Mapping protein interfaces with a fluorogenic cross-linker and mass spectrometry: application to nebulin-calmodulin complexes.
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.
Sinz
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Laboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wang
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D000595:Amino Acid Sequence; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D002147:Calmodulin / Q000032:analysis* / Q000378:metabolism; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000378:metabolism*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D001643:Bridged Bicyclo Compounds; D002147:Calmodulin; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009124:Muscle Proteins; D010446:Peptide Fragments; C035620:nebulin; C037094:dibromobimane; D005022:Ethyldimethylaminopropyl Carbodiimide