Mapping Protein Interfaces with a Fluorogenic Cross-Linker and Mass Spectrometry: Application to Nebulin−Calmodulin Complexes
- 9 June 2001
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 40 (26) , 7903-7913
- https://doi.org/10.1021/bi010259+
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 ∼6 Å 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.Keywords
This publication has 9 references indexed in Scilit:
- Identification and localisation of nebulin as a thin filament component of invertebrate chordate muscles.Journal of Comparative Physiology B, 1999
- A six-module human nebulin fragment bundles actin filaments and induces actin polymerization.Journal of Muscle Research and Cell Motility, 1998
- Nebulin is a full-length template of actin filaments in the skeletal muscle sarcomere: an immunoelectron microscopic study of its orientation and span with site-specific monoclonal antibodiesJournal of Muscle Research and Cell Motility, 1993
- Activation of enzymes by calmodulins containing intramolecular cross-linksBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1993
- Nebulin as a length regulator of thin filaments of vertebrate skeletal muscles: correlation of thin filament length, nebulin size, and epitope profile.The Journal of cell biology, 1991
- Evidence that nebulin is a protein‐ruler in muscle thin filamentsFEBS Letters, 1991
- How calmodulin binds its targets: sequence independent recognition of amphiphilic α-helicesTrends in Biochemical Sciences, 1990
- Toward a Model of the Calmodulin-Myosin Light-Chain Kinase ComplexJournal of Cardiovascular Pharmacology, 1988
- Carp Muscle Calcium-binding ProteinJournal of Biological Chemistry, 1973