On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes
- 1k Downloads
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.
Key wordsMembrane protein complexes MALDI Chemical cross-linking NHS-esters
Mass spectrometry (MS), using electrospray ionization (ESI) or matrix-assisted 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 ESI-MS . The first mass spectrum of a membrane protein complex in detergent micelles recored by native ESI-MS was the heteromeric vitamin B12 importer BtuC2D2 . Laser-induced 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 ExbB-ExbD . Alternatively, MALDI-MS with high-mass detection capabilities is a straighforward method to study integral membrane proteins . 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 .
Noncovalent interactions are easily disrupted in MALDI, either during sample preparation or ion formation. Chemical cross-linkers such as glutaraldehyde are thus often used, to stabilize noncovalent interactions before analyzing complexes by MALDI-MS [5, 6]. Although glutaraldehyde is known to react with membrane protein complexes , 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 , 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 three-dimensional structural analysis based on chemical cross-linking combined with MS.
N-hydroxysuccinimide (NHS) esters, which react specifically with Lys residues, are among the most widely applied chemical cross-linkers. They are convenient for analyzing the three-dimensional 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 . Cross-linking protocols, mass spectrometric analysis of cross-linked samples, and also data analysis are well established, as described in some recent reviews [9, 10, 11, 12].
Recently, NHS esters have been applied in structure characterization of membrane proteins [13, 14, 15, 16]. It has been reported that a NHS ester-based cross-linker, which was used in the development of the so-called protein interaction reporter (PIR) technology, was able to stabilize protein complexes in living cells, including outer membrane protein A (OmpA) [13, 14]. Another NHS-based cross-linker, BS3, has also been applied to study chloroplast F-ATPases. The results suggested relations among phosphorylation, dynamic interactions, and regulation of a transmembrane molecular motor [15, 16]. To futher subject cross-linked proteins to tandem mass spectrometry (top-down approach) or to in-solution digestion (bottom-up 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 micelles.
To 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 NHS-esters with integral membrane protein complexes from two main perspectives, the chemical properties of the cross-linker and the structural properties of the membrane proteins. All four NHS ester-based cross-linkers studied here, including bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl suberate (DSS), bis(succinimidyl) penta(ethylene glycol) (BS(PEG)5), and bis(succinimidyl) nonaethyleneglycol (BS(PEG)9) (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 cross-linking PglK in the presence of detergents including DDM and C12E8, which are frequently used above the critical micelle concentration for solubilizing membrane proteins.
2 Materials and Methods
PglK and BtuC2D2 were purified as described previously [4, 19]. After buffer exchange, PglK was in a buffer of 10 mM Bicine-NaOH at pH 8.2 containing 500 mM NaCl, 0.5 mM EDTA-NaOH, 10% (w/v) glycerol, and 0.016% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace). Dodecyl octaethylene glycol ether (C12E8) was also used as a detergent to solublilize PglK. The BtuC2D2 protein was in a final buffer with the following composition: 50 mM Na-phosphate, pH 7, 500 mM NaCl, 0.5% EDTA, 0.1% LDAO. Sinapinic acid (SA) was purchased from Sigma-Aldrich Chemie GmbH (Buchs, Switzerland). All chemical cross-linkers were purchased from Pierce Protein Research Products (Thermo Fisher Scientific, Rockford, IL, USA). Trifluoroacetic acid (TFA) was obtained from Acros Organics (Thermo Fisher Scientific, Geel, Belgium). All commercial reagents and solvents were obtained in the highest purity available and used without further purification.
2.2 Chemical Cross-Linking Protocol
Cross-linker solutions were prepared at a concentration 1000 times higher than that of the protein complexes. BS3 was dissolved in water, whereas the other cross-linkers used here were dissolved in acetonitrile. To chemically cross-link the proteins, the cross-linker solutions were mixed with the protein solution in a 1:10 volume ratio at room temperature, for 2 h. The mixture was further diluted with the original protein buffer solution or water prior to mass spectrometric analysis.
2.3 Mass Spectrometry
A commercial MALDI-TOF/TOF mass spectrometer (model 4800 plus; AB SCIEX, Darmstadt, Germany) equipped with a high-mass detector (HM2; CovalX AG, Zurich, Switzerland) was used. All measurements were performed in linear positive ion mode, with standard settings. Ionization was achieved with a Nd:YAG laser (355 nm) with the pulse energy set just above the threshold for ion formation. Each mass spectrum was the average of 1000 laser shots acquired at random sample positions. Sinapinic acid (20 mg/mL in water/acetonitrile/TFA, 49.95/49.95/0.1, v/v/v) was used as the matrix. The samples were directly mixed with the matrix solution in a 1/2 (v/v) ratio. One μL of the mixture was spotted on to a stainless steel plate and allowed to dry under ambient conditions. All mass spectra were baseline-corrected and smoothed using a Savitzky-Golay algorithm available within Igor Pro (ver. 6.2; WaveMetrics, Portland, OR, USA). Distance calculations between specific amino acid residues, based on the protein structure from the Protein Data Bank, were carried out using UCSF Chimera (ver. 1.6.2; University of California, San Francisco, CA, USA).
3 Results and Discussion
For PglK, the cross-linking efficiency was comparable for all NHS esters and for glutaraldehyde. Supplementary Table 2 presents the cross-linking efficiency of all NHS esters applied to PglK, which was solubilized in two different detergent micelle preparations. In both detergents, all the cross-linkers exhibited a similar cross-linking efficiency, 63% in DDM and 71% in C12E8. In a previous report, comparable cross-linking efficiency was observed for NCoA-1·STAT6Y, which has a KD around 30 nM . This suggests that the PglK dimer is also tightly bound, with a KD in the nanomolar range. Besides the contribution of the binding affinity of the PglK dimer, we also believe the high prevalence of lysine residues in PglK (55 lysine residues, 9.4%, higher than average, which is around 6%), to play an important role in the stabilization via chemical cross-linking.
In this study, we conducted cross-linking with chemically specific NHS-esters of two membrane protein complexes, PglK and BtuC2D2. The cross-linking experiment was carried out with a series of chemical cross-linkers with different spacer arm lengths. We found clear evidence that NHS esters can be used to stabilize or cross-link membrane protein complexes even in the presence of different detergents, such as DDM, LDAO, and C12E8. The reactivity differs among the cross-linkers applied, and depends on the protein structure, including the number of lysine residues and the amine-amine distances between different subunits. The low number of available lysine residues is probably the reason for the relatively low cross-linking efficiency in BtuC2D2, compared with the efficient stabilization of the PglK dimer. The successful cross-linking of membrane protein complexes in different detergent micelles via NHS esters highlights the possibility to map membrane protein structures by chemical cross-linking in situ, combined with mass spectrometry.
The authors acknowledge financial support from the Swiss National Science Foundation (SNF), grant no. 200020-124663 (to R.Z.) and grant no. 31003A-116191 (to K.P.L.), as well as from the National Center for Excellence in Research (NCCR) Structural Biology (to K.P.L.).
- 7.Migneault, I., Dartiguenave, C., Bertrand, M.J., Waldron, K.C.: Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. BioTechniques 37, 790–802 (2004)Google Scholar
- 9.Sinz, A.: Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein–protein interactions. Mass Spectrom. Rev. 25(4), 663–682, (2006)Google Scholar
- 13.Zhang, H., Tang, X., Munske, G.R., Tolic, N., Anderson, G.A., Bruce, J.E.: In vivo identification of the outer membrane protein OmcA–MtrC interaction network in Shewanella oneidensis MR-1 cells using novel hydrophobic chemical cross-linkers. Mol. Cell. Proteomics 8, 409–420 (2009)CrossRefGoogle Scholar
- 15.Schmidt, C., Zhou, M., Marriott, H., Morgner, N., Politis, A., Robinson, C.V.: Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation. Nat. Commun. 4, 1985 (2013)Google Scholar