry codes 5XX3, 5XX5, 5XX2, and 5XX4, respectively. We previously reported the structures of Gly14 Ala38 (2ZJX) and Gly14 Val38 (2ZVX).

Role of the IXI/V motif in oligomer assembly and function of StHsp14.0, a small heat shock protein from the acidothermophilic archaeon, Sulfolobus tokodaii strain 7.

Small heat shock proteins (sHsps) are one of the most ubiquitous molecular chaperones. They are grouped together based on a conserved domain, the alpha-crystallin domain. Generally, sHsps exist as oligomers of 9-40 subunits, and the oligomers undergo reversible temperature-dependent dissociation into smaller species as dimers, which interact with denaturing substrate proteins. Previous studies have shown that the C-terminal region, especially the consensus IXI/V motif, is responsible for oligomer assembly. In this study, we examined deletions or mutations in the C-terminal region on the oligomer assembly and function of StHsp14.0, an sHsp from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Mutated StHsp14.0 with C-terminal deletion or replacement of IIe residues in the IXI/V motif to Ala, Ser, or Phe residues could not form large oligomers and lost chaperone activity. StHsp14.0WKW, whose Ile residues in the IXI/V motif are changed to Trp, existed as an oligomer like that of the wild type. However, it dissociates to small oligomers and exhibits chaperone activity at relatively lowered temperature. Replacement of two Ile residues in the motif to relatively small residues, Ala or Ser, also resulted in the change of beta-sheet rich secondary structure and decrease of hydrophobicity. Interestingly, StHsp14.0 mutant with amino acid replacements to Phe kept almost the same secondary structure and relatively high hydrophobicity despite that it could not form an oligomeric structure. The results show that hydrophobicity and size of the amino acids in the IXI/V motif in the C-terminal region are responsible not only for assembly of the oligomer but also for the maintenance of beta-sheet rich secondary structure and hydrophobicity, which are important for the function of sHsp.