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

• Published in: Biochemistry (Easton) 2016-08, Vol.55 (32), p.4469-4475
• Main Authors: Saotome, Tomonori, Nakamura, Shigeyoshi, Islam, Mohammad M, Nakazawa, Akiko, Dellarole, Mariano, Arisaka, Fumio, Kidokoro, Shun-ichi, Kuroda, Yutaka
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Dengue Virus; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Point Mutation; Protein Multimerization; Protein Structure, Quaternary; Protein Unfolding; Temperature; Viral Envelope Proteins - chemistry; Viral Envelope Proteins - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.6b00431

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.