Ligand binding properties of myoglobin reconstituted with iron porphycene: unusual O2 binding selectivity against CO binding

Sperm whale myoglobin, an oxygen storage hemoprotein, was successfully reconstituted with the iron porphycene having two propionates, 2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis(carboxyethyl)porphycenatoiron. The physicochemical properties and ligand bindings of the reconstituted myoglobin were investigated. The ferric reconstituted myoglobin shows the remarkable stability against acid denaturation and only a low-spin characteristic in its EPR spectrum. The Fe(III)/Fe(II) redox potential (-190 mV vs NHE) determined by the spectroelectrochemical measurements was much lower than that of the wild-type. These results can be attributed to the strong coordination of His93 to the porphycene iron, which is induced by the nature of the porphycene ring symmetry. The O2 affinity of the ferrous reconstituted myoglobin is 2600-fold higher than that of the wild-type, mainly due to the decrease in the O2 dissociation rate, whereas the CO affinity is not so significantly enhanced. As a result, the O2 affinity of the reconstituted myoglobin exceeds its CO affinity (M' = K(CO)/K(O2) < 1). The ligand binding studies on H64A mutants support the fact that the slow O2 dissociation of the reconstituted myoglobin is primarily caused by the stabilization of the Fe-O2 sigma-bonding. The IR spectra for the carbon monoxide (CO) complex of the reconstituted myoglobin suggest several structural and/or electrostatic conformations of the Fe-C-O bond, but this is not directly correlated with the CO dissociation rate. The high O2 affinity and the unique characteristics of the myoglobin with the iron porphycene indicate that reconstitution with a synthesized heme is a useful method not only to understand the physiological function of myoglobin but also to create a tailor-made function on the protein.     

Preparation and O2 binding study of myoglobin having a cobalt porphycene

Sperm whale myoglobin, an oxygen-storage hemoprotein, was reconstituted with 2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis(carboxyethyl)porphycenatocobalt(II) in order to investigate the reactivities of a cobalt porphycene in a protein matrix. Similar to the previously reported finding for the myoglobin with the iron porphycene, the reconstituted myoglobin with the cobalt porphycene was also found to have an O2 affinity 2 orders of magnitude greater than that of the myoglobin possessing cobalt protoporphyrin IX. The EPR spectra of the deoxy and oxy myoglobins having the cobalt porphycene at 77 K also have features similar to those of the myoglobin with cobalt protoporphyrin IX. These spectra suggest that the porphycene cobalt in the deoxy form is coordinated by one nitrogenous ligand postulated to be the imidazole ring of His93, and that the bond configuration of CoII-O2 is regarded as the CoIII-Omicron2*- species.     

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

Coldspray ionization (CSI) mass spectrometry, a variant of electrospray ionization (ESI) operating at low temperature (20 to −80°C), has been used to characterize protein conformation and noncovalent complexes. A comparison of CSI and ESI was presented for the investigation of the equilibrium acid-induced unfolding of cytochrome c, ubiquitin, myoglobin, and cyclophilin A (CypA) over a wide range of pH values in aqueous solutions. CSI and nanoelectrospray ionization (nanoESI) were also compared in their performance to characterize the conformational changes of cytochrome c and myoglobin. Significant differences were observed, with narrower charged-state distribution and a shift to lower charge state in the CSI mass spectra compared with those in ESI and nanoESI mass spectra. The results suggest that CSI is more prone to preserving folded protein conformations in solution than the ESI and nanoESI methods. Moreover, the CSI-MS data are comparable with those obtained by other established biophysical methods, which are generally acknowledged to be the suitable techniques for monitoring protein conformation in solution. Noncovalent complexes of holomyoglobin and the protein-ligand complex between CypA and cyclosporin A (CsA) were also investigated at a neutral pH using the CSI-MS method. The results of this study suggest the ability of CSI-MS in retaining of protein conformation and noncovalent interactions in solution and probing subtle protein conformational changes. Additionally, the CSI-MS method is capable of analyzing quantitatively equilibrium unfolding transitions of proteins. CSI-MS may become one of the promising techniques for investigating protein conformation and noncovalent protein-ligand interactions in solution.     

Genetic engineering of the heme pocket in human serum albumin: modulation of O2 binding of iron protoporphyrin IX by variation of distal amino acids

Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin (rHSA) generates an artificial hemoprotein, which can bind O2 in much the same way as hemoglobin (Hb). We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O2 reversibly: (i) Ile-142-->His, which is axially coordinated to the central Fe2+ ion of the heme, and (ii) Tyr-161-->Phe or Leu, which makes the sixth coordinate position available for ligand interactions [I142H/Y161F (HF) or I142H/Y161L (HL)]. Here we describe additional new mutations designed to manipulate the architecture of the heme pocket in rHSA-heme complexes by specifically altering distal amino acids. We show that introduction of a third mutation on the distal side of the heme (at position Leu-185, Leu-182, or Arg-186) can modulate the O2 binding equilibrium. The coordination structures and ligand (O2 and CO) binding properties of nine rHSA(triple mutant)-heme complexes have been physicochemically and kinetically characterized. Several substitutions were severely detrimental to O2 binding: for example, Gln-185, His-185, and His-182 all generated a weak six-coordinate heme, while the rHSA(HF/R186H)-heme complex possessed a typical bis-histidyl hemochrome that was immediately autoxidized by O2. In marked contrast, HSA(HL/L185N)-heme showed very high O2 binding affinity (P1/2O2 1 Torr, 22 degrees C), which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb. Introduction of Asn at position 185 enhances O2 binding primarily by reducing the O2 dissociation rate constant. Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment, yielded a complex with reduced O2 binding affinity (P1/2O2 9-10 Torr, 22 degrees C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport.     

Differences between protein dynamics of hemoglobin upon dissociation of oxygen and carbon monoxide

Protein dynamics of human adult hemoglobin (HbA) upon ligand photolysis of oxygen (O(2)) and carbon monoxide (CO) was investigated using time-resolved resonance Raman (TR(3)) spectroscopy. The TR(3) spectra of the both photoproducts at 1-ns delay differed from that of the equilibrium deligated form (deoxy form) in the frequencies of the iron-histidine stretching [ν(Fe-His)] and methine wagging (γ(7)) modes, and the band intensity of pyrrole stretching and substituent bending (ν(8)) modes. Spectral changes of the O(2) photoproduct in the submicrosecond region were faster than those of the CO photoproduct, indicating that the structural dynamics following the photodissociation is ligand dependent for HbA. In contrast, no ligand dependence of the dynamics was observed for myoglobin, which has a structure similar to that of the subunit of HbA. The structural dynamics and relevance to the functionality of HbA also are discussed.     

Conformational studies on (17alpha,20Z)-21-(X-Phenyl)-19-norpregna-1, 3,5(10),20-tetraene-3,17beta-diols using 1D and 2D NMR spectroscopy and GIAO calculations of (13)C shieldings

Differences in agonist responses of the novel estrogen receptor ligands (17alpha,20Z)-(p-methoxyphenyl)vinyl estradiol (1), (17alpha, 20Z)-(o-alpha,alpha,alpha-trifluoromethylphenyl)vinyl estradiol (2), and (17alpha,20Z)-(o-hydroxymethylphenyl)vinyl estradiol (3) led us to investigate their solution conformation. In competitive binding assay studies, we observed that several phenyl-substituted (17alpha, 20E/Z)-(X-phenyl)vinyl estradiols exhibited significant estrogen receptor binding, but with variation (RBA (1) = 20; RBA (2) = 23; RBA (3) = 140 where estradiol RBA = 100) depending on the phenyl substitution pattern. Because the 17alpha-phenylvinyl substituent interacts with the key helix-12 of the ligand binding domain, we considered that differences in the preferred conformation of 1-3 could account for their varying binding affinity. 2D NMR experiments at 500 MHz allowed the complete assignment of the (13)C and (1)H spectra of 1-3. The conformations of these compounds in solution were established by 2D and 1D NOESY spectroscopy. A statistical approach of evaluating contributing conformers of 1-3 from predicted (13)C shifts correlated quite well with the NOE data. The 17alpha substituents of 1 and 2 exist in similar conformational equilibria with some differences in relative populations of conformers. In contrast, the 17alpha substituent of 3 exists in a different conformational equilibrium. The similarity in solution conformations of 1 and 2 suggests they occupy a similar receptor volume, consistent with similar RBA values of 20 and 23. Conversely, the different conformational equilibria of 3 may contribute to the significant binding affinity (RBA = 140) of this ligand.     

Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (Ω) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra- and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding.     

High-pressure ammonia adsorption and dissociation on clean fe(111) and oxygen-precovered fe(111) studied by sum frequency generation vibrational spectroscopy

The adsorption of gases N2, H2, O2, and NH3 that play a role in ammonia synthesis have been studied on the Fe(111) crystal surface by Sum Frequency Generation (SFG) vibrational spectroscopy using an integrated ultrahigh vacuum/high-pressure system. SFG spectra are presented for the dissociation intermediates, NH2 ( approximately 3325 cm-1) and NH ( approximately 3235 cm-1) under high pressure of ammonia (200 Torr) on the clean Fe(111) surface. Addition of 0.5 Torr of oxygen to 200 Torr of ammonia does not significantly change the bonding of dissociation intermediates to the surface. However, it leads to a phase change of nearly 180 degrees between the resonant and nonresonant second-order nonlinear susceptibility of the surface, demonstrated as a reversal of the SFG spectral features. Heating the surface in the presence of 200 Torr of ammonia and 0.5 Torr of oxygen reduces the oxygen coverage, which can be seen from the SFG spectra as another relative phase change of 180 degrees . The reduction of the oxide is also supported by Auger electron spectroscopy. The result suggests that the phase change of the spectral features could serve as a sensitive indicator of the chemical environment of the adsorbates. Clean Fe(111) is found to have a large SFG nonresonant signal. The magnitude of the nonresonant signal was dependent on the adsorption species; O2 and N2 decrease, while H2 and NH3 increase the SFG nonresonant signal. The change in nonresonant signal is correlated to the change in work function for Fe(111) upon adsorption. Adsorption-induced changes in the SFG nonresonant signal was used as an indicator of surface conditions and to monitor surface reactions.     

Localized changes in the structural stability of myoglobin upon adsorption onto silica particles,as studied with hydrogen/deuterium exchange mass spectrometry

A new method is presented for monitoring the conformational stability of various parts of a protein that is physically adsorbed onto nanometer-sized silica particles. The method employs hydrogen/deuterium (H/D) exchange of amide hydrogens, a process that is extremely sensitive to structural features of proteins. The resulting mass increase is analyzed with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Higher structural specificity is obtained by enzymatically cleaving the adsorbed proteins prior to mass spectrometric analysis. The mass increases of four peptic fragments of myoglobin are followed as a function of the H/D exchange time. The four peptic fragments cover 90% of the myoglobin structure. Two of the peptic fragments, located in the middle of the myoglobin sequence and close to the heme group, do not show any adsorption-induced changes in their structural stability, whereas the more stable C- and N-terminal fragments are destabilized. Interestingly, for the N-terminal fragment, comprising residues 1-29, two distinct and equally large conformational populations are observed. One of these populations has a stability similar to that in solution (-23 kJ/mol), whereas the other population is highly destabilized upon adsorption (-11 kJ/mol).     

Surface stress changes induced by the conformational change of proteins

A potential binding assay based on conformational-change-induced micromechanical motion is described. Calmodulin was used to modify a microcantilever (MCL) by a self-assembled layer-by-layer approach. The results showed that the modified MCL bent when the proteins changed their conformation upon binding with Ca2+. The cantilever deflection amplitudes were different under different ionic strengths, indicating different degrees of conformational change of the proteins in these conditions. On the contrary, cantilevers modified by proteins, such as hemoglobin and myoglobin, that do not change conformations upon binding with analytes do not cause the cantilever deflection. These results suggest that the conformational changes of proteins may be used to develop cantilever biosensors, and the MCL system has potential for use in label-free, protein-analyte screening applications.     

Conformational analysis of oligosaccharides in solution

A complete understanding of the role of carbohydrates in biological systems is to a large extent dependent on the information available about the equilibrium mixture and about the preferred conformation of the carbohydrate molecules in solution. The conformational analysis offers a tool which can determine all possible conformations which influence the solution behavior of carbohydrates. This paper attempts to survey the progress in the theoretical conformational analysis of saccharides in solution. The conformational analysis will be discussed in detail both with respect to the strategy for the investigation of conformational properties but also with regard to the quality of the method used for calculations of the energy of the isolated molecule and free energy of solvation. Finally, examples will be given to illustrate how the methods of conformational analysis can be used to estimate the solution behavior of cyclic model compounds of carbohydrates 2-methoxytetrahydropyran, monosaccharide D -glucopyranose, and two disaccharides; β-maltose and β-cellobiose.     

Negatively charged sol-gel column with stable electroosmotic flow for online preconcentration of zwitterionic biomolecules in capillary electromigration separations

A negatively charged sol-gel coating was developed for on-line preconcentration of zwitterionic biomolecules in capillary electrophoresis (CE), using asparagine and myoglobin as representative zwitterionic bioanalytes. The sol-gel coating was created by using a solution containing three precursors: mercaptopropyltrimethoxysilane (MPTMS), tetramethoxysilane (TMOS), and n-octadecyltriethoxysilane (C18-TEOS). The resulting sol-gel coating contained chemically bonded mercaptopropyl functional groups that were further oxidized by hydrogen peroxide to the corresponding sulfonic acid moieties. Such a surface-bonded sol-gel coating can carry a negative charge over a wide range of pH due to the presence of deprotonated sulfonic acid groups. Under favorable pH conditions, the negatively charged sol-gel coating can facilitate the extraction of positively charged analytes from a zwitterionic sample through electrostatic interaction. This principle was employed to extract myoglobin and asparagine by passing aqueous samples of these zwitterionic analytes through a negatively charged sol-gel column. The extracted analytes were then desorbed and focused via local pH change and stacking. The local pH change was accomplished by passing a buffer solution with a pH above the solute p/ value, while a dynamic pH junction between the sample solution and the background electrolyte was utilized to facilitate solute focusing. The sorption/desorption phenomena could, perhaps, also be explained on the basis of ion-exchange and local pH junction effects. On-line preconcentration and analysis results obtained on sulfonated sol-gel columns were compared with those obtained on an uncoated fused silica capillary of identical dimensions using conventional sample injections. Using UV detection, the presented sample preconcentration technique provided a sensitivity enhancement factor (SEF) on the order of 3 x 10(3) for myoglobin, and 7 x 10(3) for asparagine.     

Thermal dissociation of molten KHSO4: temperature dependence of Raman spectra and thermodynamics

Raman spectroscopy is used to study the thermal dissociation of molten KHSO4 at temperatures of 240-450 degrees C under static equilibrium conditions. Raman spectra obtained at 10 different temperatures for the molten phase and for the vapors thereof exhibit vibrational wavenumbers and relative band intensities inferring the occurrence of the temperature-dependent dissociation equilibrium 2HSO4(-)(l) <--> S2O7(2-)(l) + H2O(g). The Raman data are adequate for determining the partial pressures of H2O in the gas phase above the molten mixtures. A formalism for correlating relative Raman band intensities with the stoichiometric coefficients, the equilibrium constant, and the thermodynamics of the reaction equilibrium is derived. The method is used along with the temperature-dependent features of the Raman spectra to show that the studied equilibrium 2HSO4(-)(l) <--> S 2O7(2-)(l) + H2O(g) is the only process taking place to a significant extent in the temperature range of the investigation and for determining its enthalpy to be DeltaH degrees=64.9+/-2.9 kJ mol(-1). The importance of these findings for the understanding of the performance of the industrially important sulfuric acid catalyst under "wet" conditions is briefly addressed.     

Vinylic selenides

The ¹H, ¹³C and ²⁹Si NMR spectra of 1,3-dioxa-6-aza-2-silacyclooctanes (I) containing a transannular N → Si bond have been studied at different temperatures in solution. The sterically less crowded equatorial location of the Si substituent at the trigonal bipyramid (TBP) corresponds to the energetically unfavourable axial position in the eight-membered heterocycle (C₈). For this reason, the preferred orientation of the Si substituent and the conformational equilibrium of C₈ strongly depend on the energy of the N → Si bond. The C₈ of I in solution has the boat-chair (BC) and/or chair-chair (CC) conformations. The weakening of the N → Si bond leads to an increase in dihedral angle ?_ON and shifts the conformational equilibrium of C₈ to the CC form. The exchange between the Si substituent positions at the TBP, observed at low temperatures proceeds through the inversion of C₈ and requires dissociation of the N → S bond.     

Investigation of the influence of intramolecular electrostatic interactions on the conformational equilibrium in pyrimidine nucleosides by the PMR method

The influence of solvents on the PMR spectra of uridine and cytidine has been studied. Because of intramolecular electrostatic interactions (IEIs) between the 2-keto oxygen and the freely rotating 2-hydroxyl, the position of the conformational equilibrium in the pyrimidine nucleosides but not in purine and deoxy nucleosides, depends substantially on the dielectric constant of the solvent and the size of the partial negative charge on the 2-keto oxygen of the base. It has been shown that an increase in the IEI leads to an increase in the 3′-endo (N) population of the ribose ring and to an increased influence of the temperature on the state of the conformational equilibrium.     

Unusual ligand discrimination by a myoglobin reconstituted with a hydrophobic domain-linked heme

New, reconstituted horse heart myoglobins possessing a hydrophobic domain at the terminal of the two heme propionate side chains were constructed. The O2 and CO bindings for the reconstituted deoxymyoglobins were examined in detail by laser flash photolysis and stopped-flow rapid mixing techniques. The artificially created domain worked as a barrier against exogenous ligand penetration into the heme pocket, whereas the bound O2 was stabilized in the reconstituted myoglobin as well as in the native one. In contrast, the CO dissociation rate for the reconstituted myoglobin increased by 20-fold compared to the native protein, suggesting that the incorporation of the hydrophobic domain onto the heme pocket perturbs the distal-site structure of the reconstituted myoglobin. As a result, the substantial ligand selectivity for the reconstituted myoglobin significantly increases in favor of O2 over CO with the M' value (= KCO/KO2) of 0.88, whereas, to the best of our knowledge, there is no myoglobin mutant in which the O2 affinity exceeds the CO one. The present work concludes that the O2 selectivity of myoglobin over CO is markedly improved by chemically modifying the heme propionates without any mutation of the amino acid residues in the distal site.     

1H NMR spectra and conformational analysis of some 1,4-dithiepan-6-ones

¹H NMR Parameters are reported for five 1,4-dithiepan-6-ones. 1,4-Dithiepan-6-one 1-oxide exists in solution as an equilibrium involving two different twist-chair conformations, which contrasts with its conformational behaviour in the solid state. Twist-chair conformers are also adopted by other members of the series, the favoured form varying with ring substitution. The S?O bond in 1,4-dithiepan-6-one 1-oxide and in its 5,5-dimethyl analogue exhibit a preference for the pseudoaxial site.     

25℃时三元体系SrCl2-CaCl2-H2O相平衡研究

采用等温溶解平衡法,研究了三元体系SrCl2-CaCl2-H2O 25℃相平衡,同时测定了平衡溶液的物化性质(密度和折光率)。平衡液相所对应的平衡固相由湿渣法、偏光显微镜观测法和XRD法确定。根据实验研究数据,绘制了该三元体系相图。相图由3个无变量点、4条单变量曲线和4个盐类结晶区构成,4个结晶区分别对应CaCl2.6H2O、钙盐为基固溶体、锶盐为基固溶体和SrCl2.6H2O结晶区。     

On the structure and dynamics of duplex GNA

Glycol nucleic acid (GNA), with a nucleotide backbone comprising of just three carbons and the stereocenter derived from propylene glycol (1,2-propanediol), is a structural analog of nucleic acids with intriguing biophysical properties, such as formation of highly stable antiparallel duplexes with high Watson-Crick base pairing fidelity. Previous crystallographic studies of double stranded GNA (dsGNA) indicated two forms of backbone conformations, an elongated M-type (containing metallo-base pairs) and the condensed N-type (containing brominated base pairs). A herein presented new crystal structure of a GNA duplex at 1.8 ? resolution from self-complementary 3'-CTC(Br)UAGAG-2' GNA oligonucleotides reveals an N-type conformation with alternating gauche-anti torsions along its (O3'-C3'-C2'-O2') backbone. To elucidate the conformational state of dsGNA in solution, molecular dynamic simulations over a period of 20 ns were performed with the now available repertoire of structural information. Interestingly, dsGNA adopts conformational states in solution intermediate between experimentally observed backbone conformations: simulated dsGNA shows the all-gauche conformation characteristic of M-type GNA with the higher helical twist common to N-type GNA structures. The so far counterintuitive, smaller loss of entropy upon duplex formation as compared to DNA can be traced back to the conformational flexibility inherent to dsGNA but missing in dsDNA. Besides extensive interstrand base stacking and conformational preorganization of single strands, this flexibility contributes to the extraordinary thermal stability of GNA.     

Ultrafast dynamics of myoglobin probed by time-resolved resonance Raman spectroscopy

Recent experimental work carried out in this laboratory on the ultrafast dynamics of myoglobin (Mb) is summarized with a stress on structural and vibrational energy relaxation. Studies on the structural relaxation of Mb following CO photolysis revealed that the structural change of heme itself, caused by CO photodissociation, is completed within the instrumental response time of the time-resolved resonance Raman apparatus used (approximately 2 ps). In contrast, changes in the intensity and frequency of the iron-histidine (Fe-His) stretching mode upon dissociation of the trans ligand were found to occur in the picosecond regime. The Fe-His band is absent for the CO-bound form, and its appearance upon photodissociation was not instantaneous, in contrast with that observed in the vibrational modes of heme, suggesting appreciable time evolution of the Fe displacement from the heme plane. The band position of the Fe-His stretching mode changed with a time constant of about 100 ps, indicating that tertiary structural changes of the protein occurred in a 100-ps range. Temporal changes of the anti-Stokes Raman intensity of the v4 and v7 bands demonstrated immediate generation of vibrationally excited heme upon the photodissociation and decay of the excited populations, whose time constants were 1.1 +/- 0.6 and 1.9 +/- 0.6 ps, respectively. In addition, the development of the time-resolved resonance Raman apparatus and prospects in this research field are described.