Solid-state NMR on a large multidomain integral membrane protein: the outer membrane protein assembly factor BamA

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Multidomain proteins are crucial for cellular functions but difficult to structurally characterize due to size and flexibility.
• Understanding the structure of large multidomain membrane proteins is essential for elucidating their physiological roles.

Purpose of the Study:

• To develop and apply a motional-filtered solid-state NMR (ssNMR) approach for structural analysis of large multidomain membrane proteins.
• To investigate the folding and structural characteristics of the 790-residue BamA protein, a key component of the bacterial outer membrane protein assembly machinery.

Main Methods:

• Utilized motional-filtered high-resolution solid-state NMR (ssNMR) experiments.
• Applied dipolar- and scalar-based two-dimensional ssNMR techniques to uniformly (13)C,(15)N-labeled BamA variants.
• Analyzed atomic-scale structural information in a native-like environment.

Main Results:

• Demonstrated the capability of ssNMR to observe and analyze very large multidomain membrane proteins with diverse motional timescales.
• Identified characteristic secondary structure elements within the BamA transmembrane segment and its POTRA domains.
• Revealed distinct dynamics in different regions of the BamA protein.

Conclusions:

• Motional-filtered ssNMR provides a general strategy for obtaining atomic-scale structural insights into large multidomain (membrane) proteins.
• The study successfully characterized structural features and dynamics of the complex BamA protein.
• This approach facilitates the study of challenging protein targets in their native-like environments.