Hydrophobin-1 promotes thermostability of firefly luciferase

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Abstract

The thermal sensitivity of firefly luciferase limits its use in certain applications. Firefly luciferase has hydrophobic sites on its surface, which lead to aggregation and inactivation of the enzyme at temperatures over 30 °C. We have successfully stabilized firefly luciferase at high temperatures with the assistance of a unique protein, hydrophobin-1 (HFB1). HFB1 is a small secretory protein belonging to class II of hydrophobins with a low molecular weight (7.5 kDa) and distinct functional hydrophobic patch on its surface. The interaction of HFB1 with hydrophobic sites on the surface of luciferase was confirmed by extrinsic fluorescence studies using 8-anilino-1-naphthalenesulfonic acid (ANS) as a hydrophobic reporter probe. Calculation of thermodynamic parameters of heat inactivation of luciferase shows that conformational changes and flexibility of enzyme decreased in the presence of HFB1, and thermostability of the HFB1-treated enzyme increased. Furthermore, the addition of HFB1 into the enzymatic solution leads to an increase in catalytic efficiency of luciferase and subsequently improves the utility of the enzyme as an ATP detector.

Firefly luciferase (EC 1.13.12.7) is a thermolabile enzyme. Due to the existence of hydrophobic sites on the surface of firefly luciferase, its aggregation occurs at room temperature and then is followed by structural changes and further inactivation. Hydrophobin-1 (HFB1) is a small amphiphilic protein with distinct hydrophobic and hydrophilic parts on its surface, which can interact with the surface of luciferase. These interactions between HFB1 and luciferase promote thermostability of luciferase.

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