High-Resolution Protein 3D Structure Determination in Living Eukaryotic Cells

Area of Science:

• Structural Biology
• Biophysics
• Molecular Cell Biology

Background:

• Proteins interact with numerous biomolecules within cells.
• Understanding protein behavior under intracellular crowding is crucial.
• Observing 3D protein structures at atomic resolution in vivo is challenging.

Purpose of the Study:

• To demonstrate de novo protein structure determination exclusively using NMR data from living eukaryotic cells.
• To establish a standard method for in-cell protein structure analysis.

Main Methods:

• Utilized the sf9 cell/baculovirus system for protein expression in eukaryotes.
• Acquired Nuclear Magnetic Resonance (NMR) data directly from living cells.
• Applied 3D Nuclear Overhauser Effect Spectroscopy (NOESY) for structural information.

Main Results:

• Successfully obtained structural information for five proteins (rat calmodulin, human HRas, human ubiquitin, T. thermophilus HB8 TTHA1718, and Streptococcus protein G B1 domain) using in-cell NMR.
• Achieved well-resolved 3D Nuclear Overhauser Effect Spectroscopy (NOESY) data in all tested cases.
• Determined well-converged 3D structures for three proteins, with Protein G B1 domain showing the most accurate in-cell structure, revealing conformational differences from solution structures.

Conclusions:

• The developed method enables de novo protein structure determination in living eukaryotic cells.
• This approach can serve as a standard tool for analyzing protein structures within their native cellular environment.
• In-cell structural analysis revealed distinct conformations, such as a tilted helix-loop region in Protein G B1 domain, compared to solution states.