Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in...

Area of Science:

• Biochemistry
• Structural Biology
• Spectroscopy

Background:

• Proteins often contain intrinsically disordered regions (IDRs) that are challenging to study using traditional methods.
• Nuclear Magnetic Resonance (NMR) spectroscopy is ideal for characterizing dynamic IDRs but faces limitations like spectral overlap and assignment difficulties, especially for large IDRs.

Purpose of the Study:

• To develop a method for segmental isotopic labeling of IDR-containing proteins to overcome NMR challenges.
• To enable selective probing and characterization of specific segments within large IDRs.

Main Methods:

• Utilized sortase-mediated ligation (SML) for segmental isotopic labeling of a protein fragment containing a large IDR and an adjacent folded domain (C-terminus of Arabidopsis thaliana villin 4).
• Applied NMR spectroscopy to analyze the labeled protein and performed backbone residue-specific assignment of the IDR.

Main Results:

• Segmental labeling significantly reduced NMR spectral complexity, enabling group identification of signals from the labeled IDR fragment (segmental assignment).
• Demonstrated nonuniform temperature-dependent spectral alterations in the IDR adjacent to the folded domain.
• Identified distinct temperature response patterns in two specific segments within the IDR.

Conclusions:

• The SML approach facilitates selective labeling and characterization of target segments within large IDRs.
• This method simplifies NMR analysis, reduces recording time, and aids in studying the function and binding interfaces of unstable IDRs.