Unusual Reversible Oligomerization of Unfolded Dengue Envelope

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Unusual reversible oligomerization of unfolded Dengue envelope protein domain 3 at high temperature and its abolition by a point mutation Tomonori Saotome, Shigeyoshi Nakamura, Mohammad Monirul Islam, Akiko Nakazawa, Mariano Dellarole, Fumio Arisaka, Shun-ichi Kidokoro, and Yutaka Kuroda Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00431 • Publication Date (Web): 19 Jul 2016 Downloaded from http://pubs.acs.org on July 20, 2016

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Unusual reversible oligomerization of unfolded Dengue envelope protein domain 3 at high temperature and its abolition by a point mutation Tomonori Saotomea, Shigeyoshi Nakamurab, c, Mohammad M. Islama, d, Akiko Nakazawab, Mariano Dellarolee, f, Fumio Arisakag, Shun-ichi Kidokorob, Yutaka Kurodaa$ a

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan b Department of Bioengineering, Nagaoka University of Technology, Niigata 940-2188, Japan c

Department of Creative Engineering, National Institute of Technology, Kitakyushu College, Kitakyushu 802-0985, Japan d Present address: Department of Biochemistry and Molecular Biology, University of Chittagong, Bangladesh Centre de Biochimie Structurale, CNRS UMR5048, INSERM U554, Université de Montpellier, Montpellier, France 34090. f Present address: Structural Virology Department, Pasteur Institute, 28 rue du Dr. Roux, e

Paris, France 75015. g College of Bioresource Science, Nihon University, Fujisawa, Kanagawa 252-0880, Japan $

Correspondence to Y.K. Email: [email protected] Phone/Fax: +81-42-388-7794 Manuscript information: 31 Pages; 6 Figures; 5 Tables. Funding source material : This research was supported by a JSPS grant-in-aid for scientific research (KAKENHI: 15H04359) to Y.K and a JSPS long term invitation fellowship (F2014) to M.M.I. Abbreviations: DEN4 ED3: Dengue 4 envelope protein domain 3; DSC: Differential Scanning Calorimetry; PISA: Proteins Interfaces Surfaces and Assemblies; AUC: Analytical Ultracentrifugation; DLS: Dynamic Light Scattering; CD: Circular

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Dichroism; Rh: Hydrodynamic Radius; Tm: Midpoint Temperature; Keywords: Small monomeric globular protein; Micro-calorimetry; Thermodynamics; Unfolded oligomer; oligomer interface; Hydrophobicity; Reversibility; Concentration dependency.


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Abstract (237 words) Here we report 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 away with 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 temperature. In order 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 protein crystal, to 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 from 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 temperature is a truly new phenomenon, which was fully inhibited by an accurately designed single mutation.


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Introduction Reversible two-state thermal unfolding can be regarded as a biophysical hallmark of a natively folded single domain globular protein [1 - 4]. The highly cooperative unfolding transition, as observed by various spectroscopic methods such as fluorescence [5, 6] and circular dichroism [7, 8] monitoring, respectively, the protein’s sidechain environment and the secondary structure content, is a reflection of the disruption of the densely packed protein interior in an all-or-none fashion [3]. The unfolding is accompanied by a large heat absorption that is observed as a sharp peak in the Differential Scanning Calorimetry (DSC) thermogram. Such cooperativity is not observed in the thermal unfolding of structures formed by polypeptides that lack a hydrophobic core and undergo a broad thermal unfolding transition [9]. The thermal unfolding of many (if not most) small monomeric proteins is reversible and occurs under equilibrium condition, which enables a thermodynamic analysis [10, 11], and direct measurements of the heat capacity change by DSC have confirmed a two-state unfolding (N⇄ D) for many monomeric, small-to-medium size single domain globular proteins [4]. Dengue virus is a flavivirus [12] with a single stranded RNA that encodes three structural proteins: The nucleocapsid (C-protein), membrane protein (M-protein), and envelope glycoprotein (E-protein) [13, 14]. ED3 is the third domain of the E-protein, which is located in the outermost layer of the dengue virus particle [15]. ED3 is about hundred residues long and contains the main antibody recognition site and also the receptor binding sites that drive its entry into the host cell [16-18]. X-ray crystallography demonstrated that dengue ED3 remains folded in a well packed single domain native structure even when isolated from rest of the E-protein [19, 20, 21], but its thermodynamic properties remained to be determined.


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Here we report that contrary to our expectation, DEN4 ED3 undergoes a multi-step thermal unfolding transition uncharacteristic of small globular proteins [1, 12], as assessed by DSC. We show that this peculiar phenomenon is attributable to the formation of an unfolded oligomeric state at high temperature. Furthermore, Analytical Ultra Centrifuge (AUC) and Dynamic Light Scattering (DLS) indicated that DEN4 ED3 formed a monomer at low temperature, and a presumed unfolded tetramer at high temperature, which fell apart again into unfolded monomer at even higher temperature. Such peculiar thermal unfolding process has never or barely been observed for a small single domain monomeric globular protein. We further confirmed our DSC analysis by showing that a single mutation at residue Val 380 (which is residue 661 according to UNIPROT numbering employed in PDB 3we1) to hydrophilic amino acids yields variants that undergo a two-state thermal unfolding as demonstrated by DSC.

Materials and Methods Synthesis, expression and purification of protein Synthetic genes encoding the DEN4 ED3 were cloned into a pET15b vector (Novagen) at the endonuclease NdeI and BamHI sites. Mutations at Val 380 were introduced by site directed mutagenesis using a Quik Change protocol (Stratagene, USA). All variants were overexpressed in the Escherichia coli strain JM109 (DE3) pLysS as inclusion bodies and purified essentially as previously described [22]. Protein expression was induced by the addition of 1.0 mM IPTG when the optical density at 590 nm was equal to 0.6 OD. After centrifugation, the cells were lysed by sonication and the cysteines were air-oxidized for 36 h at 37°C in 6 M guanidine hydrochloride. The

hexahistidine-tagged

protein

was

purified

by


using

Ni-NTA (Qiagen)

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chromatography in the presence of 6 M guanidine hydrochloride, followed by overnight dialysis against 50 mM Tris-HCl (pH 7.0) at 4°C. The hexahistidine-tag was removed by thrombin (T6634; SIGMA, USA) cleavage and the protein was further purified by a second passage through a Ni-NTA column followed by reverse-phase HPLC (Shimadzu Ltd). The protein’s identities were confirmed by MALDI-TOF mass spectroscopy (TOF/TOF™ 5800; ABI SCIEX), and the molecular weights of all proteins were within 3 Dalton of the respective computed values (~11.5 kDa; Supplemental Table S1). The purified protein was lyophilized and stored at -30°C until use.

Differential Scanning Calorimetry (DSC) measurement Samples for DSC measurements were prepared by dissolving lyophilized protein powders in 50 mM glycine buffer (pH 3.0-3.6) or acetate buffer (pH 4.1-5.1) at 0.5-1.0 mg/mL. The protein samples were dialyzed for 18 hours at 4°C using Spectra/Por 3 membrane (MWCO of 3.5 kDa) with one time buffer exchange. After dialysis, protein samples were filtered with a 0.20 µm membrane filter (Millex-GV; Millipore, USA) to remove the aggregates. The samples were thoroughly degassed, and protein concentrations and the sample pHs were confirmed just before DSC measurements. DSC measurements were performed using a VP-DSC MicroCalorimeter (MicroCal, USA) at a scan rate of 0.5-1.0°C /min in the temperature range of 10 to 120°C. Blank measurements were taken using 50 mM glycine buffer (pH 3.0-3.6) or acetate buffer (pH 4.1-5.1) for several times before sample measurements. Reversibility of the thermal unfolding of protein was checked by repeated scans of the same sample. The thermodynamic parameters (Tm and ⊿H (Tm)) were determined by analyzing the apparent heat capacity curves using non-linear least-square fitting method DDCL [23,


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24], and assuming a linear temperature dependence of the heat capacity for the native and denatured states.

CD (circular dichroism) measurements Samples were prepared by dissolving the lyophilized protein powders at 0.1-0.5 mg/ml concentration in 20-50 mM sodium acetate buffer (pH 4.6). The samples were filtered with a 0.20 µm membrane filter (Millex-GV; Millipore, USA) to remove aggregates. CD measurements were conducted a JASCO J-820 spectropolarimeter at a 0.1 mg/ml protein concentrations and 20 mM sodium acetate buffer (pH 4.6) using a 1-mm optical path length quartz cuvette. The secondary structure content was calculated by using the CD value in the range of 200-240 nm using K2D3 [25]. Thermal stability were measured at 0.5 mg/ml protein concentrations, 50 mM sodium acetate buffer (pH 4.6), and a 1°C/min scan rate, using a 1 cm optical path length cuvette, and was monitored between 40°C and 95°C using the CD value at 235 nm. The midpoint temperatures (Tm) were computed by means of least-squares fittings of the experimental data with a two-state model using Origin 6.1 J.

Analytical Ultracentrifugation (AUC) measurements AUC’s samples were prepared in the same way as for CD at a protein concentration of 0.6 mg/ml. Sedimentation velocity experiments were carried out using an Optima XL-I analytical ultracentrifuge (Beckman–Coulter) with an eight-hole An50Ti rotor at 20°C. Before centrifugation, samples were dialyzed overnight against buffer solutions with 50 mM acetate buffer at pH 4.6. Each sample was then transferred into a 12-mm double-sector Epon cell and centrifuged at a rotor speed of 50,000 rpm. The


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concentrations were monitored at 280 nm. The sedimentation velocity data were analyzed using the SEDFIT program [26].

Dynamic light scattering (DLS) measurements Samples for DLS were prepared in the same way as for CD at a protein concentration of 1 mg/ml. The concentrations and pHs of the samples were confirmed just before performing the experiments. DLS measurements were performed by using a glass cuvette with a Zeta-nanosizer (Nano S; Malvern). The sample`s temperature was increased from 25°C to 65°C, 90°C and cooled back to 25°C for assessing reversibility. The hydrodynamic radius (Rh) was calculated from size-volume graphs by using the Stokes-Einstein equation.


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Results and Discussion Thermodynamic characterization of wt DEN4 ED3 We first analyzed the thermal unfolding of wt DEN4 ED3 by DSC at a protein concentration of 1 mg/ml and a pH range of 3.0-5.1 (Figure 1, Table 1). A single endothermic peak was observed in the DSC thermogram at acidic pH (3.0-3.6), with a melting temperature increasing together with pHs. However, two endothermic peaks appeared in mild acidic conditions (pH 4.1-5.1), where the second peak moved to a higher temperature (peak temperature of 63.2°C to 92.9°C) as pH increased, whereas the position of the first peak remained unchanged (61.6-64.4°C). Small single domain monomeric globular proteins usually exhibit a single DSC peak that can be analyzed using a two-state thermal unfolding model (N⇄D). DEN4 ED3‘s high temperature peak is thus a novel and peculiar phenomenon as discussed thereafter. In order to determine the nature of the high temperature heat capacity peak, we further analyzed the thermal unfolding of wt DEN4 ED3 at pH 4.6 where the thermal unfolding was almost perfectly reversible and two peaks were clearly distinguished within the experimentally reachable temperature: The first at 63.6°C and the second at 88.1°C (Figure S1, Table 1). The first peak barely shifted to lower/higher temperature upon increasing protein concentration, but the second peak moved to higher temperature (Figure 2 (B)). A global fitting using self-dissociation/association model [23, 24] indicated that the concentration dependent denaturation curves were best fitted with a N ⇄I4⇄D model (Figure 2 (A), Suppl. Fig S2, Table 2) where the native state was

hypothesized as a monomer based on AUC measurements. Fitting of DSC curves, assuming dimeric to hexameric intermediate states, were compared using the standard


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error between the experimental and calculated values, indicated a tetrameric intermediate state (Suppl. Fig S3, Suppl. Table S2). On the other hand, the thermal denaturation of wt ED3 monitored by Circular Dichroism (CD) indicated a reversible two-state transition with a midpoint temperature of approximately 65°C (Figure 3; Table 3), and the CD signal remained constant between 70 and 90°C temperature range indicating that the high temperature transition observed in the DSC thermogram did not involve changes in secondary structure contents. We thus associated the first DSC peak to the unfolding of native monomeric ED3 into unfolded tetrameric intermediates, and the second one to its dispersion into unfolded monomers. Altogether, DSC combined with AUC and DLS data computed that a native monomer unfolded to a tetrameric intermediate (presumably with very low or no secondary structure content according to CD), which further dissociated to a denatured monomer at very high temperature in a reversible manner. The denatured monomer states were not directly measured by AUC, but inferred from the global fitting of the DSC thermograms (Supplemental Table S2).

Design of DEN4 ED3 variants undergoing two-state unfolding Small monomeric globular proteins usually unfold according to a two-state model and their DSC thermograms exhibit thus a single endothermic peak [1]. In order to experimentally confirm whether the second endothermic peak observed in the DSC thermogram corresponds to the dissociation/oligomerization of wt DEN4 ED3, we designed mutations at the dimeric interface of our previously reported DEN4 ED3 structure (PDB ID: 3WE1) [19] expecting that it could simultaneously inhibit the high temperature oligomerization and produce a variant that unfolds according to a classical two-state transition. To this end, we analyzed chain A and chain B in the asymmetric


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unit of 3WE1 and calculated that Val 380‘s BSA/ASA ratio (buried surface area upon dimerization (BSA) and accessible surface area as monomer (ASA)) and its solvation free energy (∆G) were the highest among all residues (Suppl. Table S3) using PISA (Proteins Interfaces Surfaces and Assemblies [27]; Figure 4 (A)). We further reasoned that Val 380 might be responsible for triggering the formation of wt ED3’s high temperature oligomers. We thus substituted Val 380 to five less hydrophobic amino acids (Ala, Ser, Thr, Asn and Lys) (Figure 4 (B), Suppl. Table. S4). CD clearly indicated that all variants retained their secondary structure contents at 20°C (Suppl. Fig. S4, Suppl. Table. S5), which was close to values calculated from the crystal structure, and thus suggested that the point mutations did not affect the native DEN4 ED3 structure. In addition, wt DEN4 ED3 and all variants showed two-state thermal denaturation (Figure 3, Suppl. Fig S5).

Oligomeric state by AUC and DLS Analytical ultracentrifugation (AUC) of wt DEN4 ED3 and

DEN4 V380K was

performed under the identical conditions at 25°C, which showed a sharp single peak with the sedimentation coefficient of 1.37 S and 1.38 S, respectively, which correspond to a molecular weight of 12.4 kDa and 12.2 kDa, equivalent to wt DEN4 ED3 monomer (Suppl. Fig S6). Hence, we concluded that all DEN4 ED3 formed monomers in the native state under the DSC condition at 25°C. Dynamic Light Scattering (DLS) 25°C indicated nearly identical Rh for wt ED3 and all of its variants, suggesting that they remained monomer at this temperature. At 65°C the Rh of wt DEN4 ED3 was about 50% larger than those of the variants (Suppl. Fig S7). Furthermore, the hydrodynamic radius of the wt DEN4 ED3 at 90°C were significantly


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larger than those of its variants ((Table 4). These results were in line with DSC and CD analysis, which showed that a native wt DEN4 ED3 monomer was reversibly converted upon increasing temperature to unfolded tetramers, and dispersed into unfolded monomers (Suppl. Fig. S2), whereas the mutated variants unfolded according to a two-state model. Namely, Rh of the variants slightly increased at 90°C because of thermal unfolding (Suppl. Table S1), and not because of the formation of an oligomeric intermediate. On the other hand, Rh of wt ED3 was 2.62nm at 65°C, which was in line with a N⇄I4⇄D model because at this temperature about half of the wt ED3 were folded and most of the remaining formed unfolded tetramers (Suppl. Fig. S2). Finally, The Rh of wt DEN4 ED3 at 90°C was substantially larger than those of the variants, which can be rationalized with fractions computed from DSC because at this temperature wt DEN4 ED3 was mostly in the I4 and D states.

Thermodynamic characterization of DEN4 ED3 mutants Thermal denaturation monitored by CD at 235 nm indicated a single transition for all variants (DEN4 V380A, DEN4 V380S, DEN4 V380T, DEN4 V380K, and DEN4 V380N) essentially in line with a two-state transition model (N⇄D), which was also the case for wt DEN4 ED3 (Figure 3, Suppl. Fig. S5). Furthermore, all variants remained monomeric between 25°C and 90°C, and exhibited a single heat absorption peak (Figure 6; Table 5). The peak temperatures of the first endothermic transition observed by DSC (T>S>~A>K>N; Table 5) agreed qualitatively with the midpoint temperature (Tm) estimated from CD (T>S>~A>K>N; Table 3), which suggested that the low temperature


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DSC peak corresponded to the melting of the secondary structures of native monomeric DEN4 ED3’s. For the purpose of discussion, we performed a detailed DSC analysis of DEN4 V380K. DEN4 V380K showed a fully reversible single endothermic peak at 65°C (Suppl. Fig. S1), and the thermal denaturation was independent of protein concentration (Figure 2 (B), Table 5). These observations suggested that the unfolding of DEN4 V380K was reversible, thermally equilibrated, and a global fitting of the DSC denaturation curves using DDCL3 [23, 24] indicated a two state unfolding ( N⇄D ) (Figure 2 (A)), where the monomeric native state was confirmed by AUC. This model qualitatively agrees with DLS measurements, which showed that the Rh values of all variants remained essentially unchanged between 25°C and 65°C, and increased moderately at 90°C temperature range (Figure 5 (B), Table 4).

Reversible oligomerization at high temperature Irreversible aggregation of unfolded protein is a well-known (and often detrimental) phenomenon [28]. In contrast, reversible oligomerization in the unfolded state is a very unique phenomenon, which we observed recently for a natural protein at extreme pH (N.S. et al submitted), but has so far been largely overlooked. A possible reason for this lack of notice is that the second peak becomes clearly identifiable only at temperatures above 80 to 90°C. The second peak can be confused during the analysis with the baseline when data are measured only under 100°C as it is usually the case, or it may appear as a mere shoulder of the main endothermic peak (as observed at pH 4.1 for DEN4 ED3; Figure 1) or broaden the peak (Figure 1). Broad reversible DSC peaks and shoulders are also observed for molten globules and other partially folded states [29],


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but as discussed above the high temperature peak reported in the present study is clearly related to the reversible formation/dislocation of oligomers. Furthermore, the concentration dependency of ED3’s melting temperature is rather unique and opposite to most of the previous observations ([30-33]). This is because, ED3 is monomeric at low temperature, and oligomeric (tetrameric) at high temperature. Thus when the protein concentration is increased, protein unfolding is promoted and Tm drops as described in earlier reviews [30].

Conclusion In summary, wt DEN4 ED3’s thermal denaturation monitored by DSC exhibited two endothermic peaks and was atypical of a single domain globular protein. The high temperature endothermic peak was associated with a reversible association and dispersion of (mostly) unfolded oligomers. The thermal unfolding of five single mutation variants, designed to inhibit the high temperature oligomerization by replacing Val 380 to hydrophilic amino acids, exhibited a two-state unfolding typical of a globular protein. Overall, this study suggested that very unique reversible oligomerization can occur at high temperature and can be inhibited by substituting a single amino acid. This phenomenon might have been overlooked in other proteins, because it is seen only by DSC and because the second peak becomes clearly distinguishable only when the scanning temperature is extended to up to 120°C.

Authors Contributions T.S. and Y.K. designed and performed the research analyzed data and wrote the manuscript. S.K. and S.N. analyzed and supervised DSC measurements and wrote the


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manuscript. M.M.I advised with DLS measurements and revised manuscript. F.A. performed the AUC analysis. A.N and M.D. assisted DSC experiments, and M.D. revised the manuscript.

Acknowledgements We are thankful to Prof. Catherine A. Royer for discussion, and Dr. Montasir Elahi for advice on wt DEN4 ED3 expression and purification. We are grateful to Profs. Tsuyoshi Tanaka, Tomoko Yoshino and Atsushi Arakaki for access to Zeta-nanosizer equipment and to Ms. Patricia McGahan for English proofreading. We thank Manjiri R Kulkarni and all members of the Kuroda Laboratory for discussion and technical advices.

Supporting Information Available: 7 Supplementary figures; 5 Supplementary Tables


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References [1] Privalov PL. (1979). Stability of proteins: small globular proteins. Adv Protein Chem. 33, 167-241. [2] Ohgushi M, Wada A. (1983). 'Molten-globule state': a compact form of globular proteins with mobile side-chains. FEBS Lett. 164, 21-24. [3] Kuwajima K. (1989). The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins. 6, 87-103. [4] Privalov PL, Tiktopulo EI, Venyaminov SY, Griko YV, Makhatadze GI, Khechinashvili NN. (1989). Heat capacity and conformation of proteins in the denatured state. J Mol Biol. 205,

737-750.

[5] Brandts JF, Hunt L. (1967). Thermodynamics of protein denaturation. III. Denaturation of ribonuclease in water and in aqueous urea and aqueous ethanol mixtures. JACS. 89, 4826-4838. [6] Eftink MR, Ghiron CA, Kautz RA, Fox RO. (1991). Fluorescence and Conformational Stability Studies of Staphylococcus Nuclease and Its Mutants, Including the Less Stable Nuclease Concanavalin, A Hybrids. Biochemistry. 30, 1193–1199. [7] Ikeguchi M, Kuwajima K, and Sugai S. (1986). Cat+-Induced Alteration in the Unfolding Behavior of a-Lactalbumin.

Biochem. 99, 1191-1201.

[8] Kuroda Y, Kidokoro S, Wada A. (1992). Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process. J Mol Biol. 223, 1139-1153. [9] Scholtz JM, Marqusee S, Baldwin RL, York EJ, Stewart JM, Santoro M, Bolen DW. (1991). Calorimetric determination of the enthalpy change for the alpha-helix to coil transition of an alanine peptide in water. Proc Natl Acad Sci U S A. 88, 2854-2858.[10] Privalov PL, Khechinashvili NN. (1974). A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J Mol Biol. 86, 665-684. [11] Kato A, Yamada M, Nakamura S, Kidokoro S, Kuroda Y. (2007). Thermodynamic properties of BPTI variants with highly simplified amino acid sequences. J Mol Biol.372, 737-746. [12] Bhatt S, Hay S.I. (2013). The global distribution and burden of dengue. Nature. 496,


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504–507. [13] Zhang W, Chipman PR, Corver J, Johnson PR, Zhang Y, Mukhopadhyay S, Baker TS, Strauss JH, Rossmann MG, Kuhn RJ. (2003). Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol. 10, 907-912. [14] Kuhn R.J, Zhang W, Baker T.S, Strauss J.H. (2002). Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell. 108, 717–725. [15] Modis Y, Ogata S, Clements D, Harrison S.C. (2004). Structure of the dengue virus envelope protein after membrane fusion. Nature. 427, 313–319. [16] Gromowski GD, Roehrig JT, Diamond MS, Lee JC, Pitcher TJ, Barrett AD. (2010). Mutations of an antibody binding energy hot spot on domain III of the dengue 2 envelope glycoprotein exploited for neutralization escape. Virology. 407,237–246. [17] Lisova O, Hardy F, Petit V, Bedouelle H. (2007). Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus. J. Gen. Virol. 88, 2387–2397. [18] Liao M, Kielian M. (2005). Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion. J Cell Biol. 171, 111-120. [19] Elahi M, Islam MM, Noguchi K, Yohda M, Toh H, Kuroda Y. (2014). Computational prediction and experimental characterization of a "size switch type repacking" during the evolution of dengue envelope protein domain III (ED3). Biochim Biophys Acta. 1844, 585-592. [20] Kulkarni MR, Numoto N, Ito N, Kuroda Y. (2016). Modeling and experimental assessment of a buried Leu-Ile mutation in dengue envelope domain III. Biochem Biophys Res Commun. 471, 163-168. [21] Kulkarni MR, Islam MM, Numoto N, Elahi M, Mahib MR, Ito N, Kuroda Y. (2015). Structural and biophysical analysis of sero-specific immune responses using epitope grafted Dengue ED3 mutants. Biochim Biophys Acta. 1854, 1438-1443. [22] Islam MM, Sohya S, Noguchi K, Yohda M, Kuroda Y. (2008). Crystal structure of an extensively simplified variant of bovine pancreatic trypsin inhibitor in which over one-third of the residues are alanines. Proc Natl Acad Sci U S A.105, 15334-15339. [23] Kidokoro S, Wada A. (1987). Determination of thermodynamic functions from scanning calorimetry data. Biopolymers. 26, 213-229.


Biochemistry

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[24] Kidokoro S, Uedaira H, Wada A. (1987). Determination of thermodynamic functions from scanning calorimetry data II. For the system that includes self-dissociation/association process. Biopolymers. 27, 271-297. [25] Louis-Jeune C, Andrade-Navarro MA, Perez-Iratxeta C. (2012). Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins. 80, 374-381. [26] Schuck P. (2000). Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J. 78, 1606–1619. [27] Krissinel E, Henrick,K. (2007). Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774-797. [28] Morris AM, Watzky MA, Finke RG. (2009). Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature. Biochim Biophys Acta. 1794, 375-397. [29] Hamada D, Kidokoro S, Fukada H, Takahashi K, Goto Y. (1994). Salt-induced formation of the molten globule state of cytochrome c studied by isothermal titration calorimetry. Proc Natl Acad Sci. 91, 10325–10329. [30] Sturtvant JM (1987), Biochemical applications of differential scanning calorimetry, Ann. Rev. Phys. Chem., 1987, 38:463-88 [31] Kitakuni, E, Kuroda Y, Oobatake M, Tanaka T, Nakamura H. (1994). Thermodynamic characterization of an artificially designed α-helical peptide containing periodic prolines: Observation of high thermal stability and cold denaturation. Protein Science. 3, 831-837. [33] Hagihara Y, Oobatake M, Goto Y. (1994). Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c. Protein Science. 3, 1418-1429. [34] Ali SA, Iwabuchi N, Matsui T, Hirota K, Kidokoro S, Arai M, Kuwajima K, Schuck P, and Arisaka F. (2003). Reversible and Fast Association Equilibria of a Molecular Chaperone, gp57A, of Bacteriophage T4, Biophys. J., 85, 2605-2618.


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Biochemistry

Tables pH

Peak temperature of first peak (°C )

Peak temperature of second peak (°C )

3.0

54.2

3.6

65.1

4.1

61.6

63.2

4.6

63.6

88.1

5.1

64.4

92.9

Table 1. Peak temperature of wt DEN4 ED3 in DSC thermograms at 1 mg/ml, pH 3.0-5.1 and 1°C /min scan rate. Protein concentration

Transition

T (°C )

∆Hcal (T ) (kJ/mol)

N-I4

63.63

190.01

I4-D

81.66

59.66

N-D

69.27

249.68

N-I4

66.31

195.74

I4-D

71.55

59.45

N-D

68.83

255.19

m

m

(mg/ml) 1.0

0.5

Table 2. Midpoint temperature (Tm) and calorimetric enthalpy (⊿Hcal (Tm)) of each transition step of wt DEN4 ED3 by DDCL3 analysis in N-I4-D model. Tm was defined as the temperature where the molar fractions of the two considered states were equal.

Name

T (°C)

∆Hvan’t hoff (T ) (kJ/mol)

wt DEN4 ED3

63.87

312.75

DEN4 V380A

65.87

293.21

DEN4 V380S

66.51

296.57

DEN4 V380T

68.58

266.94

DEN4 V380N

63.43

214.28

DEN4 V380K

65.31

262.08

m

m

Table 3. Midpoint temperature (Tm) and van’t Hoff enthalpy (⊿Hvan’t Hoff (Tm)) of wt DEN4 ED3 and mutants by two-state analysis in CD measurement at 0.5 mg/ml, pH 4.6 and 1°C /min scan rate.


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Name

25°C

65°C

90°C

25°C (after heating)

wt DEN4 ED3

1.79

2.62

4.07

2.05

DEN4 V380A

1.89

1.78

2.31

1.79

DEN4 V380S

1.91

1.73

2.41

1.80

DEN4 V380T

1.72

1.57

1.91

1.65

DEN4 V380N

1.80

1.98

1.77

1.81

DEN4 V380K

1.76

1.71

2.11

1.77

Table 4. Hydrodynamic radius (nm, Rh) of wt DEN4 ED3 and variants at 25°C , 65°C , 90°C and 25°C after heating. Concentration

T (°C )

∆Hcal (T ) (kJ/mol)

∆Hcal (T )/∆Hvan’t Hoff (T )

m

m

m

m

(mg/ml) DEN4 V380A

1.0

67.08

269.32

1.04

DEN4 V380S

1.0

67.90

277.89

0.90

DEN4 V380T

1.0

69.23

285.90

0.91

DEN4 V380N

1.0

66.05

240.86

0.88

DEN4 V380K

1.0

66.34

275.08

0.99

DEN4 V380K

0.5

65.97

271.37

0.95

Table 5. Midpoint temperature (Tm), calorimetric enthalpy (⊿Hcal (Tm)) and van’t Hoff enthalpy (⊿Hvan’t Hoff (Tm)) of DEN4 ED3 variants at pH 4.6 determined by fitting the DSC thermogram using DDCL3 in N-D model.


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Biochemistry

Figure legends Figure 1: DSC thermogram of wt DEN4 ED3 at pH 3.0-5.1. Protein concentration was 1 mg/ml and the scan rate was 1 °C /min.

Figure 2: (A) DDCL3 analysis of DSC thermogram of wt DEN4 ED3 (N-I4-D model) and DEN4 V380K (N-D model) at 1 mg/ml, pH 4.6 and 1°C /min scan rate. Black dots represent the raw data and gray lines represent the fitting curve. (B) The concentration dependence of DSC thermogram of wt DEN4 ED3 and DEN4 V380K at 0.5-1 mg/ml, pH 4.6 and 1°C /min scan rate. Open black circle (●) and open white circle (○) showed DSC thermogram at 1 mg/ml and 0.5 mg/ml.

Figure 3: The CD thermal denaturation curves of wt DEN4 ED3 and DEN4 V380K. The protein concentration was 0.1 mg/ml. Black dots and gray lines represent the raw CD value at 235nm and the fitting curves.

Figure 4: Ribbon model (a) and sequence (b) of wt DEN4 ED3. The ribbon model was drawn using Pymol and the PDB structure 3WE1. V380 at the interface of the dimeric unit is shown. The beta strand positions as defined by DSSP are indicated by black arrows on top of the sequence. β1: 304-312 (FSIDKEMAE), β2: 318-324 (TVVKVKY), β3: 331-332 (CK), β4: 335-338(IEIR), β5: 348-349 (RI), β6: 355-356 (LA), β7: 362-367 (VTNIEL), β8: 372-378 (GDSYIVI), β9: 385-391 (LHLHWFR)

Figure 5: The DLS graphs of wt DEN4 ED3 and DEN4 V380K and at 1 mg/ml, pH 4.6 and 25-90°C. 25°C (solid line); 65°C (one-chain dashed line); 90°C (dashed line); and 25°C after heating (dotted line).

Figure 6: The DSC thermogram of DEN4 V380A, DEN4 V380S, DEN4 V380S, DEN4 V380N, DEN4 V380K, and wt DEN4 ED3 at 1 mg/ml, pH 4.6 and 1°C /min scan rate.


Biochemistry

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Figures

Figure 1.

Figure 2.

Figure 3.


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Biochemistry

Figure 4.

Figure 5.


Biochemistry

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Figure 6.


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Biochemistry

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Unusual Reversible Oligomerization of Unfolded Dengue Envelope

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