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Amino Acids

, Volume 46, Issue 11, pp 2615–2625 | Cite as

The functional role of Cys3–Cys4 loop in hydrophobin HGFI

  • Baolong Niu
  • Yanbo Gong
  • Xianghua Gao
  • Haijin Xu
  • Mingqiang Qiao
  • Wenfeng LiEmail author
Original Article

Abstract

Hydrophobins are a large group of low-molecular weight proteins. These proteins are highly surface-active and can form amphipathic membranes by self-assembling at hydrophobic–hydrophilic interfaces. Based on physical properties and hydropathy profiles, hydrophobins are divided into two classes. Upon the analysis of amino acid sequences and higher structures, some models suggest that the Cys3–Cys4 loop regions in class I and II hydrophobins can exhibit remarkable difference in their alignment and conformation, and have a critical role in the rodlets structure formation. To examine the requirement for the Cys3–Cys4 loop in class I hydrophobins, we used protein fusion technology to obtain a mutant protein HGFI-AR by replacing the amino acids between Cys3 and Cys4 of the class I hydrophobin HGFI from Grifola frondosa with those ones between Cys3 and Cys4 of the class II hydrophobin HFBI from Trichoderma reesei. The gene of the mutant protein HGFI-AR was successfully expressed in Pichia pastoris. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the purified HGFI-AR could form amphipathic membranes by self-assembling at mica and hydrophobic polystyrene surfaces. This property enabled them to alter the surface wettabilities of polystyrene and mica and change the elemental composition of siliconized glass. In comparison to recombinant class I hydrophobin HGFI (rHGFI), the membranes formed on hydrophobic surfaces by HGFI-AR were not robust enough to resist 1 % hot SDS washing. Atomic force microscopy (AFM) measurements indicated that unlike rHGFI, no rodlet structure was observed on the mutant protein HGFI-AR coated mica surface. In addition, when compared to rHGFI, no secondary structural change was detected by Circular Dichroism (CD) spectroscopy after HGFI-AR self-assembled at the water–air interface. HGFI-AR could not either be deemed responsible for the fluorescence intensity increase of Thioflavin T (THT) and the Congo Red (CR) absorption spectra shift (after the THT(CR)/HGFI-AR mixed aqueous solution was drastically vortexed). Remarkably, replacement of the Cys3–Cys4 loop could impair the rodlet formation of the class I hydrophobin HGFI. So, it could be speculated that the Cys3–Cys4 loop plays an important role in conformation and functionality, when the class I hydrophobin HGFI self-assembles at hydrophobic–hydrophilic interfaces.

Keywords

Hydrophobin HGFI Self-assembly Cys3–Cys4 loop Rodlets structure 

Notes

Acknowledgments

This research was financially supported by the National Natural Science Foundation of China (31170066), Tianjin Key Research Program of Application Foundation and Advanced Technology (12JCZDJC22600) and the Natural Science Foundation of Shanxi Province (2014021020-3).

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Baolong Niu
    • 1
    • 2
  • Yanbo Gong
    • 3
  • Xianghua Gao
    • 1
    • 2
  • Haijin Xu
    • 4
  • Mingqiang Qiao
    • 4
  • Wenfeng Li
    • 1
    • 2
    Email author
  1. 1.Key Laboratory of Interface Science and Engineering in Advanced Materials, College of Materials Science and EngineeringTaiyuan University of Technology, Ministry of EducationTaiyuanPeople’s Republic of China
  2. 2.College of Materials Science and EngineeringTaiyuan University of TechnologyTaiyuanPeople’s Republic of China
  3. 3.College of Light Textile Engineering and ArtTaiyuan University of TechnologyTaiyuanPeople’s Republic of China
  4. 4.State Key Laboratory of Medicinal Chemical Biology, College of Life SciencesNankai UniversityTianjinPeople’s Republic of China

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