title: On the efficiency of NHS ester cross-linkers for stabilizing integral membrane protein complexes.
abstract: We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes. Here, we investigated the capability of N-hydroxysuccinimide (NHS) esters, which react much more specifically, to cross-link membrane protein complexes such as PglK and BtuC(2)D(2). We present clear evidence that NHS esters are capable of stabilizing membrane protein complexes in situ, in the presence of detergents such as DDM, C12E8, and LDAO. The stabilization efficiency strongly depends on the membrane protein structure (i.e, the number of primary amine groups and the distances between primary amines). A minimum number of primary amine groups is required, and the distances between primary amines govern whether a cross-linker with a specific spacer arm length is able to bridge two amine groups.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zürich, 8093, Zürich, Switzerland.
authors.forename: Fan
authors.initials: F
authors.lastname: Chen
authors.forename: Sabina
authors.initials: S
authors.lastname: Gerber
authors.forename: Volodymyr M
authors.initials: VM
authors.lastname: Korkhov
authors.forename: Samantha
authors.initials: S
authors.lastname: Mireku
authors.forename: Monika
authors.initials: M
authors.lastname: Bucher
authors.forename: Kaspar P
authors.initials: KP
authors.lastname: Locher
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D004952:Esters / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013388:Succinimides / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D004952:Esters; D008565:Membrane Proteins; D013388:Succinimides; C001426:N-hydroxysuccinimide