Thermal Unfolding Transition Research Articles

Exploring the role of preferential solvation in the stability of globular proteins through the study of ovalbumin interaction with organic additives

<abstract> <p>The impact of denaturing and stabilizing osmolytes on protein conformational dynamics has been extensively explored due to the significant contribution of protein solvation to the stability, function, malfunction and regulation of globular proteins. We studied the effect of two nonspecific organic molecules, urea, which is a conventional denaturant, and dimethyl sulfoxide (DMSO), which is a multilateral organic solvent, on the stability and conformational dynamics of a non-inhibitory serpin, ovalbumin (OVA). A differential scanning microcalorimetry (DSC) experimental series conducted in the phosphate buffer solutions containing 0–30% of additives revealed the destabilizing impact of both urea and DMSO in a mild acidic media, manifested in the gradual decrease of thermal unfolding enthalpy and transition temperature. These findings differ from the results observed in our study of the mild alkaline DMSO buffered solutions of OVA, where the moderate stabilization of OVA was observed in presence of 5–10% of DMSO. However, the overall OVA interaction patterns with urea and DMSO are consistent with our previous findings on the stability and conformational flexibility of another model globular protein, α-chymotrypsin, in similar medium conditions. The obtained results could be explained by preferential solvation patterns. Positive preferential solvation of protein by urea in urea/water mixtures mainly weakens the hydrophobic interactions of the protein globule and eventually leads to the disruption of the tertiary structure within the whole range of urea concentrations. Alternatively, under certain experimental conditions in DMSO/water mixtures, positive preferential solvation by water molecules can be observed. We assume that the switch to the positive preferential solvation by DMSO, which is shown to have a soft maximum around 20–30% DMSO, could be shifted towards lower additive concentrations due to the intrinsic capability of ovalbumin OVA to convert into a heat-stable, yet flexible set of conformations that have increased the surface hydrophobicity, characteristic to molten-globule-like states.</p> </abstract>

Protein Engineering and HDX Identify Structural Regions of G-CSF Critical to Its Stability and Aggregation.

The protein engineering and formulation of therapeutic proteins for prolonged shelf-life remain a major challenge in the biopharmaceutical industry. Understanding the influence of mutations and formulations on the protein structure and dynamics could lead to more predictive approaches to their improvement. Previous intrinsic fluorescence analysis of the chemically denatured granulocyte colony-stimulating factor (G-CSF) suggested that loop AB could subtly reorganize to form an aggregation-prone intermediate state. Hydrogen deuterium exchange mass spectrometry (HDX-MS) has also revealed that excipient binding increased the thermal unfolding transition midpoint (Tm) by stabilizing loop AB. Here, we have combined protein engineering with biophysical analyses and HDX-MS to reveal that increased exchange in a core region of the G-CSF comprising loop AB (ABI, a small helix, ABII) and loop CD packed onto helix B and the beginning of loop BC leads to a decrease in Tm and higher aggregation rates. Furthermore, some mutations can increase the population of the aggregation-prone conformation within the native ensemble, as measured by the greater local exchange within this core region.

The effects of intramolecular and intermolecular electrostatic repulsions on the stability and aggregation of NISTmAb revealed by HDX-MS, DSC, and nanoDSF.

The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX-MS), differential scanning calorimetry (DSC), and nano-differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pHs (pH5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX-MS results showed that NISTmAb is more conformationally stable at a pH near its isoelectric point (pI) in the presence of NaCl than a pH far from its pI in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the CH 2 domain of the protein is more conformationally stable at a pH near its pI. On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a pH far from its pI, particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the pI than far away from the pI, can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry-Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a pH from the pI, the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at pH far from the pI.

Stability of the Retinoid X Receptor-α Homodimer in the Presence and Absence of Rexinoid and Coactivator Peptide.

Differential scanning calorimetry and differential scanning fluorimetry were used to measure the thermal stability of human retinoid X receptor-α ligand binding domain (RXRα LBD) homodimer in the absence or presence of rexinoid and coactivator peptide, GRIP-1. The apo-RXRα LBD homodimer displayed a single thermal unfolding transition with a Tm of 58.7 °C and an unfolding enthalpy (ΔH) of 673 kJ/mol (12.5 J/g), much lower than average value (35 J/g) of small globular proteins. Using a heat capacity change (ΔCp) of 15 kJ/(mol K) determined by measurements at different pH values, the free energy of unfolding (ΔG) of the native state was 33 kJ/mol at 37 °C. Rexinoid binding to the apo-homodimer increased Tm by 5 to 9 °C and increased the ΔG of the native homodimer by 12 to 20 kJ/mol at 37 °C, consistent with the nanomolar dissociation constant (Kd) of the rexinoids. GRIP-1 binding to holo-homodimers containing rexinoid resulted in additional increases in ΔG of 14 kJ/mol, a value that was the same for all three rexinoids. Binding of rexinoid and GRIP-1 resulted in a combined 50% increase in unfolding enthalpy, consistent with reduced structural fluidity and more compact folding observed in other published structural studies. The complexes of UAB110 and UAB111 are each more stable than the UAB30 complex by 8 kJ/mol due to enhanced hydrophobic interactions in the binding pocket because of their larger end groups. This increase in thermodynamic stability positively correlates with their improved RXR activation potency. Thermodynamic measurements are thus valuable in predicting agonist potency.

The effect of cysteine oxidation on DJ-1 cytoprotective function in human alveolar type II cells

DJ-1 is a multifunctional protein with cytoprotective functions. It is localized in the cytoplasm, nucleus, and mitochondria. The conserved cysteine residue at position 106 (Cys106) within DJ-1 serves as a sensor of redox state and can be oxidized to both the sulfinate (-SO2−) and sulfonate (-SO3−) forms. DJ-1 with Cys106-SO2− has cytoprotective activity but high levels of reactive oxygen species can induce its overoxidation to Cys106-SO3−. We found increased oxidative stress in alveolar type II (ATII) cells isolated from emphysema patients as determined by 4-HNE expression. DJ-1 with Cys106-SO3− was detected in these cells by mass spectrometry analysis. Moreover, ubiquitination of Cys106-SO3− DJ-1 was identified, which suggests that this oxidized isoform is targeted for proteasomal destruction. Furthermore, we performed controlled oxidation using H2O2 in A549 cells with DJ-1 knockout generated using CRISPR-Cas9 strategy. Lack of DJ-1 sensitized cells to apoptosis induced by H2O2 as detected using Annexin V and propidium iodide by flow cytometry analysis. This treatment also decreased both mitochondrial DNA amount and mitochondrial ND1 (NADH dehydrogenase 1, subunit 1) gene expression, as well as increased mitochondrial DNA damage. Consistent with the decreased cytoprotective function of overoxidized DJ-1, recombinant Cys106-SO3− DJ-1 exhibited a loss of its thermal unfolding transition, mild diminution of secondary structure in CD spectroscopy, and an increase in picosecond–nanosecond timescale dynamics as determined using NMR. Altogether, our data indicate that very high oxidative stress in ATII cells in emphysema patients induces DJ-1 overoxidation to the Cys106-SO3− form, leading to increased protein flexibility and loss of its cytoprotective function, which may contribute to this disease pathogenesis.

Macromolecular properties and partial amino acid sequence of a Kunitz-type protease inhibitor from okra (Abelmoschus esculentus) seeds

A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra (Abelmoschus esculentus) seeds by a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circular dichroism spectrum showed that the protein contains approx. 39% beta-sheets but only approx. 5% alpha-helices. The protein is thermally quite stable, and exhibits a cooperative thermal unfolding transition at approx. 70 degree C, as determined by circular dichroism spectroscopy and differential scanning fluorimetry. De novo sequencing of OPI by nanoESI-Q-ToF mass spectrometry (MS) allowed the assignment of about 83% of its primary structure, which indicated that the protein shares 43% sequence identity with a putative 21 kDa trypsin inhibitor from Theobroma bicolor. An intramolecular disulfide linkage between Cys149 and Cys156 was also detected. The protein showed approx 24 and approx 25% sequence identity with alpha-amylase/subtilisin inhibitor from barley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structural fold similar to other Kunitz-type TIs. The presence of Cys149-Cys156 disulfide bond as detected by MS and a second disulfide bond connecting Cys44-Cys91, conserved in all Kunitz-type TIs, is also identified in the model.

Heterogeneity in the Folding of Villin Headpiece Subdomain HP36.

Small single domain proteins that fold on the microsecond time scale have been the subject of intense interest as models for probing the complexity of folding energy landscapes. The villin headpiece subdomain (HP36) has been extensively studied because of its simple three helix structure, ultrafast folding lifetime of a few microseconds, and stable native fold. We have previously shown that folding as measured by a single 13C═18O isotopic label on residue A57 in helix 2 occurs at a different rate than that measured by global probes of folding, indicating noncooperative complexity in the folding of HP36. In order to determine whether this complexity reflects intermediates or parallel pathways over a small activation barrier, 13C═18O labels were individually incorporated at six different positions in HP36, including into all 3 helices. The equilibrium thermal unfolding transitions and the folding/unfolding dynamics were monitored using the unique IR signature of the 13C═18O label by temperature dependent FTIR and temperature jump IR spectroscopy, respectively. Equilibrium experiments reveal that the 13C═18O labels at different positions in HP36 show drastic differences in the midpoint of their transitions ( Tm), ranging from 45 to 67 °C. Heterogeneity is also observed in the relaxation kinetics; there are differences in the microsecond phase when different labeled positions are probed. At a final temperature of 45 °C, the relaxation rate for 13C═18O A57 is 2.4e + 05 s-1 whereas for 13C═18O L69 HP36 the relaxation rate is 5.1e + 05 s-1, two times faster. The observation of site-dependent midpoints for the equilibrium unfolding transitions and differences in the relaxation rates of the labeled positions enables us to probe the progressive accumulation of the folded structure, providing insight into the microscopic details of the folding mechanism.

Design of a Short Thermally Stable α-Helix Embedded in a Macrocycle.

Although helices play key roles in peptide-protein and protein-protein interactions, the helical conformation is generally unstable for short peptides (10-15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain-to-side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non-hydrogen-bonded amide groups to solvent. Here, we develop a "capped-strapped" peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end-capping interactions. We have designed a ten-residue capped-strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and Tm ≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X-ray crystallography revealed a novel quaternary structure with implications for foldamer design.

"Cooperative collapse" of the denatured state revealed through Clausius-Clapeyron analysis of protein denaturation phase diagrams.

Protein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non-2-state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two-state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs-Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147-60). We resolve this thermodynamic conundrum using a Clausius-Clapeyron analysis of the urea-temperature phase diagram that defines how and the urea m-value interconvert through the slope of cm versus T, . This relationship permits the calculation of at low temperature from m-values obtained through iso-thermal urea denaturation and high temperature m-values from obtained through iso-urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of and the m-value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea-expanded and thermally-compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide-intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼-13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this "cooperative collapse" of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall free energy.

Kinetics and thermodynamics of the thermal inactivation and chaperone assisted folding of zebrafish dihydrofolate reductase

The maintenance of thermal stability is a major issue in protein engineering as many proteins tend to form inactive aggregates at higher temperatures. Zebrafish DHFR, an essential protein for the survival of cells, shows irreversible thermal unfolding transition. The protein exhibits complete unfolding and loss of activity at 50 °C as monitored by UV-Visible, fluorescence and far UV-CD spectroscopy. The heat induced inactivation of zDHFR follows first–order kinetics and Arrhenius law. The variation in the value of inactivation rate constant, k with increasing temperatures depicts faster inactivation at elevated temperatures. We have attempted to study the chaperoning ability of a shorter variant of GroEL (minichaperone) and compared it with that of conventional GroEL-GroES chaperone system. Both the chaperone system prevented the aggregation and assisted in refolding of zDHFR. The rate of thermal inactivation was significantly retarded in the presence of chaperones which indicate that it enhances the thermal stability of the enzyme. As minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon.

Critical structural fluctuations of proteins upon thermal unfolding challenge the Lindemann criterion

Internal subnanosecond timescale motions are key for the function of proteins, and are coupled to the surrounding solvent environment. These fast fluctuations guide protein conformational changes, yet their role for protein stability, and for unfolding, remains elusive. Here, in analogy with the Lindemann criterion for the melting of solids, we demonstrate a common scaling of structural fluctuations of lysozyme protein embedded in different environments as the thermal unfolding transition is approached. By combining elastic incoherent neutron scattering and advanced molecular simulations, we show that, although different solvents modify the protein melting temperature, a unique dynamical regime is attained in proximity of thermal unfolding in all solvents that we tested. This solvation shell-independent dynamical regime arises from an equivalent sampling of the energy landscape at the respective melting temperatures. Thus, we propose that a threshold for the conformational entropy provided by structural fluctuations of proteins exists, beyond which thermal unfolding is triggered.

Differential scanning calorimetric and spectroscopic studies on the thermal and chemical unfolding of cucumber (Cucumis sativus) phloem exudate lectin

In plants, chitooligosaccharide-binding phloem exudate lectins play an important role in the defense mechanism against parasites. Here, we investigated the thermal and chaotrope-induced unfolding of cucumber (Cucumis sativus) phloem exudate lectin (CPL). Circular dichroism (CD) spectroscopic studies indicate that the secondary and tertiary structures of CPL are essentially unaltered up to 90°C. Consistent with this, differential scanning calorimetric studies revealed that CPL is highly thermostable and undergoes a cooperative thermal unfolding transition centered at 97.6°C. The unfolding process was calorimetrically irreversible, and could be described by a non-two-state model, suggesting that upon undergoing a reversible unfolding transition the protein attains a final state in an irreversible step. The ratio of calorimetric and van’t Hoff enthalpies (ΔHc/ΔHv) was >1.0, suggesting that the two monomers in the dimeric protein unfold at the same temperature. CD spectra recorded at different pH indicated that the secondary and tertiary structures of the protein are nearly unaltered in the pH range 3.0–10.0. Guanidine hydrochloride-induced unfolding studies indicate that chemical denaturation of CPL can also be described by a two-state process, without involving any intermediate. The stability of CPL to high temperatures and large variations of pH appear to be particularly suited for its role in plant defense.

Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions.

We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9M urea or 6M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.

Folding and unfolding pathway of chaperonin GroEL monomer and elucidation of thermodynamic parameters

The conformation and thermodynamic stability of monomeric GroEL were studied by CD and fluorescence spectroscopy. GroEL denaturation with urea and dilution in buffer leads to formation of a folded GroEL monomer. The monomeric nature of this protein was verified by size-exclusion chromatography and native PAGE. It has a well-defined secondary and tertiary structure, folding activity (prevention of aggregation) for substrate protein and is resistant to proteolysis. Being a properly folded and reversibly refoldable, monomeric GroEL is amenable for the study of thermodynamic stability by unfolding transition methods. We present the equilibrium unfolding of monomeric GroEL as studied by urea and heat mediated unfolding processes. The urea mediated unfolding shows two transitions and a single transition in the heat mediated unfolding process. In the case of thermal unfolding, some residual structure unfolds at a higher temperature (70–75°C). The process of folding/unfolding is reversible in both cases. Analysis of folding/unfolding data provides a measure of ΔGNUH2O, Tm, ΔHvan and ΔSvan of monomeric GroEL. The thermodynamic stability parameter ΔGNUH2O is similar with both CD and intrinsic fluorescence i.e. 7.10±1.0kcal/mol. The calculated Tm, ΔHvan and ΔSvan from the thermal unfolding transition is 46±0.5°C, 43.3±0.1kcal/mol and 143.9±0.1cal/mol/k respectively.

Interactions and cooperativity between P-glycoprotein structural domains determined by thermal unfolding provides insights into its solution structure and function

Structural changes in mouse P-glycoprotein (Pgp) induced by thermal unfolding were studied by differential scanning calorimetry (DSC), circular dichroism and fluorescence spectroscopy to gain insight into the solution conformation(s) of this ABC transporter that may not be apparent from current crystal structures. DSC of reconstituted Pgp showed two thermal unfolding transitions in the absence of MgATP, suggesting that each transition involved the cooperative unfolding of two or more interacting structural domains. A low calorimetric unfolding enthalpy and minimal structural changes were observed, which are hallmarks of the thermal unfolding of α-helical membrane proteins, because generally only the extramembranous regions undergo significant unfolding. Nucleotide binding increased the unfolding temperature of both transitions to the same extent, suggesting that one nucleotide binding domain (NBD) unfolds with each transition. Combined with the results from the two isolated NBDs, we propose that each DSC transition represents the cooperative unfolding of one NBD and the two contacting intracellular loops. Further, the presence of two transitions in both apo and MgATP bound wild-type Pgp suggests the NBD-dimeric conformation is transient, and that Pgp resides predominantly in the crystallographically observed inward-facing conformation with NBDs separated, even under conditions supporting continuous MgATP hydrolysis. In contrast, DSC of the vanadate-trapped MgADP·Pgp complex and the MgATP-bound catalytically inactive mutant, E552A/E1197A, show an additional transition at much higher temperature, corresponding to the unfolding of the nucleotide-trapped NBD-dimeric outward-facing conformation. The collective results indicate a strong preference for an NBD dissociated, inward-facing conformation of Pgp.

Effect of mechanical denaturation on surface free energy of protein powders

Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms.

Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease

Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR–DMP323 complex, and distinctively for PRD25N–DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer–inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism.

Biochemical characterization and functional analysis of the POU transcription factor POU-M2 of Bombyx mori

POU-M2 is a homeodomain transcription factor which plays important roles in the development and silk synthesis of Bombyx mori. In this study, we expressed, purified and characterized POU-M2 and studied its transcription regulation on fibroin heavy chain gene of Bombyx mori. Gel filtration showed POU-M2 existed as a dimer in solution. Far-UV circular dichroism spectra indicated POU-M2 had a well-defined α-helix structure and the α-helix content was about 26.4%. The thermal unfolding transition of POU-M2 was a cooperative process. Tm, οH and οS were 45.15±0.2°C, 138.4±0.5KJ/mol and 0.4349±0.04KJ/(mol·K), respectively. Western blotting analysis indicated the expression level of POU-M2 increased slightly from day 3 to day 7 of the fifth instar larvae in the posterior silk gland. POU-M2 was positioned in the nucleus of cells. The luciferase reporter assay demonstrated POU-M2 could stimulate the promoter activity of fibroin heavy chain gene, and the activation effect was dependent on the amount of POU-M2. Our study suggested POU-M2 may be involved in the transcriptional regulation of fibroin heavy chain gene. These findings expand toward a better understanding of the structure of POU-M2 and its function in silk synthesis of Bombyx mori.

Determinants of Domain Swapping in Staphylococcal Nuclease

Previous studies have shown that the deletion of residues 114-119 (comprising Val-114, Tyr-115, Lys-116, Gly-117, Asn-118, and Asn-119) in wild type staphylococcal nuclease (SNase) results in a C-terminal domain swapped dimer. The 114-119 segment constitutes the loop that precedes that C-terminal helix that is swapped. In the crystal structure of the domain swapped state the dimers make a well-packed interface. The domain swapped dimer is identifiable as a dimer crystallographically, by native gel electrophoresis, by light scattering, by analytical ultracentrifugation, and by NMR spectroscopy. Chemical denaturation of the domain swapped dimer leads to a single transition between folded and unfolded states, suggesting that the monomeric form is unstable. This is consistent with the effect of the 114-119 truncation on the midpoints of the thermal and acid unfolding transitions, which show that the domain swapped dimer is considerably less stable than the parent protein. In the highly stable Δ+PHS form of SNase, which is 6.4 kcal/mol more stable than the wild type, the 114-119 truncation also leads to a domain swapped state, suggesting that the tendency to swap is governed more by the local conformational properties of the 114-119 turn than by the propensity of the swappable helix to exist in the “open” conformation necessary for swapping. The 114-119 truncation is less destabilizing in Δ+PHS SNase than in the wild type SNase. A series of constructs in which residues 114 to 119 were removed systematically suggest that swapping is promoted by shortening of this segment, regardless of which residues are removed, and that only 2 to 3 residues need to be removed for swapping to be possible. However, so long as flexibility in the loop is retained through the presence of Gly-117, the protein appears to be capable of populating the monomeric state.

Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors

Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.