Binding Of CN Research Articles

Exogenous ligand binding kinetics of globin X.

Intrinsically hexa-coordinate vertebrate globins have been shown to carry various cellular functions including neuroprotective and antitumor properties but the precise mechanism of their action remains unclear. Here we focus on the molecular mechanism of diatomic ligand interactions with globin X (GbX) which is a hexa-coordinate globin expressed in lower vertebrate such as fish, amphibians, and reptiles. GbX was proposed to have a variety of enzymatic activities including nitrite reductase, and nitric oxide dioxygenase along with the ability to bind several exogeneous ligands such as O2, CO, and CN-. The wide array of enzymatic functions present in GbX along with its low intracellular concentration in vivo and its hexa-coordination has led to the theory that GbX may serve to scavenge reactive oxygen and nitrogen species. Another interesting feature of GbX is the presence of an internal disulfide bond between residues Cys63 and Cys141 and myristoylation of its N-terminus with an as of yet unknown role in the functional properties of GbX. In this study interactions of exogenous ligands, CO and CN- with GbXWT and GbXH90V and GbXC60A mutants were investigated by combining in vitro and in silica approaches. The results of flash photolysis and stop flow experiments demonstrate multiphasic ligand rebinding to all three constructs with the slowest ligand binding to GbXWT. Time-resolved UV-Vis measurements also revealed multiphasic binding of CN- to GbX. The structures of GbX constructs were determined using homology modeling and subsequent MD simulations and the possible ligand migration pathways were probed using Implicit Ligand Sampling.

Selective and Sensitive Fluorescence Turn-On Detection of Cyanide Ions in Water by Post Metallization of a MOF.

Owing to detrimental impact of cyanide ion (CN- ) towards the entire living system as well as its availability in drinking water, it has become very important developing potential sensory materials for the selective and sensitive recognition of CN- ions in water. In the domain of sensory materials, luminescent metal-organic frameworks (LMOFs) have been considered as a promising candidate owing to their unique host-guest interaction, where MOFs can serve as an ideal scaffold for encapsulating relevant guest molecules rendering specific functionality. In this study, a post-synthetically modified MOF (viz., CuCl2 @MOF-867) was applied to recognize cyanide (CN- ) ions in water via "turn-on" response. The bipyridyl functionalities in MOF-867 were used to perform post-synthetic metalation to infiltrate CuCl2 inside porous architecture of the MOF. Moreover, a CuCl2 @MOF-867 based probe demonstrated highly selective and sensitive aqueous phase recognition of CN- ions even in the presence of other interfering anions such as Br- , NO3- , I- , SO42- , OAc- , SCN- , NO2- , etc. The selective binding of CN- ions to the copper-metal center has led to the generation of stable Cu(CN)2 species. This phenomenon has further resulted in a fluorescence turn-on response. The aqueous phase cyanide detection by the rationally modified MOF system exhibited very low limit of detection (0.19 μM), which meets the standardized limit stated by World Health Organization (WHO) that is 1.9 μM.

Stepwise Assembly of Turn‐on Fluorescence Sensors in Multicomponent Metal–Organic Frameworks for in Vitro Cyanide Detection

AbstractThe controlled synthesis of multicomponent metal–organic frameworks (MOFs) allows for the precise placement of multiple cooperative functional groups within a framework, leading to emergent synergistic effects. Herein, we demonstrate that turn‐on fluorescence sensors can be assembled by combining a fluorophore and a recognition moiety within a complex cavity of a multicomponent MOF. An anthracene‐based fluorescent linker and a hemicyanine‐containing CN−‐responsive linker were sequentially installed into the lattice of PCN‐700. The selective binding of CN− to hemicyanine inhibited the energy transfer between the two moieties, resulting in a fluorescence turn‐on effect. Taking advantage of the high tunability of the MOF platform, the ratio between anthracene and the hemicyanine moiety could be fine‐tuned in order to maximize the sensitivity of the overall framework. The optimized MOF‐sensor had a CN−‐detection limit of 0.05 μm, which is much lower than traditional CN− fluorescent sensors (about 0.2 μm).

Stepwise Assembly of Turn-on Fluorescence Sensors in Multicomponent Metal-Organic Frameworks for in Vitro Cyanide Detection.

The controlled synthesis of multicomponent metal-organic frameworks (MOFs) allows for the precise placement of multiple cooperative functional groups within a framework, leading to emergent synergistic effects. Herein, we demonstrate that turn-on fluorescence sensors can be assembled by combining a fluorophore and a recognition moiety within a complex cavity of a multicomponent MOF. An anthracene-based fluorescent linker and a hemicyanine-containing CN- -responsive linker were sequentially installed into the lattice of PCN-700. The selective binding of CN- to hemicyanine inhibited the energy transfer between the two moieties, resulting in a fluorescence turn-on effect. Taking advantage of the high tunability of the MOF platform, the ratio between anthracene and the hemicyanine moiety could be fine-tuned in order to maximize the sensitivity of the overall framework. The optimized MOF-sensor had a CN- -detection limit of 0.05 μm, which is much lower than traditional CN- fluorescent sensors (about 0.2 μm).

Anion Sensing by Novel Triarylboranes Containing Boraanthracene: DFT Functional Assessment, Selective Interactions, and Mechanism Demonstration.

Analytical methods often involve expensive instrumentation and tedious sample pretreatment for an analyte detection. Being toxic and detrimental to human health, sensing of cyanide (CN–), fluoride (F–), chloride (Cl–), bromide (Br–), nitrate (NO3–), acetate (CH3COO–), and bisulfate (HSO4–) is performed by a boron-based molecular receptor, N,N,N,3,5-pentamethyl-4-{2-thia-9-boratricyclo[8.4.0.03,8]tetradeca-1(10),3(8),4,6,11,13-hexaen-9-yl}anili-nium (1), and the three newly designed receptors from it. Thermodynamics, electronic structure, and photophysical properties are computed by employing density functional theory (DFT) and time-dependent density functional theory (TD-DFT) to explore selective sensing of these anions and its mechanism. Free-energy changes (ΔG) and binding energies (ΔE) suggest that among these anions, only binding of CN– and F– is thermodynamically feasible with a very strong binding affinity with the receptors. Boron atoms containing positive natural charges act as the electrophilic centers to bind the anions involving a 2p–2p orbital overlap resulting in charge transfer. In the receptor–analyte complexes with CN– and F–, fluorescence is quenched due to the intramolecular charge transfer transitions (π–π* transitions in the case of the receptors lead to fluorescence), internal conversion, and associated configurational changes. Among the six tested functionals, CAM-B3LYP/6-31G(d) is found to be the most accurate one. The designed receptors are better fluorescent probes for F– and CN–, demonstrating their importance for the practical utility.

Selective Complexation of Cyanide and Fluoride Ions with Ammonium Boranes: A Theoretical Study on Sensing Mechanism Involving Intramolecular Charge Transfer and Configurational Changes.

The anion binding selectivity and the recognition mechanism of two isomeric boranes, namely, 4-[bis(2,4,6-trimethylphenyl)boranyl]-N,N,N-trimethylaniline ([p-(Mes2B)C6H4(NMe3)]+, 1, where "Mes" represents mesitylene and "Me" represents methyl) and 2-[bis(2,4,6-trimethylphenyl)boranyl]-N,N,N-trimethylaniline ([o-(Mes2B)C6H4(NMe3)]+, 2) has been investigated using density functional theory (DFT) and time dependent-density functional theory (TD-DFT) methods. Natural population analysis indicates that the central boron atoms in 1 and 2 are the most active centers for nucleophilic addition of anions. The negative magnitude of free energy changes (ΔG) reveals that out of CN-, F-, Cl-, Br-, NO3-, and HSO4- only the binding of CN- and F- with 1 and 2 is thermodynamically feasible and spontaneous. In addition, the calculated binding energies reveal that the CN- is showing lesser binding affinity than F- both with 1 and 2, while other ions, viz. NO3-, HSO4-, Br-, and Cl-, either do not bind at all or show very insignificant binding energy. The first excited states (S1) of 1 and 2 are shown to be the local excited states with π → σ* transition by frontier molecular orbital analysis, whereas fourth excited states (S4) of 4-[bis(2,4,6-trimethylphenyl)boranyl]-N,N,N-trimethylaniline cyanide ([p-(Mes2B)C6H4(NMe3)] CN, 1CN, the cyano form of 1) and 4-[bis(2,4,6-trimethylphenyl)boranyl]-N,N,N-trimethylaniline fluoride ([p-(Mes2B)C6H4(NMe3)] F, 1F, the fluoro form of 1) and fifth excited state (S5) of 2-[bis(2,4,6-trimethylphenyl)boranyl]-N,N,N-trimethylaniline fluoride ([o-(Mes2B)C6H4(NMe3)] F, 2F, the fluoro form of 2) are charge separation states that are found to be responsible for the intramolecular charge transfer (ICT) process. The synergistic effect of ICT and partial configuration changes induce fluorescence quenching in 1CN, 1F, and 2F after a significant internal conversion (IC) from S4 and S5 to S1.

Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the HMR, HML, telomere and rDNA regions are silenced. Silencing at the rDNA region requires Sir2, and silencing at the HMR, HML and telomere regions requires binding of a protein complex, consisting of Sir2, Sir3 and Sir4, that mediates repression of gene expression. Here, several novel Sir3 binding domains, termed CN domains (Chromosomal Novel Sir3 binding region), were identified using chromatin immunoprecipitation (ChIP) on chip analysis of S. cerevisiae chromosomes. Furthermore, analysis of G1-arrested cells demonstrated that Sir3 binding was elevated in G1-arrested cells compared with logarithmically growing asynchronous cells, and that Sir3 binding varied with the cell cycle. In addition to 14 CN regions identified from analysis of logarithmically growing asynchronous cells (CN1-14), 11 CN regions were identified from G1-arrested cells (CN15-25). Gene expression at some CN regions did not differ between WT and sir3Δ strains. Sir3 at conventional heterochromatic regions is thought to be recruited to chromosomes by Sir2 and Sir4; however, in this study, Sir3 binding occurred at some CN regions even in sir2Δ and sir4Δ backgrounds. Taken together, our results suggest that Sir3 exhibits novel binding parameters and gene regulatory functions at the CN binding domains.

Modulation of Axial-Ligand Binding and Releasing Processes onto the Triazole-Bearing Nickel(II) Picket-Fence Porphyrins: Steric Repulsion versus Hydrogen-Bonding Effects.

N-(p-Methoxycarbonylbenzyl) triazole (BTz) substituents have been introduced to Ni(II) porphyrins (NiPs), in which their modulated axial-coordination processes have been investigated. For this study, the two types of ligands, neutral pyridine versus anionic cyanide, were employed to investigate an effect of BTz substituents. The unique microenvironments given by the BTz substituents provided two different effects on the axial-coordination processes of NiPs on the ground and excited states: (1) steric shielding and (2) donation of hydrogen-bonding sites. The steric shielding diminished the binding affinity of pyridine, while the cooperation of hydrogen bonds extraordinarily strengthened the binding affinity of CN(-). Interestingly, it was observed that the binding of CN(-) with the supporting of BTz substituents accompanied nonplanar distortion of NiPs. Such conformational change perturbed the electronic structure of NiPs, which gave rise to the modulation of coordination processes of NiPs in the excited state. As a consequence, photoinudced ligand binding and releasing processes of four- and six-coordinated NiPs were changed into the dominant photoinduced ligand releasing process.

Enhanced Solubilization of Carbon Nanotubes in Aqueous Suspensions of Anionic–Nonionic Surfactant Mixtures

Single-walled carbon nanotubes (SWCNTs) suspensions in aqueous solutions of sodium dodecyl sulfate (SDS) and saturated fatty acids (Cn) are studied. The quality of the dispersions is analyzed by photoluminescence spectroscopy (PL) as a function of the Cn chain length. Resonant Raman scattering (RRS) measurements and molecular dynamics (MD) simulations were also carried out in order to study the effect of the surrounding medium on SWCNTs properties in suspensions. Both PL and RRS data indicate an increased individualization of SWCNTs in the dispersions for Cn’s having an alkyl chain longer than SDS. MD simulations showed the formation of mixed Cn-SDS aggregates around a nanotube in water and a Cn binding energy to the nanotube wall that increases linearly with chain length. The enhanced solubilization of SWCNTs is thus interpreted in terms of the reduced electrostatic repulsion within the surfactant aggregates and the increased binding energy to the nanotube wall. Powders prepared by the evaporation of dis...

Investigations of Two Bidirectional Carbon Monoxide Dehydrogenases from Carboxydothermus hydrogenoformans by Protein Film Electrochemistry

Carbon monoxide dehydrogenases (CODHs) catalyse the reversible conversion between CO and CO2 . Several small molecules or ions are inhibitors and probes for different oxidation states of the unusual [Ni-4 Fe-4 S] cluster that forms the active site. The actions of these small probes on two enzymes-CODH ICh and CODH IICh -produced by Carboxydothermus hydrogenoformans have been studied by protein film voltammetry to compare their behaviour and to establish general characteristics. Whereas CODH ICh is, so far, the better studied of the two isozymes in terms of its electrocatalytic properties, it is CODH IICh that has been characterised by X-ray crystallography. The two isozymes, which share 58.3% sequence identity and 73.9% sequence similarity, show similar patterns of behaviour with regard to selective inhibition of CO2 reduction by CO (product) and cyanate, potent and selective inhibition of CO oxidation by cyanide, and the action of sulfide, which promotes oxidative inactivation of the enzyme. For both isozymes, rates of binding of substrate analogues CN(-) (for CO) and NCO(-) (for CO2 ) are orders of magnitude lower than turnover, a feature that is clearly revealed through hysteresis of cyclic voltammetry. Inhibition by CN(-) and CO is much stronger for CODH IICh than for CODH ICh, a property that has relevance for applying these enzymes as model catalysts in solar-driven CO2 reduction.

The effect of KCN passivation on IR spectra of a-Si based structures

From the analysis of IR spectra of double layer structures after KCN passivation the presence of four absorption bands at 720–810cm−1 (assigned as SiC bond), 870–1060cm−1 (assigned as SiHn and SiO bonds), 1930–2200cm−1 (assigned as SiHn bonds) and absorption band around 2360cm−1 (assigned as SiNCO bond) was found. As IR spectra between 1400 and 1600cm−1 (assigned as CH, NH2) and 3200–3300cm−1 (assigned as CH and NH) under influence of KCN passivation did not change we suggest that formation of HCN, CH and NH was eliminated (polymeric HCN). From analysis of absorption bands assigned as SiHn, CN and SiNC (1950–2200cm−1) and absorption bands assigned as SiO bond (1000–1200cm−1) it is evident the different influence of passivation on samples <n mc-Si/i-a-Si/n high doped c-Si> and <i-a-Si/p-a-SiC/n doped c-Si>. In the spectra typical features of OH group, hydrogen bridge bonding and SiSiOCN formation were observed. From these results can be drawn the binding of CN to Si through oxygen atoms.

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

The superfamily of cyclic nucleotide (cN) phosphodiesterases (PDEs) is comprised of 11 families of enzymes. PDEs break down cAMP and/or cGMP and are major determinants of cellular cN levels and, consequently, the actions of cN-signaling pathways. PDEs exhibit a range of catalytic efficiencies for breakdown of cAMP and/or cGMP and are regulated by myriad processes including phosphorylation, cN binding to allosteric GAF domains, changes in expression levels, interaction with regulatory or anchoring proteins, and reversible translocation among subcellular compartments. Selective PDE inhibitors are currently in clinical use for treatment of erectile dysfunction, pulmonary hypertension, intermittent claudication, and chronic pulmonary obstructive disease; many new inhibitors are being developed for treatment of these and other maladies. Recently reported x-ray crystallographic structures have defined features that provide for specificity for cAMP or cGMP in PDE catalytic sites or their GAF domains, as well as mechanisms involved in catalysis, oligomerization, autoinhibition, and interactions with inhibitors. In addition, major advances have been made in understanding the physiological impact and the biochemical basis for selective localization and/or recruitment of specific PDE isoenzymes to particular subcellular compartments. The many recent advances in understanding PDE structures, functions, and physiological actions are discussed in this review.

Probing the influence of steric bulk on anion binding by triarylboranes: comparative studies of FcB(o-Tol)2, FcB(o-Xyl)2 and FcBMes2

Steric crowding brought about on pyramidalization at boron has been predicted computationally to be of central importance to the strength and selectivity of anion binding by triarylboranes. The role of steric factors in systems containing a ferrocenyl reporter unit has been systematically probed in the current study by comparison of the F(-)/CN(-) binding properties of FcB(o-Tol)(2) (1, o-Tol = C(6)H(4)Me-2), FcB(o-Xyl)(2) (2, o-Xyl = C(6)H(3)Me(2)-2,6) and FcBMes(2) (3, Mes = C(6)H(2)Me(3)-2,4,6)), both in solution and in the solid state. Somewhat surprisingly, the inclusion of an extra ortho-methyl aryl substituent (e.g. for 2/3vs.1) is found to have a relatively small effect on the binding affinities of these boranes (e.g. log(10)K(CN) = 5.94(0.02), 4.73(0.01), 5.56(0.02), for 1, 2 and 3 respectively). Consistent with this observation, the degree of pyramidalization at boron determined for the cyanide adducts [1·CN](-), [2·CN](-) and [3·CN](-) in the solid state is also found to be essentially invariant (∠C(aryl)-B-C(aryl) = 338, 337, 337°, respectively), as are the B-CN and mean B-C(aryl) distances. In the solid state at least, it is apparent that the adverse steric effects potentially brought about by increasing ortho substitution are mitigated by a greater degree of synchronous rotation of the aryl substituents about the B-C(aryl) bonds. Thus a mean inter-plane angle of 71° is observed for [1·CN](-) while the corresponding values for [2·CN](-) and [3·CN](-) are 78° and 79°.

Density Functional Theory Calculations on the Mononuclear Non-Heme Iron Active Site of Hmd Hydrogenase: Role of the Internal Ligands in Tuning External Ligand Binding and Driving H2 Heterolysis

DFT calculations on active-site models of the non-heme Fe site of Hmd hydrogenase are reported. Binding of several biologically relevant ligands (e.g., CN(-), CO, H(-), H(2), and O(2)) to the active site of Hmd was investigated using a method that reproduced the geometric and vibrational properties of the resting site. The results indicate that this neutral ferrous active site has higher affinity toward anionic ligands (e.g., H(-) and CN(-)) than π-acidic ligands (e.g., CO and O(2)). Natural population analysis and molecular orbital analysis revealed that this is due to extensive delocalization of electron density into the low-lying unoccupied orbitals of the CO, acyl, and pyridinol ligands present in the active site. In addition to normal d-π back-bonding, metal 3d orbital-mediated charge transfer from occupied ligand orbitals to the unoccupied orbitals of the internal ligands was observed. This charge transfer leads to systematic variations in the experimentally observed C-O stretching frequencies. Protonation of the thiolate ligand present in the active site significantly enhances these anion ligand binding affinities. In fact, the calculated vibrational frequencies indicate that CN(-) binding is possibly associated with protonation of the thiolate ligand. The high affinity for binding of the anionic H(-) ligand (where 81% of the electron density of H(-) is delocalized into the active site) is calculated to play a dominating role in the H-H bond heterolysis step during catalysis. The binding energies of these ligands relative to the substrate, H(2), highlight the importance of a proposed structural reorganization during catalysis.

Engineering of the Heme Pocket of an H-NOX Domain for Direct Cyanide Detection and Quantification

A new cyanide sensing system, the Heme-Nitric oxide and/or OXygen binding domain (H-NOX domain) from Thermoanaerobacter tengcongensis (Tt H-NOX), has been investigated. With straightforward absorbance-based detection, we have achieved a cyanide detection limit of 0.5 microM (approximately 10 ppb) with an upper detection range that is adjustable with protein concentration. We find a linear correlation of multiple spectroscopic features with cyanide concentration. These spectroscopic features include the Soret band maximum and absorbance changes in both the Soret and alpha/beta band regions of the spectrum. Multiple probes for cyanide detection makes sensing with Tt H-NOX unique compared to other cyanide sensing methods. Furthermore, using site-directed mutagenesis, we have rationally engineered the heme pocket of Tt H-NOX to improve its cyanide sensing properties. Using a mutant that alters the heme structure of Tt H-NOX (P115A) we were able to introduce colorimetric detection of cyanide. Through substituting phenylalanine 78 with a smaller (valine, F78V) or a larger residue (tyrosine, F78Y), we demonstrate a correlation with distal pocket steric crowding and affinity for cyanide. In particular, F78V Tt H-NOX shows a significant increase in CN(-) binding affinity and selectivity. Thus, we demonstrate the ability to fine-tune the affinity and specificity of Tt H-NOX for cyanide, suggesting that Tt H-NOX can be readily tailored into a practical cyanide sensor.

Inhibition of the Calcineurin-NFAT Signalling Cascade in the Treatment of Heart Failure

Calcineurin (Cn), a serine/threonine phosphatase, plays a crucial role in the development of myocardial hypertrophy. Cn is a cytosolic phosphatase which dephosphorylates various target molecules, e.g. the transcriptional factor nuclear factor of activated T cells (NFAT), thereby enabling its nuclear translocation. Recently, it was demonstrated that not only NFAT, but also Cn is translocated into the nucleus. The nuclear coexistence of Cn and NFAT is important for the full transcriptional activity of the Cn-NFAT signalling cascade. Once Cn and NFAT have entered the nucleus of cardiomyocytes, the transcription of genes characteristic for myocardial hypertrophy (e.g. BNP, ANP) is initiated. The nuclear localization sequence (NLS), a region spanning amino acids 172-183 of calcineurin Abeta (CnAbeta) is essential for recognition and shuttling of Cn into the nucleus by importinbeta (1). A synthetic import blocking peptide (IBP) that mimics the NLS of Cn was tested recently. The NLS analogon IBP saturates the Cn binding site of importinbeta(1) thereby preventing binding of Cn and importin. This inhibits the translocation of Cn into the nucleus. Inhibiting the Cn/importin interaction with competing synthetic peptides is one of several new approaches to prevent the development of myocardial hypertrophy. Several patents have also been filed on molecules related to inhibition of Cn-NFAT signalling.

Novel calcineurin interacting protein-2: the functional characterization of CNP-2 in Caenorhabditis elegans

Calcineurin (Cn) is a serine/threonine phosphatase implicated in a wide variety of biological responses. To identify proteins that mediate Cn signaling pathway effects, we used yeast two-hybrid assays to screen for Cn interacting proteins, discovering a protein encoded by the gene, cnp-2 (Y46G5A.10). Utilizing serially deleted forms of Cn as baits, we demonstrated that the catalytic domain of Cn (TAX-6) binds with CNP-2, and this physical interaction was able to be reconstituted in vitro, supporting our yeast two-hybrid results. cnp-2 is a nematode-specific novel gene found in C. elegans as well as its closest relative, C. briggsae. CNP-2 was strongly expressed in the intestine of C. elegans. To study the function of cnp-2, we performed cnp-2 RNAi knock-down and characterized phenotypes associated with Cn mutants. However, no gross defects were revealed in these RNAi experiments. CNP-2 was proven to be a Cn binding protein; however, its role remains to be elucidated.

Protein Conformation Changes of HemAT-Bs upon Ligand Binding Probed by Ultraviolet Resonance Raman Spectroscopy

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector (O2) from other gas molecules (CO and NO), although all of them bind to a heme. To monitor the conformational changes in the protein moiety upon binding of different ligands, we have investigated ultraviolet resonance Raman (UVRR) spectra of the ligand-free and O2-, CO-, and NO-bound forms of full-length HemAT-Bs and several mutants (Y70F, H86A, T95A, and Y133F) and found that Tyr70 in the heme distal side and Tyr133 and Trp132 from the G-helix in the heme proximal side undergo environmental changes upon ligand binding. In addition, the UVRR results confirmed our previous model, which suggested that Thr95 forms a hydrogen bond with heme-bound O2, but Tyr70 does not. It is deduced from this study that hydrogen bonds between Thr95 and heme-bound O2 and between His86 and heme 6-propionate communicate the heme structural changes to the protein moiety upon O2 binding but not upon CO and NO binding. Accordingly, the present UVRR results suggest that O2 binding to heme causes displacement of the G-helix, which would be important for transduction of the conformational changes from the sensor domain to the signaling domain.

Ligand-induced monomerization of Allochromatium vinosum cytochrome c′ studied using native mass spectrometry and fluorescence resonance energy transfer

Cytochrome c' from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN(-) to the iron of its covalently attached heme group. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c' and even showed the presence of a CO-bound monomer. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. Comparison of ligand binding kinetics as observed with UV-vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization.

Density Functional Theory Study of Acetonitrile -Water Clusters: Structures and Infrared Frequency Shifts

We present calculations for the acetonitrile - water clusters to examine the nature of interactions in the mixed clusters. We calculate conformers of various composition, either of σ -type (-OH and -CN binding linearly) or π -type (-OH and -CN interacting perpendicularly) structures for the acetonitrile - water clusters. We predict that the IR frequency of the proton-accepting C≡N stretching mode red-shifts in the σ -type clusters and blueshifts in π -type conformers, whereas the proton-donating ?OH stretching frequency red-shifts in all cases. We find that this intriguing pattern also applies to the acetonitrile - water clusters of various molar ratio.