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Method Article
Shaoning Yu
Yu's Lab, Fudan University
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
In biological systems, protein function is dependent on spatial and temporal changes known as protein dynamics, which can be probed by amide hydrogen/deuterium (H/D) exchange. Fourier-transform infrared (FT-IR) spectroscopy is a convenient and efficient tool for determining the H/D exchange rate of proteins on a global scale. The H/D exchange process is monitored by following the apparent changes in FT-IR intensity at the amide II band maxima near 1550 cm−1. This protocol covers the principles underlying the determination of protein dynamics by FT-IR, as well as the basic steps involved in protein sample preparation and FT-IR spectra collection and detailed methods for analyzing spectra to determine the H/D exchange rate of proteins. This is a semi-quantitative method and can only be used for comparing protein dynamics under certain condition. Applications include the effects of protein mutation or protein and metal ion or ligand interactions on the protein H/D exchange rate. Typically, the procedure can be completed in 2-3 days.
Keywords
hydrogen/deuterium exchange,Fourier-transform infrared spectroscopy,protein dynamics
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The authors declare no competing financial interests
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.docx
Table2 Troubleshooting table
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Posted 04 Jun, 2018
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An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Shaoning Yu
Yu's Lab, Fudan University
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 04 Jun, 2018
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
In biological systems, protein function is dependent on spatial and temporal changes known as protein dynamics, which can be probed by amide hydrogen/deuterium (H/D) exchange. Fourier-transform infrared (FT-IR) spectroscopy is a convenient and efficient tool for determining the H/D exchange rate of proteins on a global scale. The H/D exchange process is monitored by following the apparent changes in FT-IR intensity at the amide II band maxima near 1550 cm−1. This protocol covers the principles underlying the determination of protein dynamics by FT-IR, as well as the basic steps involved in protein sample preparation and FT-IR spectra collection and detailed methods for analyzing spectra to determine the H/D exchange rate of proteins. This is a semi-quantitative method and can only be used for comparing protein dynamics under certain condition. Applications include the effects of protein mutation or protein and metal ion or ligand interactions on the protein H/D exchange rate. Typically, the procedure can be completed in 2-3 days.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
The authors declare no competing financial interests
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.docx
Table2 Troubleshooting table
Loading...