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Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy
Published on: August 20, 2018
Single-Molecule Force Spectroscopy Reveals that the Fe-N Bond Enables Multiple Rupture Pathways of the 2Fe2S Cluster
Guobin Song1, Xuan Ding2, Huaxing Liu1
1State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210023, P. R. China.
Abstract:
The mitochondrial outer membrane protein, mitoNEET (mNT), is an iron-sulfur protein containing an Fe2S2(His)1(Cys)3 cluster with a unique single Fe-N bond. Previous studies have shown that this Fe(III)-N(His) bond is essential for metal cluster transfer and protein function. To further understand the effect of this unique Fe-N bond on the metal cluster and protein, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to investigate the mechanical unfolding mechanism of an mNT monomer, focusing on the rupture pathway and kinetic stability of the cluster. We found that the Fe-N bond was the weakest point of the cluster, the rupture of which occurred first, and could be independent of the cluster break. Moreover, this Fe-N bond enabled a dynamic and labile iron-sulfur cluster, as multiple unfolding pathways of mNT with a unique Fe2S2(Cys)3 intermediate were observed accordingly.
Insights
The unique Fe-N bond in mitoNEET (mNT) is the weakest point, breaking first and enabling a dynamic iron-sulfur cluster. This finding reveals new insights into mNT
Area of Science:
- Biochemistry
- Biophysics
- Structural Biology
Background:
- MitoNEET (mNT) is a mitochondrial outer membrane protein.
- It contains a unique Fe2S2(His)1(Cys)3 iron-sulfur cluster.
- The Fe(III)-N(His) bond is crucial for cluster transfer and protein function.
Purpose of the Study:
- To investigate the mechanical unfolding mechanism of mNT monomer.
- To understand the role of the unique Fe-N bond in cluster stability and protein function.
- To determine the rupture pathway and kinetic stability of the iron-sulfur cluster.
Main Methods:
- Atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS).
- Analysis of mechanical unfolding pathways and cluster rupture dynamics.
Main Results:
- The Fe-N bond is the weakest point in the iron-sulfur cluster and ruptures first.
- Fe-N bond rupture can occur independently of the overall cluster break.
- Multiple unfolding pathways were observed, highlighting a dynamic and labile cluster with a unique Fe2S2(Cys)3 intermediate.
Conclusions:
- The unique Fe-N bond dictates the lability and dynamic nature of the mNT iron-sulfur cluster.
- This bond's early rupture influences the mechanical unfolding pathway of mNT.
- Findings provide novel insights into the structure-function relationship of mitoNEET.
