Details zur Publikation

Referenztyp Zeitschriften
DOI / URL Link
Titel (primär) Chemical cross-linking and high-performance Fourier transform ion cyclotron resonance mass spectrometry for protein interaction analysis: Application to a calmodulin/target peptide complex
Autor Kalkhof, S.; Ihling, C.; Mechtler, K.; Sinz, A.;
Journal / Serie Analytical Chemistry
Erscheinungsjahr 2005
Department PROTEOM;
Band/Volume 77
Heft 2
Sprache englisch;
Abstract Chemical cross-linking has proved successful in combination with mass spectrometry as a tool for low-resolution structure determination of proteins. The integration of chemical cross-linking with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to determine protein interfaces was tested on the calcium-dependent complex between calmodulin (CaM) and a 26-amino acid peptide derived from the skeletal muscle myosin light chain kinase (M13). Different amine-reactive, homobifunctional cross-linkers and a "zero-length" cross-linker were employed. The covalently attached complexes were separated from nonreacted proteins by one-dimensional gel electrophoresis, and the bands of interest were excised and in-gel digested with trypsin. Digestion of the crosslinked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. The distance constraints obtained by chemical cross-linking were in agreement with the published NMR structure of the CaM/M13 complex, pointing to residues Lys-18 and Lys-19 of M13 being cross-linked with the central a-helix of CaM. Thus, the integrated approach described herein has proven to be efficient tool for mapping the topology of the CaM/M13 complex. As such it is applicable as a general strategy for the investigation of the spatial organization of protein complexes and complements existing techniques, such as X-ray crystallography and NMR spectroscopy
ID 3491
dauerhafte UFZ-Verlinkung https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=3491
Kalkhof, S., Ihling, C., Mechtler, K., Sinz, A. (2005):
Chemical cross-linking and high-performance Fourier transform ion cyclotron resonance mass spectrometry for protein interaction analysis: Application to a calmodulin/target peptide complex
Anal. Chem. 77 (2), 495 - 503