Carborane as an Alternative Efficient Hydrophobic Tag for Protein Degradation.

Carboranes 1 and 2 were designed and synthesized for hydrophobic tag (HyT)-induced degradation of HaloTag fusion proteins. The levels of the hemagglutinin (HA)-HaloTag2-green fluorescent protein (EGFP) stably expressed in Flp-In 293 cells were significantly reduced by HyT13, HyT55, and carboranes 1 and 2, with expression levels of 49, 79, 43, and 65%, respectively, indicating that carborane is an alternative novel hydrophobic tag (HyT) for protein degradation under an intracellular environment. To clarify the mechanism of HyT-induced proteolysis, bovine serum albumin (BSA) was chosen as an extracellular protein and modified with maleimide-conjugated m-carborane (MIC). The measurement of the ζ-potentials and the lysine residue modification with fluorescein isothiocyanate (FITC) of BSA-MIC conjugates suggested that the conjugation of carborane induced the exposure of lysine residues on BSA, resulting in the degradation via ubiquitin E3 ligase-related proteasome pathways in the cell.

Structural Characterization of 6-Thioguanosine and Its Monohydrate in the Gas Phase.

Detailed structural analysis of 6-thioguanosine (6TGs) in relation to its tautomerization and sugar conformation is performed in the gas phase using UV and IR spectroscopy combined with ab initio calculations. We have observed a thiol tautomer of 6TGs with its sugar moiety in the syn conformation that is stabilized by a strong intramolecular H-bonding between O5'H of the sugar and N3 atom of the guanine moiety. This observation is consistent with previous results for guanosine (Gs) in which the corresponding enol form is solely detected. We have also identified a monohydrate of 6TGs consisting of a thiol tautomer with the water linking guanine moiety and sugar OH group. It is demonstrated that hydration behavior of 6TGs is significantly different from that of Gs as a result of a weaker H-bonding ability of the thiol group.

Gas-Phase Resonance Raman Spectroscopy Combined with IR-Laser Ablation of a Droplet Beam: Local Structural Analysis of Myoglobin.

Gas phase spectroscopy is a powerful tool for examining fundamental chemical structures and properties free from solvent molecules. We developed a gas-phase resonance Raman spectroscopy combined with IR-laser ablation of a droplet beam, which allowed us to elucidate local structures around chromophores in gas-phase proteins and DNAs. To demonstrate the potential of this approach, we applied this method to myoglobin, one of the heme proteins, and elucidated its structures in the gas phase and in aqueous solution. The experimental spectra are compared with calculated spectra of stable heme structures for the structural determination. These results show the oxidation/spin states of the Fe atom in myoglobin in the gas phase and were compared with the aqueous solution from the obtained resonant Raman spectra. The present method gives an important tool to investigate the gas-phase structure of large biomolecules.

Design of S-S bond containing maleimide-conjugated closo-dodecaborate (SSMID): identification of unique modification sites on albumin and investigation of intracellular uptake.

An S-S bond containing maleimide-conjugated closo-dodecaborate (SSMID) was synthesised for identification of albumin binding sites. Three Lys residues, Lys221, Lys413 and Lys431, were identified as SSMID modification sites in addition to Cys34 in bovin serum albumin (BSA). Fluorescent-labelled MID-BSA was found to be accumulated in the cytosol of HeLa cells.

Effective Strategy for Conformer-Selective Detection of Short-Lived Excited State Species: Application to the IR Spectroscopy of the N1H Keto Tautomer of Guanine.

The ultrafast deactivation processes in the excited state of biomolecules, such as the most stable tautomers of guanine, forbid any state-of-the-art gas phase spectroscopic studies on these species with nanosecond lasers. This drawback can be overcome by grafting a chromophore having a long-lived excited state to the molecule of interest, allowing thus a mass-selective detection by nanosecond R2PI and therefore double resonance IR/UV conformer-selective spectroscopic studies. The principle is presently demonstrated on the keto form of a modified 9-methylguanine, for which the IR/UV double resonance spectrum in the C═O stretch region, reported for the first time, provides evidence for extensive vibrational couplings within the guanine moiety. Such a successful strategy opens up a route to mass-selective IR/UV spectroscopic investigations on molecules exhibiting natural chromophores having ultrashort-lived excited states, such as DNA bases, their complexes as well as peptides containing short-lived aromatic residues.

Structural identification of uric acid and its monohydrates by IR-UV double resonance spectroscopy.

We have employed IR-UV double resonance spectroscopy to identify the tautomeric and isomeric structures of uric acid and its monohydrated clusters which are produced by the techniques of laser-desorption and supersonic-jet cooling. The IR spectrum obtained for bare uric acid exhibits four distinct NH stretching transitions assignable to those of the most stable triketo form. We have also observed the two most stable monohydrated clusters, each with uric acid in the triketo form and water bonded to either the N3H or N9H site. It is demonstrated that the R2PI spectra of these monohydrates can be separated by using the IR-purified spectroscopic method.

Gas-phase hydration of the lysozyme ion produced by infrared-laser ablation of a droplet beam studied by photodissociation and fluorescence spectroscopy.

Biomolecules function in an aqueous environment. Elucidation of the hydration structures of biomolecules is hence important to understand their functions. Here, we investigated the hydration structure of lysozyme (Lys) in the gas phase by photodissociation and fluorescence spectroscopy in combination with droplet-beam laser ablation mass spectrometry. We found that water molecules are held inside and on the surface of the Lys molecule, and the hydration structure around the tryptophan residue changes by photoexcitation. This study provides a novel method to observe the hydration structures of large biomolecules at the molecular level.

Gas-phase isolation of diethyl guanosine 5'-monophosphate and its conformational assignment.

We show that intact neutral molecules of guanosine 5'-monophosphate can be vaporized by laser desorption when its phosphate group is esterified. The UV and IR spectroscopic measurements of this nucleotide reveal the existence of a novel internal hydrogen-bonding conformation of the phosphate group and guanine moiety.

Microhydration of the guanine-guanine and guanine-cytosine base pairs.

Monohydration structures of the guanine-guanine and guanine-cytosine base pairs have been elucidated by IR-UV double resonance spectroscopy combined with ab initio calculations. The systems studied consist of the homodimer of 9-methylguanine and the heterodimer of 9-methylguanine and 1-methylcytosine in which the methyl group is introduced to mimic the presence of the sugar-phosphate backbone and to block specific tautomerization. The monohydrate of the homodimer is identified as that of the most stable symmetric structure formed by the keto tautomers of guanine, which demonstrates that the base pair structure is not influenced by the hydration. It is also shown that at least two structural isomers, one of which retains the Watson-Crick GC pair structure, contribute the monohydrated cluster of the heterodimer. Although stacked base pairs are suggested to be significantly stabilized by the addition of water, the result shows no clear indication for the presence of stacked monohydrates in either homodimer or heterodimer case.

Hydration structures of 2'-deoxyguanosine studied by IR-UV double resonance spectroscopy: comparison with guanosine.

Infrared spectra of mono- and dihydrated clusters of 2'-deoxyguanosine (2'-dGs), which are formed by laser desorption combined with supersonic jet-cooling, have been measured by the technique of IR-UV double resonance spectroscopy. The structures of these hydrates are compared with those reported for guanosine (Gs) to elucidate the importance of the 2'-hydroxy group in the hydration. It is shown that monohydrated structures observed for 2'-dGs are similar to those of Gs, indicating no significant influence by the absence of 2'-OH group. For the dihydrated cluster, two structural isomers are identified and assigned to the dihydrates with the guanine moiety is in the keto form, which are consistent with the lowest-energy structures derived from theoretical calculations. Comparison with the results for Gs suggests that the presence of 2'-OH group leads to the stabilization of specific dihydrate structures involving the sugar group.

IR-UV double resonance spectroscopy of the hydrated clusters of guanosine and 9-methylguanine: evidence for hydration structures involving the sugar group.

Infrared spectra of mono- and dihydrated clusters of guanosine (Gs) formed by laser desorption combined with supersonic jet cooling have been measured by the technique of IR-UV double resonance spectroscopy. The results are compared with those of 9-methylguanine (9MG), in which the sugar group of Gs is replaced with a methyl group, to elucidate the importance of the sugar group in the hydration structures. It is shown that the UV spectrum observed for the monohydrated cluster of Gs is composed of multiple structural isomers of larger stabilities. One of the monohydrates is identified to possess a hydration structure involving the 5'-OH group of the sugar and the amino group of the guanine moiety. The IR spectrum of the dihydrated clusters reveals that the 2'-OH group is significantly influenced by the addition of the second water, which suggests the possibility of specific dihydrate structures for Gs.

Evaporation and Subsequent Adsorption of Alcohol Molecules at Aqueous Droplet Surface Observed by Cavity-Enhanced Raman Spectroscopy.

Mass transfer toward and across liquid surfaces is important for the interpretation of various interfacial phenomena, such as evaporation, adsorption, and mass accommodation, which have been investigated by the use of various methods. These studies, however, have focused on only one of the mass-transfer processes occurring at the surface. We investigate the surface concentration of alcohol molecules at aqueous droplet surfaces on the several-millisecond time scale using cavity-enhanced droplet Raman spectroscopy. A decrease and subsequent increase of the alcohol concentration are observed in a set of measurements, which arise from an evaporation and subsequent adsorption of the alcohol molecules at the surface. This facilitates an understanding of the surface kinetics of molecules at the liquid surfaces.

Multiple hydrogen-bonding interactions of uric acid/9-methyluric acid with melamine identified by infrared spectroscopy.

Hydrogen-bonded complexes of uric acid/9-methyluric acid (UA/9MUA) with melamine (MEL) are prepared by the combined technique of laser desorption and supersonic-jet expansion, and their stable structures are investigated by infrared spectroscopy and theoretical calculations. It is shown that the 1:1 complex formed between UA and MEL is of nonplanar type, in which the two chromophore planes are significantly folded and thus allow for triple hydrogen-bonding interactions. An anomalously broad IR band is observed in the low-frequency range 2500-2800 cm(-1), which is taken as evidence for the formation of a strong hydrogen bond between one of the NH sites of UA and MEL. In the case of 9MUA, in which hydrogen bonding to the N9H site of UA is blocked by the methyl group, two planar pairs formed of 9MUA and MEL are found to coexist. The nature of the multiple hydrogen-bonding interactions in these complexes is discussed based on the natural bond orbital analysis and compared with those of the guanine-cytosine base pair. The results are expected to provide important information on the structural characterization of urinary stones developed in infants after ingesting MEL-contaminated formula.

Controlling Glycosyl Bond Conformation of Guanine Nucleosides: Stabilization of the anti Conformer in 5'-O-Ethylguanosine.

Nucleosides that consist of base and sugar moieties can adopt two main conformations, syn and anti, about the glycosidic bond. We have investigated the conformational properties of guanine nucleosides in the gas phase by using laser desorption combined with IR-UV double resonance spectroscopy. In guanosine, syn conformation is preferred as a result of internal hydrogen bonding between the 5'-OH group of the sugar and the N3 site of the guanine moiety. We have therefore employed a chemically modified nucleoside 5'-O-ethylguanosine, in which possible glycosyl bond conformations are restricted upon ethylation of the 5'-OH group. The result shows that anti conformer is stabilized by the formation of hydrogen bonding involving the 2'-OH group.