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

Abstract

Nebulin is a giant multifunctional protein that is thought to serve as both a length-regulating protein ruler and calcium/CaM-mediated regulatory protein on the thin filaments of the skeletal muscle sarcomere. To define molecular interfaces between nebulin and CaM, we thiolated lysines of CaM and ND66, a four-module cloned fragment from the C-terminus of nebulin, with 2-iminothiolane and cross-linked the complex with dibromobimane, which alkylates thiol pairs within approximately 6 A of each other to form a fluorescent adduct. Such a two-stage cross-linking generated mainly 1:1 complexes of ND66 and CaM, with a limited extent of intramolecular cross-linking. In-gel chymotryptic digestion of the dibromobimane-cross-linked complexes yielded peptides that were first screened by HPLC with fluorescence detection and then scored for cross-linking with mass spectrometry. Several inter- and intramolecular sites were identified and confirmed further by ESI-MS/MS experiments, defining molecular interfaces and patterns of protein folding. In particular, five intermolecular cross-linking products of sequences within the region of amino acids 83-99 (YKENMGKGTPLPVTPEM) in ND66 and several sequences of CaM indicate that the nebulin-CaM interface is close to, and may overlap with, the nebulin-actin interface. This proximity suggests a potential competition between CaM and actin for this nebulin interface. Intramolecular cross-linking of amino acids 13-16 (KEAF) and 13-18 (KEAFSL) with amino acids 145-148 (MTAK) and 146-148 (TAK) in CaM suggests the interaction of two lobes across the central helix. The cross-linking of amino acids 1-6 (MKTPEM) with amino acids 114-129 (YKENVGKATATPVTPE) and 115-129 (KENVGKATATPVTPE) in ND66 hints at an association of noncontiguous nebulin modules in solution.