On the Efficiency of NHS Ester CrossLinkers for S

Authors: Fan Chen Sabina Gerber Volodymyr M Korkhov Samantha Mireku Monika Bucher Kaspar P Locher Renato Zenobi

Publish Date: 2014/11/18

Volume: 26, Issue: 3, Pages: 493-498

PDF Link

Abstract

We have previously presented a straightforward approach based on highmass matrixassisted laser desorption/ionization MALDI mass spectrometry MS to study membrane proteins In addition the stoichiometry of integral membrane protein complexes could be determined by MALDIMS following chemical crosslinking 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 Nhydroxysuccinimide NHS esters which react much more specifically to crosslink 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 ie 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 crosslinker with a specific spacer arm length is able to bridge two amine groupsMass spectrometry MS using electrospray ionization ESI or matrixassisted laser desorption/ionization MALDI is a powerful method for studying macromolecular complexes Membrane proteins however are difficult to study by MS because detergents are required for solubilizing them which often compromises efficient ionization Marcoux and Robinsin have written a good review on recent progress in studying membrane proteins and their complexes by native ESIMS 1 The first mass spectrum of a membrane protein complex in detergent micelles recored by native ESIMS was the heteromeric vitamin B12 importer BtuC2D2 2 Laserinduced liquid bead ion desorption LILBID MS a highly specialized technique was also applied to study membrane protein complexes specifically the oligomeric state of ExbB and ExbBExbD 3 Alternatively MALDIMS with highmass detection capabilities is a straighforward method to study integral membrane proteins 4 It allows rapid determination of their molecular weights pinpointing glycosylation sites and elucidation of the subunit stoichiometry of membrane protein complexes without the need for extensive sample purification and optimization of the sample preparation 4Noncovalent interactions are easily disrupted in MALDI either during sample preparation or ion formation Chemical crosslinkers such as glutaraldehyde are thus often used to stabilize noncovalent interactions before analyzing complexes by MALDIMS 5 6 Although glutaraldehyde is known to react with membrane protein complexes 4 the structure of glutaraldehyde in aqueous solution is not well defined because it polymerizes Moreover glutaraldehyde reacts unspecifically with a number of functional groups of proteins 7 which severely limits its application in structure determination For instance different polymeric forms of glutaraldehyde compromise mapping the distances between different amino acid side chains which is at the core of threedimensional structural analysis based on chemical crosslinking combined with MSNhydroxysuccinimide NHS esters which react specifically with Lys residues are among the most widely applied chemical crosslinkers They are convenient for analyzing the threedimensional structure of proteins because of the high prevalence of lysine residues in proteins about 6 Under carefully controlled reaction conditions side reactions of NHS esters with amino acids other than Lys can be largely avoided 8 Crosslinking protocols mass spectrometric analysis of crosslinked samples and also data analysis are well established as described in some recent reviews 9 10 11 12Recently NHS esters have been applied in structure characterization of membrane proteins 13 14 15 16 It has been reported that a NHS esterbased crosslinker which was used in the development of the socalled protein interaction reporter PIR technology was able to stabilize protein complexes in living cells including outer membrane protein A OmpA 13 14 Another NHSbased crosslinker BS3 has also been applied to study chloroplast FATPases The results suggested relations among phosphorylation dynamic interactions and regulation of a transmembrane molecular motor 15 16 To futher subject crosslinked proteins to tandem mass spectrometry topdown approach or to insolution digestion bottomup approach for structure determination it is thus critical to estabilish under which conditions NHS esters react effectively with membrane proteins or their complexes in particular in the presence of detergent micellesTo answer this question we used a series of NHS esters and two membrane protein complexes specifically the ATP binding cassette ABC transporters 17 18 PglK and BtuC2D2 In the following we look at the reactivity of NHSesters with integral membrane protein complexes from two main perspectives the chemical properties of the crosslinker and the structural properties of the membrane proteins All four NHS esterbased crosslinkers studied here including bissulfosuccinimidyl suberate BS3 disuccinimidyl suberate DSS bissuccinimidyl pentaethylene glycol BSPEG5 and bissuccinimidyl nonaethyleneglycol BSPEG9 Supplementary Table 1 were found to be capable of stabilizing membrane protein complexes in situ The stabilization efficiency strongly depended on the protein structure including the primary and tertiary structure We succeeded in crosslinking PglK in the presence of detergents including DDM and C12E8 which are frequently used above the critical micelle concentration for solubilizing membrane proteins

Keywords: