Improved in-gel approaches to generate peptide maps of integral membrane proteins with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry van Montfort, B.A.; Canas, B.; Duurkens, R.H.T.; Godovac-Zimmermann, J.; Robillard, G.T. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. This paper reports studies of in-gel digestion procedures to generate MALDI-MS peptide maps of integral membrane proteins. The methods were developed for the membrane domain of the mannitol permease of E. coli. In-gel digestion of this domain with trypsin, followed by extraction of the peptides from the gel, yields only 44% sequence coverage. Since lysines and arginines are seldomly found in the membrane-spanning regions, complete tryptic cleavage will generate large hydrophobic fragments, many of which are poorly soluble and most likely contribute to the low sequence coverage. Addition of the detergent octyl-b-glucopyranoside (OBG), at 0.1% concentration, to the extraction solvent increases the total number of peptides detected to at least 85% of the total protein sequence. OBG facilitates the recovery of hydrophobic peptides when they are SpeedVac dried during the extraction procedure. Many of the newly recovered peptides are partial cleavage products. This seems to be advantageous since it generates hydrophobic fragments with a hydrophilic solubilizing part. In-gel CNBr cleavage resulted in 5-10-fold more intense spectra, 83% sequence coverage, fully cleaved fragments and no effect of OBG. In contrast to tryptic cleavage sites, the CNBr cleavage sites are found in transmembrane segments; cleavage at these sites generates smaller hydrophobic fragments, which are more soluble and do not need OBG. With the results of both cleavages, a complete sequence coverage of the membrane domain of the mannitol permease of E. coli is obtained without the necessity of using HPLC separation. The protocols were applied to two other integral membrane proteins, which confirmed the general applicability of CNBr cleavage and the observed effects of OBG in peptide recovery after tryptic digestion. Copyright 2002 John Wiley & Sons, Ltd. KEYWORDS: membrane protein; peptide mapping; octyl glucoside; matrix-assisted laser desorption/ionization; sodium dodecyl sulfate polyacrylamide gel electrophoresis Abbreviations: MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; MS, mass spectrometry; OBG, octyl-ˇ-glucopyranoside; SDS, sodium dodecyl sulfate; PAGE, polyacrylamidegel electrophoresis; CNBr, cyanogen bromide; HPLC, high-performance liquid chromatography; C domain, the membrane domain of the mannitol transporter without the soluble A and B domain; MscL, mechanosensitive channel of l...
A limitation of the in-gel approaches for the generation of peptides of membrane proteins is the size and hydrophobicity of the fragments generated. For membrane proteins like the lactose transporter (LacS) of Streptococcus thermophilus, tryptic digestion or CNBr cleavage yields several hydrophobic fragments larger than 3.5 kDa. As a result, the sequence coverage of the membrane domain is low when the in-gel tryptic-digested or CNBr-cleaved fragments are analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). The combination of tryptic digestion and subsequent CNBr cleavage on the same gel pieces containing LacS approximately doubled the coverage of the hydrophobic membrane domain compared to the individual cleavage methods, while the coverage of the soluble domain remained complete. The fragments formed are predominantly below m/z 2500, which allows accurate mass measurement.
Part of the dimer and B/C domain interface of theThe uptake and concomitant phosphorylation of a wide variety of carbohydrates into bacterial cells is, in many cases, accomplished by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) (1). In a cascade of phosphorylation reactions (Fig. 1) Mannitol in the periplasm is bound by the C domain, transported into the cell via C and, while bound at the cytoplasmic site of C, phosphorylated by the B domain. EII mtl is most likely a dimeric protein and the subunit interactions occur in the C domain (3-8).Domain interactions and in particular the B/C domain interface play an important role in the catalytic cycle of EII mtl . The energy coupling mechanism involves conformational interaction between the B and C domain. The evidence for this notion is manifold. 1) Phosphorylation of the B domain increases the rate of transport 2-3 orders of magnitude (9, 10). 2) Modification or mutagenesis of the phosphorylation site in the B domain as well as removal of the cytoplasmic domains changes the mannitol binding kinetics of the C domain (11,12). 3) Timeresolved fluorescence and phosphorescence spectroscopy showed that, upon phosphorylation of the B domain, Trp 109 in the C domain becomes immobilized whereas Trp 30 in the C domain becomes more flexible (13,14). 4) Differential scanning calorimetry showed that the thermal stability of the C domain is higher in the presence of the B domain (15). 5) Isothermal titration calorimetry experiments indicated that a significant part of the structural changes upon the binding of mannitol to the C domain reside in the B domain. Approximately 50 -60 residues are removed from the bulk water upon binding of mannitol, which was much less when the same measurements were done after removal of the B domain (16). 6). Close proximity of the B and C domain has been suggested for another PTS transporter, that is the BglF system of E. coli. 3To date, there is no structural information about the B/C domain or dimer interface of EII mtl or any other EII. The topological model of the C domain predicts 6 membrane-span-
A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline) 3 -catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning ␣-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys 73 ). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys 134 ), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys 134 becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for ␣-helix packing in the mannitol permease.In bacteria, the phosphoenolpyruvate-dependent phosphotransferase system is involved in the uptake and concomitant phosphorylation of a wide variety of carbohydrates (1). The system consists of two general components EI and HPr and several carbohydrate-specific enzymes II (EII). In a cascade of phosphorylation reactions, the phosphoryl group is transferred from the energy donor phosphoenolpyruvate via EI and HPr to EII, as is illustrated in Fig. 1 for the mannitol-specific phosphotransferase system (reviewed in Ref. 2). All EII's have a similar architecture and consist of the cytoplasmic A and B domains and a membrane-embedded C domain. In the mannitol-specific EII (EII mtl ) 1 of Escherichia coli the three domains are covalently linked. The C domain transports mannitol into the cell and the A and B domains transfer the phosphoryl group from HPr to mannitol. The imported mannitol is phosphorylated by B while being bound by C. The association state of EII mtl is most likely the dimer with contacts between two subunits being provided by the C domain. The evidence for this oligomeric state is manifold, but the strongest indications are from in vitro and in vivo complementation studies. The formation of heterodimers from inactive subunits with defects in different domains restores the activity of EII mtl (3-5). Moreover, subunits of EII mtl could be covalently cross-linked to a dimer with bifunctional sulfhydryl-specific reagents, lysine-specific cross-linkers, or by oxidative disulfide bond formation (6 -9). The 5-Å projection structure of the C domain in two-dimensional crysta...
Penicillins and cephalosporins are an efficacious group of antibiotics produced by fungi such as Penicillium chrysogenum and Acremonium chrysogenum. The last step in their biosynthesis is catalyzed by acyl coenzyme A:isopenicillin N transferase (AT). This enzyme is produced as a single-chain proenzyme, which is activated by autocatalytic cleavage of the Gly102-Cys103 peptide bond, resulting in a heterodimeric protein with subunits of 11 and 29 kDa. The Cys103Ala mutant of the proenzyme, which does not undergo this cleavage, was purified and crystallized. Diffraction-quality crystals of the mutant and an L-SeMet-substituted mutant were obtained by vapour diffusion against solutions containing (NH(4))(2)SO(4), NaCl and HEPES-NaOH pH 7.5. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 231.36, b = 68.27, c = 151.31 A and beta = 129.56 degrees. They diffract to 2.8 A resolution with X-rays from a rotating-anode generator.
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