Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Area of Science:

• Structural biology
• Biophysics
• Biochemistry

Background:

• High-resolution structures of membrane proteins are crucial for understanding their function.
• Studying membrane proteins within a cellular environment presents significant challenges.
• In-cell NMR approaches offer potential for 3D structural information of proteins in living cells.

Purpose of the Study:

• To develop and demonstrate a novel NMR approach for high-resolution structural studies of membrane proteins in a cellular context.
• To overcome challenges associated with in-cell NMR studies of membrane-bound proteins.
• To investigate electron transfer processes mediated by membrane proteins.

Main Methods:

• Utilized dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy.
• Employed ¹³C-labeled membrane-anchored cytochrome-b5 in native Escherichia coli cells.
• Performed 2D ¹³C/¹³C chemical shift correlation MAS experiments.

Main Results:

• Achieved a ~16-fold DNP signal enhancement for membrane-anchored cytochrome-b5.
• Demonstrated effective suppression of background signals from cellular contents.
• Showcased the feasibility of high-resolution structural studies on membrane proteins in situ.

Conclusions:

• Dynamic nuclear polarization magic angle spinning NMR is a feasible technique for in-cell structural studies of membrane proteins.
• This approach enables high-resolution structural insights into membrane proteins within their native cellular environment.
• The study opens new avenues for understanding the functional roles of membrane-associated proteins and their complexes in physiological processes.