Characterization of leucine zipper complexes by electrospray ionization mass spectrometry

Wendt, H.; Dürr, Eberhard; Thomas, Richard M.; Przybylski, Michael; Bosshard, Hans Rudolf

doi:10.1002/pro.5560040814

Cited by 32 publications

(35 citation statements)

References 35 publications

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“…Therefore, we conclude that the hydrophobic effect plays the dominant role in stabilizing the LZ dimer in solution. Small amounts (Ͻ25%) of LZ dimer have previously been detected for a modified LZ peptide in combination with a different buffer and instrument [34]. These results are consistent with our findings, since the small amount of complex detected does not reflect the situation in solution, where 78% of complex should be present.…”

Section: Resultssupporting

confidence: 91%

Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

Bich

Baer

Jecklin

et al. 2010

J. Am. Soc. Mass Spectrom.

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The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real "snap-shot" of the situation in solution can be obtained. (J Am Soc Mass Spectrom 2010, 21, 286 -289) © 2010 American Society for Mass Spectrometry E lectrospray is an exceptionally soft ionization technique, which has allowed the observation of numerous noncovalent complexes, including protein-protein, protein-small molecule, and protein-DNA complexes [1,2]. A major question is still whether the information obtained from gas-phase results is representative of the species present in solution. The term "noncovalent bonding" in biochemistry generally summarizes three types of intermolecular forces: electrostatic interactions (e.g., salt bridges), dipolar interactions (e.g., hydrogen bonds), and van der Waals interactions (e.g., hydrophobic interactions) [3]. In addition, protein-water interactions often play a major role in complex stabilization. In solution, hydrophobic interactions are very weak compared with the hydrogen bonds between water molecules. Hence, it is not the hydrophobic forces that are the actual reason for the interaction of nonpolar binding partners, but rather the surrounding water molecules that "force" hydrophobic binding partners to aggregate. This solvent-driven process is called the hydrophobic effect, and plays a major role in protein folding, adhesion of lipid bilayers, partitioning effects of drugs and metabolites, and many other aggregation phenomena in chemistry and biology [4][5][6]. The hydrophobic effect is defined by the thermodynamics of mixing hydrophobic compounds with water, and is dominated by large, entropic factors [4,7]. Obviously, in the absence of a hydr...

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Section: Resultssupporting

confidence: 91%

Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

Bich

Baer

Jecklin

et al. 2010

J. Am. Soc. Mass Spectrom.

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“…In some studies it was shown that the gas-phase stability reflects the binding properties in solution [37][38][39]. More frequently, however, a correlation between the gas-phase stability and the solution-phase stability is absent [25,26,28,[40][41][42][43][44][45][46], for example for leucine-zippers and acyl-CoA binding protein (ACBP) and a series of acyl CoA derivatives [28]. If binding properties in solution correlate with gas-phase stability it has to be assumed that the dominant interactions are very similar in solution and in the gas phase, and that solvent mediation play only a minor role (see Daniel at al.…”

Section: Introductionmentioning

confidence: 99%

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

Cubrilovic

Biela

Sielaff

et al. 2012

J. Am. Soc. Mass Spectrom.

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NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (K D ) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in K D and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the K D values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (K D ) measured via nanoESI-MS were compared with kinetic inhibition constants (K i ) in solution.

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“…Interactions between the acyl CoA-binding protein and ligands were not observed in conditions normally used for non-covalent complexes because hydrophobic interactions are critical to the preservation of these complexes in solution (30). Also leucine zipper complexes are poorly retained in the mass spectrometer because of the prominent role hydrophobic interactions play in these complexes (31 (13). We here observe that, under the experimental conditions used, only the hydrogen bond interactions and salt bridges are not sufficient to preserve the interactions between CprK1 and its effectors in the mass spectrometer.…”

Section: The Quaternary Structure Of Cprk1 Does Not Depend On the Promentioning

confidence: 99%

Transcriptional Activation by CprK1 Is Regulated by Protein Structural Changes Induced by Effector Binding and Redox State

Mazon

Gábor

Leys

et al. 2007

Journal of Biological Chemistry

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The transcriptional activator CprK1 from Desulfitobacteriumhafniense, a member of the ubiquitous cAMP receptor protein/ fumarate nitrate reduction regulatory protein family, activates transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. 3-Chloro-4-hydroxyphenylacetate is a known effector for CprK1, which interacts tightly with the protein, and induces binding to a specific DNA sequence ("dehalobox," TTAAT--ATTAA) located in the promoter region of chlorophenol reductive dehalogenase genes. Despite the availability of recent x-ray structures of two CprK proteins in distinct states, the mechanism by which CprK1 activates transcription is poorly understood. In the present study, we have investigated the mechanism of CprK1 activation and its effector specificity. By using macromolecular native mass spectrometry and DNA binding assays, analogues of 3-chloro-4-hydroxyphenylacetate that have a halogenated group at the ortho position and a chloride or acetic acid group at the para position were found to be potent effectors for CprK1. By using limited proteolysis it was demonstrated that CprK1 requires a cascade of structural events to interact with dehalobox dsDNA. Upon reduction of the intermolecular disulfide bridge in oxidized CprK1, the protein becomes more dynamic, but this alone is not sufficient for DNA binding. Activation of CprK1 is a typical example of allosteric regulation; the binding of a potent effector molecule to reduced CprK1 induces local changes in the N-terminal effector binding domain, which subsequently may lead to changes in the hinge region and as such to structural changes in the DNA binding domain that are required for specific DNA binding.Halogenated hydrocarbons are often toxic molecules. They are widespread environmental pollutants because of their numerous applications, for example in industry (degreasers), agriculture (pesticides), and private households (flame retardants). Although these compounds are generally very stable, they can be converted in many sediments and soils by reductive dehalogenation. Several strictly anaerobic bacteria capable of dehalogenation have been isolated including Desulfomonile, Dehalobacter, and Desulfitobacterium. These organisms use chlorinated compounds as terminal electron acceptors (halorespiration) and thus remove the chloride atom while energy is conserved via electron transport phosphorylation (1-3). The prospect of using these organisms in bioremediation of sediments contaminated with a variety of chlorinated aromatic compounds is promising. Desulfitobacterium dehalogenans can reductively dechlorinate phenolic compounds at the ortho position (4, 5), and the closely related Desulfitobacterium hafniense DCB-2 (6) can dechlorinate phenolic compounds at the ortho and meta position; the latter reaction, however, is described only for 3,5-dichlorophenol (3,5-DCP) 2 as a substrate (7).A large number of the proteins involved in halorespiration are encoded in the chlorophenol reductive dehalogenase (cpr...

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Characterization of leucine zipper complexes by electrospray ionization mass spectrometry

Cited by 32 publications

References 35 publications

Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

Probing the hydrophobic effect of noncovalent complexes by mass spectrometry

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

Transcriptional Activation by CprK1 Is Regulated by Protein Structural Changes Induced by Effector Binding and Redox State

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