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.

Direct Analysis of Mitochondrial Damage Caused by Misfolded/Destabilized Proteins.

Protein quality control is essential for cellular homeostasis. In this study, we examined the effect of improperly folded proteins that do not form amyloid fibrils on mitochondria, which play important roles in ATP production and cell death. First, we prepared domain 3 of the dengue envelope protein in wild type and four mutants with widely different biophysical properties in misfolded/aggregated or destabilized states. The effects of the different proteins were detected using fluorescence microscopy and Western blotting, which revealed that three of the five proteins disrupted both inner and outer membrane integrity, while the other two proteins, including the wild type, did not. Next, we examined the common characteristics of the proteins that displayed toxicity against mitochondria by measuring oligomer size, molten globule-like properties, and thermal stability. The common feature of all three toxic proteins was thermal instability. Therefore, our data strongly suggest that thermally unstable proteins generated in the cytosol can cause cellular damage by coming into direct contact with mitochondria. More importantly, we revealed that this damage is not amyloid-specific.

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.

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.

EGFR extracellular domain III expressed in Escherichia coli with SEP tag shows improved biophysical and functional properties and generate anti-sera inhibiting cancer cell growth.

The epidermal growth factor receptor extracellular domain III (EGFR-ECDIII) protein is a promising target of anti-cancer research, and its production in Escherichia coli would thus represent significant benefits. However, despite its moderate size (19 kDa), the expression of EGFR-ECDIII in E.coli is hampered by the presence of multiple cysteines producing misfolded proteins with incorrect S-S bonds. In our study, we show that a short 12-residue solubility enhancing peptide (SEP) tag containing nine arginines (C9R) attached at the C-terminus of EGFR-ECDIII reduces the inclusion body formation and increases the final yield by six times (20 mg/L). EGFR-ECDIII-C9R purified from the soluble fraction eluted as a sharp single RP-HPLC peak, suggesting a single S-S bond pairing. Biophysical characterization using circular dichroism, fluorescence, and light scattering confirmed its native-like properties together with reversible thermal denaturation. The binding activity of EGFR-ECDIII-C9R to anti-EGFR-VHH7D12, a single-domain antibody with specific binding to the ECDIII, was assessed by sandwich ELISA. Further, we produced anti-EGFR-ECDIII-C9R antisera in mouse models and anti-sera inhibited A431 cancer cells' growth. These results demonstrate that the SEP tag enables the rapid production of the multiple disulfide-bonded EGFR-ECDIII in E. coli having native-like biophysical properties and producing neutralizing antibodies.

Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR.

Gaussia luciferase (GLuc) is a small luciferase (18.2 kDa; 168 residues) and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a Solubility Enhancement Petide (SEP) tag. Almost perfect assignments of GLuc's 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10-18, 36-81, 96-145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å ± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.

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.

Folding of the Ig-Like Domain of the Dengue Virus Envelope Protein Analyzed by High-Hydrostatic-Pressure NMR at a Residue-Level Resolution.

Dengue fever is a mosquito-borne endemic disease in tropical and subtropical regions, causing a significant public health problem in Southeast Asia. Domain III (ED3) of the viral envelope protein contains the two dominant putative epitopes and part of the heparin sulfate receptor binding region that drives the dengue virus (DENV)'s fusion with the host cell. Here, we used high-hydrostatic-pressure nuclear magnetic resonance (HHP-NMR) to obtain residue-specific information on the folding process of domain III from serotype 4 dengue virus (DEN4-ED3), which adopts the classical three-dimensional (3D) ß-sandwich structure known as the Ig-like fold. Interestingly, the folding pathway of DEN4-ED3 shares similarities with that of the Titin I27 module, which also adopts an Ig-like fold, but is functionally unrelated to ED3. For both proteins, the unfolding process starts by the disruption of the N- and C-terminal strands on one edge of the ß-sandwich, yielding a folding intermediate stable over a substantial pressure range (from 600 to 1000 bar). In contrast to this similarity, pressure-jump kinetics indicated that the folding transition state is considerably more hydrated in DEN4-ED3 than in Titin I27.

Misfolding of a Single Disulfide Bonded Globular Protein into a Low-Solubility Species Conformationally and Biophysically Distinct from the Native One.

In practice and despite Anfinsen's dogma, the refolding of recombinant multiple SS-bonded proteins is famously difficult because misfolded species with non-native SS-bonds appear upon the oxidization of their cysteine residues. On the other hand, single SS-bond proteins are thought to be simple to refold because their cysteines have only one SS-bond partner. Here, we report that dengue 4 envelope protein domain 3 (DEN4 ED3), a single SS-bonded protein can be irreversibly trapped into a misfolded species through the formation of its sole intramolecular SS-bond. The misfolded species had a much lower solubility than the native one at pHs higher than about 7, and circular dichroism measurements clearly indicated that its secondary structure content was different from the native species. Furthermore, the peaks in the Heteronuclear Single Quantum Correlation spectroscopy (HSQC) spectrum of DEN4 ED3 from the supernatant fraction were sharp and well dispersed, reflecting the beta-sheeted native structure, whereas the spectrum of the precipitated fraction showed broad signals clustered near its center suggesting no or little structure and a strong tendency to aggregate. The two species had distinct biophysical properties and could interconvert into each other only by cleaving and reforming the SS-bond, strongly suggesting that they are topologically different. This phenomenon can potentially happen with any single SS-bonded protein, and our observation emphasizes the need for assessing the conformation and biophysical properties of bacterially produced therapeutic proteins in addition to their chemical purities.

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.