On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes



Fan Chen1, Sabina Gerber2, Volodymyr M. Korkhov2, Samantha Mireku2, Monika Bucher2, Kaspar P. Locher2, and Renato Zenobi1


  1. Department of Chemistry and Applied Biosciences, ETH Zürich, 8093 Zürich, Switzerland
  2. Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland


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 BtuC2D2. 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.

CovalX Technology Used (Click each option to learn more)



Proteins were subject to cross-linking at room temperature for 2 hours in a 1:10 volume ratio. After the 2 hours, the mixture was diluted using the original protein buffer solution or water. Then, samples were mixed with the matrix solution before 1 μL of each was spotted on a stainless steel plate before being dried in ambient conditions. MALDI-TOF/TOF analysis was performed using a mass spectrometer that had been modified with a CovalX HM2 detection system.



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