Hans-Dieter Barth

ANTI-HYDROGEN BOND BETWEEN CHLOROFORM AND FLUOROBENZENE

Abstract Accurate theoretical calculations (ab initio MP2/6-31G * counterpoise-corrected gradient optimization, harmonic and anharmonic vibrational analysis) on the fluorobenzene⋯chloroform complex predict a new type of bonding, termed the anti-hydrogen bond. This bond distinguishes itself by the contraction of the C–H bond of chloroform and a blue shift of the corresponding stretching frequency, i.e. features opposite to those characteristic for a hydrogen bond. The predicted blue shift was confirmed experimentally by double-resonance infrared ion-depletion spectroscopy. The calculated blue shift of the chloroform C–H stretching frequency (12 cm −1 ) agrees with the experimental value of 14 cm −1 . The anti-hydrogen bond originates from the dispersive interaction between molecules (contrary to the hydrogen bond which is of electrostatic origin). It plays a significant role in benzene-containing molecular clusters and is expected to be of consequence for the structure of biomolecules.

A New Way To Detect Noncovalently Bonded Complexes of Biomolecules from Liquid Micro-Droplets by Laser Mass Spectrometry

A new version of laser mass-spectrometry is presented, which allows the quantitative analysis of specific biocomplexes in native solution. On-demand micro droplets, injected into vacuum, are irradiated by mid IR-laser pulses. Above a certain intensity threshold they explode due to the transmitted energy, setting free a fraction of the charged biomolecules which are then mass-analyzed. Amounts of analyte in the attomolar range may be detected with the ion intensity being linear over a wide range of molarity. Evidence is given that this method is soft, tolerant against various buffers, reflects properties of the liquid phase, and suitable for studying noncovalently bonded specific complexes. This is highlighted by results from antibiotics specifically binding into the minor groove of duplex DNA.

Subunit mass fingerprinting of mitochondrial complex I.

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Hydrogen bonding in (substituted benzene)·(water)n clusters with n≤4

Abstract Infrared ion-depletion spectroscopy, a double resonance method combining vibrational predissociation with resonant two-photon ionization (R2PI) spectroscopy, has been applied to study mixed clusters of the type (substituted benzene)·(H 2 O) n with n ≤4. The UV chromophores were p -difluorobenzene, fluorobenzene, benzene, toluene, p -xylene and anisole. From the IR depletion spectra in the region of the OH stretching vibrations it could be shown that the water molecules are attached as subclusters to the chromophores. Size and configuration of the subclusters could be deduced from the IR depletion spectra. In the anisole·(H 2 O) 1 and 2 complexes the water clusters form an ordinary hydrogen bond to the oxygen atom of the methoxy group. In all other mixed complexes a π-hydrogen bond is formed between one of the free OH groups of a water subcluster and the π-system of the chromophore. According to the strength of this interaction the frequency of the respective absorption band exhibits a characteristic red-shift which could be related to the total atomic charges in the aromatic ring.

Functional Dissection of the Proton Pumping Modules of Mitochondrial Complex I

A catalytically active subcomplex of respiratory chain complex I lacks 14 of its 42 subunits yet retains half of its proton-pumping capacity, indicating that its membrane arm has two pump modules.

Studying noncovalent protein complexes in aqueous solution with laser desorption mass spectrometry

Abstract The study of noncovalent aggregation with mass spectrometry has been largely the domain of electrospray ionization mass spectrometry (ESI-MS). In contrast, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been applied to this field to a much lesser extent. The main drawback of MALDI-MS is that the sample preparation requires a crystalline matrix. This disrupts the solution environment and often leads to dissociation of noncovalent complexes. A new laser desorption method, developed in our group, promises to circumvent this shortcoming. It is called laser induced liquid beam ionization/desorption mass spectrometry (LILBID-MS). The major advantage of this new method is the use of a liquid beam in vacuum for sample preparation and as target. The beam is directly injected into the mass spectrometer, using the solvent (mostly water) as the natural matrix substance, thus allowing for a softer probe preparation and desorption. In this article we present examples for the application of this new desorption method for detecting noncovalent aggregates of proteins in aqueous solutions. Using ribonuclease S, calmodulin/melittin, and bovine pancreatic trypsin inhibitor as model systems, evidence is given that LILBID-MS is capable of desorbing intact noncovalent complexes into the gas phase. Even water bound into cavities of a protein structure can be detected. In addition, it will be shown that solution parameters (e.g. pH, temperature) have a decisive influence on the mass spectra obtained, thus confirming earlier observations that the ions detected by LILBID-MS are formed in the solution phase and are not gas phase artifacts produced by the detection process.

Ionic clathrates from aqueous solutions detected with laser induced liquid beam ionization/desorption mass spectrometry

Abstract Clathratelike structures of the alkali metal ions M + = Li + , Na + , K + Cs + , and ammonium with water molecules have been investigated with laser induced liquid beam ionization/desorption (LILBID) mass spectrometry. The LILBID process implies the desorption of ions from a thin jet of supercooled water in high vacuum by means of an IR laser pulse. Magic numbered water clusters are observed in the cation mass spectra of K + and Cs + halides, but not for Li + and Na + . Predominant cluster sizes for K + (H 2 O) n are n = 16, 20, 25, 27, and for Cs + (H 2 O) n , n = 18, 20, 22, 24, 27, 29, with the highest signal intensity for n = 20. Water clusters of this size correspond to deformed dodecahedral cage structures (clathrates) with a cation in the cavity, which are known to exist in the gas phase and in the solid state. The possible occurrence of clathratelike clusters in the liquid phase is discussed in the context of the physical chemistry of electrolyte solutions and the LILBID experiment.

Laser-induced liquid bead ion desorption-MS of protein complexes from blue-native gels, a sensitive top-down proteomic approach

We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN‐PAGE) and laser‐induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent‐tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 μg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small‐scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.

Copper-binding abilities of the tripeptide diglycylhistidine studied by laser-induced liquid beam ionization/desorption mass spectrometry in aqueous solution

Laser-induced liquid beam ionization/desorption (LILBID) mass spectrometry is capable of desorbing ions directly from a liquid beam into the gas phase by means of an IR laser pulse tuned to an absorption band of the solvent used. Up to now, it has been restricted to the use of alcoholic solutions owing to the limitations of the desorbing IR laser system. This paper presents the first results using an Nd:YAG–optical parametric oscillator laser system that enables us to study aqueous solutions. Using this laser system, studies on the Cu(II)-binding abilities of the tripeptide diglycylhistidine are presented. Complex formation with various concentrations of Cu(II) ions was observed in water at basic pH. The use of buffer solutions did not affect the signal intensity of the peptide-related peaks. In acidic solutions, the complex partially dissociates. The free tripeptide and the released Cu(II) can be observed in the mass spectrum. The results obtained with this method were compared with measurements undertaken with electrospray ionization mass spectrometry.

Binding Sites of the Viral RNA Element TAR and of TAR Mutants for Various Peptide Ligands, Probed with LILBID: A New Laser Mass Spectrometry

A new laser-based mass spectrometry method, called laser induced liquid bead ion desorption (LILBID), was applied to investigate RNA:ligand interactions. As model system the HIV Tat:TAR transactivation complex and its binding behavior were analyzed. TARwt of HIV Type 1 and Type 2 and different artificial mutants were compared regarding their binding to Tat and different peptide ligands. Specific and nonspecific association to TAR was deduced, with the bulge being the well known specific binding site of TAR. In the case of triple arginine (RRR) as a nonspecific ligand, multiple electrostatic binding to TAR was found at higher concentration of RRR. This contrasted with the formation of only ternary complexes in competitive binding studies with TAR, Tat, and potential inhibitors. The fact that the stoichiometries of the complexes can be determined is a major advantage of MS methods over the widely applied fluorimetric methods. A quantitative evaluation of the spectra by a numeric model for ternary complex formation allows conclusions about the role and strength of the binding sites of the RNAs, the specificity and affinity of different ligands, the determination of apparent IC50 and KD values, and a comparison of the binding efficiencies of potential inhibitors.