Unusual reversible oligomerization of unfolded Dengue envelope protein domain 3 at high temperature and its abolition by a point mutation

By Tomonori Saotome, Shigeyoshi Nakamura, Mohammad Monirul Islam, Akiko Nakazawa, Mariano Dellarole, Fumio Arisaka, Shun-ichi Kidokoro, Yutaka Kuroda

July 28, 2017

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Title

Unusual reversible oligomerization of unfolded Dengue envelope protein domain 3 at high temperature and its abolition by a point mutation

Author

Tomonori Saotome, Shigeyoshi Nakamura, Mohammad Monirul Islam, Akiko Nakazawa, Mariano Dellarole, Fumio Arisaka, Shun-ichi Kidokoro, Yutaka Kuroda

Year

2016

Journal

Biochemistry

Abstract

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.

Full Article

http://pubs.acs.org/doi/abs/10.1021/acs.biochem.6b00431

Instrument

J-820

Keywords

Circular dichroism, Secondary structure, Biochemistry