Konstantin Barylyuk

Hexameric supramolecular scaffold orients carbohydrates to sense bacteria.

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated β-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus β-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.

Fragmentation of Benzylpyridinium Thermometer Ions and Its Effect on the Accuracy of Internal Energy Calibration

Electrospray ionization mass spectrometry (ESI-MS) is a powerful analytical method to study biomolecules and noncovalent complexes. The prerequisite for their intact observation is soft ionization. In ESI, the internal energy of ions is primarily influenced by collisional activation in the source. The survival yield method is frequently used to probe the energy deposition in ions during the electrospray process. In the present work, we investigate the fragmentation pathways of para-substituted benzylpyridinium ions, the most widely used “thermometer ions” in the survival yield method. In addition to the C-N bond cleavage, alternative fragmentation channels were found for the compounds studied. We consider these pathways to result from intramolecular rearrangements. The effect of these additional fragments on the accuracy of the internal energy calibration is estimated for both collision-cell and in-source collision-induced dissociation (CID). Altogether, results presented suggest that a correction of the energy scale is necessary for the method based on benzylpyridinium ions to precisely quantify ion internal energies.

Quantifying protein-protein interactions within noncovalent complexes using electrospray ionization mass spectrometry.

Several electrospray-mass spectrometry (ESI-MS)-based methods are available for determining the constant of association (K(a)) between a protein and a small ligand, but current MS-based strategies are not fully adequate for measuring K(a) of protein-protein interactions accurately. We expanded the application of ESI-MS-based titration to determine the strength of noncovalent interactions between proteins, forming a complex. Taking into account relative response factors (probability of being ionized, transmitted, and detected), we determined K(a) values of an equilibrium between dimers and tetramers at three different pH values (6.8, 3.4, and 8.4). We investigated the association of the lectin concanavalin A, whose dimer-tetramer ratio in the gas phase is affected by solution concentration and by pH. To calculate the constants of association in solution, we also utilized isothermal titration calorimetry (ITC) for a comparison with MS-based titration. At pH 6.8 and pH 8.4, the K(a) values measured by MS and by ITC were in agreement. ITC results allowed us to restrain the response factor to a value close to 4. At pH 3.4, we were able to measure the K(a) only by MS, but not by ITC because of limited sensitivity of calorimetry. Our investigation illustrates the great potential MS for calculating the binding strength of protein-protein interactions within noncovalent complexes. The main advantages of MS over ITC are its sensitivity (i.e., the required amount of sample is >100 times less than the one necessary for ITC), and the possibility to obtain precise information on composition of protein complexes, their stoichiometry, their subunit interactions, and their assembly pathway. Compared to previous investigations, our study shows the strong influence of response factors on determining accurate protein-protein association constants by MS.

Determination of protein-ligand binding constants of a cooperatively regulated tetrameric enzyme using electrospray mass spectrometry.

This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (KD) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the allosteric mechanism in the binding of inhibitors to the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their KDs by nanoESI-MS. The KDs determined by ESI-MS correlate very well with IC50 values in solution. The Hill coefficients derived from nanoESI-MS suggest positive cooperativity. From single-point measurements we could obtain information on relative potency, stoichiometry, conformational changes, and mechanism of cooperativity. A new X-ray crystal structure of FBPase tetramer binding ligand 3 in a 4:4 stoichiometry is also reported. NanoESI-MS-based results match the current understanding of the investigated system and are in agreement with the X-ray structural data, but provide additional mechanistic insight on the ligand binding, due to the better dynamic resolution. This method offers a powerful approach for studying other proteins with allosteric binding sites, as well.

Optical properties of protonated Rhodamine 19 isomers in solution and in the gas phase.

Visible light absorption and fluorescence of three positional isomers of protonated Rhodamine 19 (o-, m- and p-R19H(+)) were studied in solution and in the gas phase. In solution, strong solvatochromic effects lead to spectral shifts between rhodamine isomers. In contrast, in the gas phase, these species were found to exhibit very similar fluorescence, while pronounced differences were observed in the absorption spectra. The o-R19H(+) was found to have the largest Stokes shift in the gas phase (around 10 nm), suggesting that an intramolecular relaxation operates in the excited electronic state for this isomer. Several mechanisms for this relaxation are proposed, such as the change of the dihedral angle between the carboxyphenyl group and the xanthene chromophore or that between the carboxylic group and the phenyl ring.

Dynamic Assembly and Disassembly of Functional β-Endorphin Amyloid Fibrils

Neuropeptides and peptide hormones are stored in the amyloid state in dense-core vesicles of secretory cells. Secreted peptides experience dramatic environmental changes in the secretory pathway, from the endoplasmic reticulum via secretory vesicles to release into the interstitial space or blood. The molecular mechanisms of amyloid formation during packing of peptides into secretory vesicles and amyloid dissociation upon release remain unknown. In the present work, we applied thioflavin T binding, tyrosine intrinsic fluorescence, fluorescence anisotropy measurements, and solid-state NMR spectroscopy to study the influence of physiologically relevant environmental factors on the assembly and disassembly of β-endorphin amyloids in vitro. We found that β-endorphin aggregation and dissociation occur in vitro on relatively short time scales, comparable to times required for protein synthesis and the rise of peptide concentration in the blood, respectively. Both assembly and disassembly of amyloids strongly depend on the presence of salts of polyprotic acids (such as phosphate and sulfate), while salts of monoprotic acids are not effective in promoting aggregation. A steep increase of the peptide aggregation rate constant upon increase of solution pH from 5.0 to 6.0 toward the isoelectric point as well as more rapid dissociation of β-endorphin amyloid fibrils at lower pH indicate the contribution of ion-specific effects into dynamics of the amyloid. Several low-molecular-weight carbohydrates exhibit the same effect on β-endorphin aggregation as phosphate. Moreover, no structural difference was detected between the phosphate- and carbohydrate-induced fibrils by solid-state NMR. In contrast, β-endorphin amyloid fibrils obtained in the presence of heparin demonstrated distinctly different behavior, which we attributed to a dramatic change of the amyloid structure. Overall, the presented results support the hypothesis that packing of peptide hormones/neuropeptides in dense-core vesicles do not necessarily require a specialized cellular machinery.

Determination of thermodynamic and kinetic properties of biomolecules by mass spectrometry.

Over the past two decades, mass spectrometry (MS) has transformed the life sciences. The advances in understanding biomolecule structure and function by MS is progressing at an accelerated pace. MS has also largely been applied to study thermodynamic and kinetic structure of biomolecules. Herein, we highlight the recent discussions about native mass spectrometry and studies about determining stable gas phase structures, hydrogen/deuterium exchange studies about reaction kinetics and determination of binding constants of biomolecules with their ligands.

Rhodamines in the gas phase

Optical spectroscopy of biological molecules in the gas phase has recently gained considerable attention, being able to provide complementary structural information in the absence of native matrix. Biomolecules can change their properties when brought into the gas phase, and so can chromophores associated with them. Understanding the photophysics of chromophore labels is central for the correct interpretation of experimental data. In this report, the structure and the optical properties of Rhodamine 19 (R19) in the gas phase were examined by a combination of Fourier-transform ion cyclotron resonance mass spectrometry and visible-light laser spectroscopy. While R19 in solution is found either in neutral (R19(n)) or protonated (R19+H(+)) forms, other structures can be generated in the gas phase, such as anions (R19-H(-)) and adducts with metal cations (R19+M(+)). Experimental evidence for the lactone structure of neutral gas-phase R19 is presented for the first time. The different properties of gas-phase compared to solution-phase R19 are discussed in view of structural analysis of labeled gas-phase biological molecules by optical spectroscopy.

Probing the mechanisms of ambient ionization by laser‐induced fluorescence spectroscopy

The ionization mechanisms of several atmospheric pressure ion sources based on desorption and ionization of samples deposited on a surface were studied. Home-built desorption electrospray ionization (DESI), laserspray ionization (LSI), and atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) sources were characterized using low-molecular-weight compounds, in particular fluorescent dyes. Detection of the desorbed and ionized species was performed by laser-induced fluorescence and ion cyclotron resonance mass spectrometry. The dependences of the signal intensities on various experimental parameters were studied. The data obtained reveals common features, such as formation of solvated species and clusters in the ionization processes, in all of the techniques considered.

Native biomolecules in the gas phase? The case of green fluorescent protein.

Green fluorescent protein (GFP) was ionized by native electrospray ionization and trapped for many seconds in high vacuum, allowing fluorescence emission to be measured as a probe of its biological function, to answer the question whether GFP exists in the native form in the gas phase or not. Although a narrow charge-state distribution, a collision cross-section very close to that expected for correctly folded GFP, and a large stability against dissociation all support a near-native gas-phase structure, no fluorescence emission was observed. The loss of the native form is attributed to the absence of residual water in the gas phase, which normally stabilizes the para-hydroxybenzylidene imidazolone chromophore of GFP.