Mapping protein interfaces with a fluorogenic cross-linker and mass spectrometry: application to nebulin-calmodulin...

Area of Science:

• Muscle physiology
• Protein biochemistry
• Molecular biology

Background:

• Nebulin is a giant protein regulating skeletal muscle thin filament length and calcium sensitivity.
• Calmodulin (CaM) is a key calcium-binding protein involved in muscle regulation.
• Understanding nebulin-CaM interactions is crucial for elucidating muscle function.

Purpose of the Study:

• To define the molecular interfaces between nebulin and CaM.
• To investigate the structural basis of nebulin-CaM complex formation.

Main Methods:

• Chemical cross-linking of CaM and a nebulin fragment (ND66) using 2-iminothiolane and dibromobimane.
• In-gel chymotryptic digestion followed by HPLC and mass spectrometry (MS/MS) for site identification.
• Fluorescence detection to screen cross-linked peptides.

Main Results:

• Established 1:1 complexes of ND66 and CaM with limited intramolecular cross-linking.
• Identified specific intermolecular cross-linking sites between nebulin (amino acids 83-99) and CaM.
• Indicated that the nebulin-CaM interface is near or overlaps with the nebulin-actin interface.
• Revealed intramolecular cross-linking within CaM, suggesting lobe interactions.
• Hinted at associations between noncontiguous nebulin modules in solution.

Conclusions:

• The nebulin-CaM interface is identified, suggesting potential competition with actin binding.
• Structural insights into CaM's interaction with nebulin and its own folding are provided.
• This research clarifies nebulin's regulatory role in skeletal muscle contraction.