Toward a synthesis of Clostridium butyricum apoferredoxin: Two tetradecapeptides comprising half the sequence (residues 7–20 and 21–34)

The two protected tetradecapeptides Z·Ser·Cys[Bzl(OMe)]·Val·Ser·Cys[Bzl(OMe]·Gly·Ala·Cys[Bzl(OMe)]·Ala·Gly·Glu(OBu^t)· Cys[Bzl(OMe)]·Pro·Val·NH·NH·Boc and Z·Ser·Ala·Ile·Thr·Gln·Gly·Asp(OBu^t)·Thr(Bu^t)·Gln·Phe·Val·Ile·Asp(OBu^t)·Ala·NH·NH·Boc, corresponding to residues 7–20 and 21–34 in the amino acid sequence of Clostridium butyricum apoferredoxin have been synthesized as a first stage in a total synthesis of the apoferredoxin. The former peptide has been deprotected to the tetra-thiol peptide H·Ser·Cys·Val·Ser·Cys·Gly·Ala·Cys·Ala·Gly·Glu·Cys·Pro·Val·NH·NH₂, and two tri-thiol and three di-thiol peptide components of this have also been synthesized for iron-sulfur complexing studies.     

Free energy of CO binding to iron(II) protoporphyrin IX in water

A series of CO binding constants to two iron porphyrins in different solvents have been determined spectrophotometrically in an effort to estimate the free energy of CO binding to heme in water. The free energy of CO binding to iron(II) protoporphyrin IX dimethylester(1,2-dimethylimidazole), FePPIXMe(DMI), in water has been estimated by determining the CO binding constant for the water-soluble heme iron(II) tetra(p-trimethlyammoniumphenyl)porphyrin(DMI), FeTAP(DMI), in phosphate buffer and assuming that the difference in free energies for binding CO to FeTAP(DMI) and to FePPIXMe(DMI) is the same in water as in DMSO solvent (this is equivalent to assuming that the FePPIXMe(DMI)-to-FeTAP(DMI) ratio of CO binding constants is the same in DMSO as in water). These studies estimate the CO binding constant to FePPIXMe(DMI) in phosphate buffer to be (9.1 ± 2.4) × 10⁶ M⁻¹ (P_1/2^CO=0.082±0.022 Torr). Using reported CO affinity to T-state hemoglobin (J.P. Collman, Inorg. Chem. (1997) 5145 and references therein), this leads to the smaller estimate of distal and proximal protein contributions to CO binding in T-state hemoglobin of +0.55 kcal/mol.     

Direct electrochemistry of protein-protein complexes involving cytochrome c, cytochrome b5, and plastocyanin

The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.     

The Reactions of O2 and NO with Mixed-Valence ba3 Cytochrome c Oxidase from Thermus thermophilus

Earlier CO flow-flash experiments on the fully reduced Thermus thermophilus ba₃ (Tt ba₃) cytochrome oxidase revealed that O₂ binding was slowed down by a factor of 10 in the presence of CO (Szundi et al., 2010, PNAS 107, 21010–21015). The goal of the current study is to explore whether the long apparent lifetime (∼50 ms) of the Cu_B⁺-CO complex generated upon photolysis of the CO-bound mixed-valence Tt ba₃ (Koutsoupakis et al., 2019, Acc. Chem. Res. 52, 1380–1390) affects O₂ and NO binding and the ability of Cu_B to act as an electron donor during O-O bond splitting. The CO recombination, NO binding, and the reaction of mixed-valence Tt ba₃ with O₂ were investigated by time-resolved optical absorption spectroscopy using the CO flow-flash approach and photolabile O₂ and NO carriers. No electron backflow was detected after photolysis of the mixed-valence CO-bound Tt ba₃. The rate of O₂ and NO binding was two times slower than in the fully reduced enzyme in the presence of CO and 20 times slower than in the absence of CO. The purported long-lived Cu_B⁺-CO complex did not prevent O-O bond splitting and the resulting P_M formation, which was significantly faster (5–10 times) than in the bovine heart enzyme. We propose that O₂ binding to heme a₃ in Tt ba₃ causes CO to dissociate from Cu_B⁺ in a concerted manner through steric and/or electronic effects, thus allowing Cu_B⁺ to act as an electron donor in the mixed-valence enzyme. The significantly faster O₂ binding and O-O bond cleavage in Tt ba₃ compared to analogous steps in the aa₃ oxidases could reflect evolutionary adaptation of the enzyme to the microaerobic conditions of the T. thermophilus HB8 species.     

Binding site of α2-plasmin inhibitor to plasminogen

Peptide T-11, a carboxyl terminal tryptic fragment of α₂-plasmin inhibitor, inhibits the reversible first step of the reaction between plasmin and α₂-plasmin inhibitor. To elucidate which amino-acid residues played a important role in the inhibitory activity of peptide T-11, we prepared the various synthetic derivatives of peptide T-11 and determined the peptide concentration that inhibited the apparent rate constant of the reaction between plasmin and α₂-plasmin inhibitor by 50% (IC₅₀). Peptide III, which lacked the residues Gly-1 to Pro-7 of peptide I (peptide T-11), had a strong inhibitory activity, like peptide I (IC₅₀: peptide 1, 7 μM; peptide III, 13 μM). The peptides that lacked the Leu-9 and Lys-10 or Lys-26 of peptide III showed much weaker activity, and the loss of amidation of the C-terminal lysine of peptide III also markedly reduced the inhibitory activity, Peptide III competitivef inhibited the binding of [¹⁴C]tranexamic acid to kringle 1 + 2 + 3 (K1–3) and kringle 4 (K4) in a binding assay performed by the gel-diffusion method. The respectively dissociation constants (K_d) of peptide III for K1–3 and K4 were 0.85 μM and 35.2 μM. These data suggest that the amino residue of Lys-10 and the carboxylic acid of Lys-26 in peptide T-11 play crucial roles in the ionic binding of α₂-plasmin inhibitor to the tranexamic acid-binding site (lysine-binding site) of plasminogen. Peptide T-11: H-G-D-K-L-F-G-P-D-L-K-L-V-P-P-M-E-E-D-Y-P-Q-F-G-S-P-K-OH.     

Synthesis and reactivity of imido niobium complexes containing the functionalized (dichloromethylsilyl)cyclopentadienyl ligand

The niobium complex [NbCp^ClCl₄] (Cp^Clη⁵-C₅H₄(SiCl₂Me)) (1) with a functionalized (dichloromethylsilyl)cyclopentadienyl ligand was isolated by the reaction of [NbCl₅] with C₅H₄(SiCl₂Me)(SiMe₃). Complex 1 was a precursor for the imido silylamido derivative [NbCp^NCl₂(NtBu)] (Cp^Nη⁵-C₅H₄[SiClMe(NHtBu)]) (2) after addition of LiNHtBu, which subsequently gave the dichlorosilyl compound [NbCp^ClCl₂(NtBu)] (3) when reacted with SiCl₃Me. Addition of LiNHtBu to complex 2 gave the niobium amido complex [NbCp^NCl(NHtBu)(NtBu)] (4), which slowly evolved with exchange of the niobium-amido and the silicon-chloro groups to give the dichloroniobium complex [NbCp^NNCl₂(NtBu)] (Cp^NNη⁵-C₅H₄[SiMe(NHtBu)₂]) (5). Reaction of 2 with excess LiNHtBu gave the silyl-η-amido constrained geometry complexes [Nb{η⁵-C₅H₄[SiMe(NHtBu)(-η-NtBu)]}(NHtBu)(NtBu)] (6) and [Nb{η⁵-C₅H₄[SiClMe(-η-NtBu)]}(NHtBu)(NtBu)] (7), whereas addition of one equimolecular amount of LiNHtBu to 5 in C₆D₆ afforded complex [NbCp^NNCl(NHtBu)(NtBu)] (8). All of the new complexes were characterized by ¹H, ¹³C and ²⁹Si NMR spectroscopy.     

Pyrazolate-bridged group 11 metal(I) complexes: Substituent effects on the supramolecular structures and physicochemical properties

Polynuclear homoleptic pyrazolate-bridged group 11 metal(I) complexes with three different alkyl substituted pyrazolate anions, 3,5-diisopropylpyrazolate (3,5-iPr₂pz⁻ = L1⁻), 3-tert-butyl-5-isopropylpyrazolate (3-tBu-5-iPrpz⁻ = L3⁻), and 3,5-di-tert-butylpyrazolate (3,5-tBu₂pz⁻ = L4⁻), i.e. [Cu(μ-3,5-iPr₂pz)]₃ (CuL1), [Ag(μ-3,5-iPr₂pz)]₃ (AgL1), [Au(μ-3,5-iPr₂pz)]₃ (AuL1), [Cu(μ-3-tBu-5-iPrpz)]₄ (CuL3), [Ag(μ-3-tBu-5-iPrpz)]₃ (AgL3), [Au(μ-3-tBu-5-iPrpz)]₄ (AuL3), [Cu(μ-3,5-tBu₂pz)]₄ (CuL4), [Ag(μ-3,5-tBu₂pz)]₄ (AgL4), and [Cu(μ-3,5-tBu₂pz)]₄ (AuL4), were systematically synthesized in order to investigate the influence of pyrazole bulkiness on their structures and physicochemical properties. The structural characterization indicates that the geometries are greatly influenced by the steric hindrance exerted by the substituent groups of the pyrazolyl rings and the differences of the central metal (I) ionic radius (Cu⁺ < Au⁺ < Ag⁺). These complexes were also characterized by spectroscopic techniques, namely, UV-Vis, IR/far-IR, Raman, and luminescence spectroscopy.     

Interaction of gramicidin S and its aromatic amino-acid analog with phospholipid membranes

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (K_b). The K_b1 and K_b2 values for endothermic two-step binding processes corresponded to high and low binding affinities (K_b1 ≥ 100 K_b2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.     

Synthesis and characterization of organotin Schiff base chelates

The synthesis and characterization of five organotin compounds containing Salophen(^tBu) [Salophen(^tBu)=N,N′-phenylene-bis(3,5-di-tert-butylsalicylideneimine)], Salomphen(^tBu) [Salomphen(^tBu)=N,N′-(4,5-dimethyl)phenylene-bis(3,5-di-tert-butylsalicylideneimine)] and Phensal(^tBu) [Phensal(^tBu)=3,5-di-tert-butylsalicylidene(1-aminophenylene-2-amine)] ligands is described. These compounds include the monomeric complexes LSnCl₂ (where L=Salophen(^tBu), L=Salomphen(^tBu)), L(ⁿBu)SnCl (where L=Salophen(^tBu), Salomphen(^tBu)), L(ⁿBu)SnCl₂ (where L=Phensal(^tBu)). Spectroscopic techniques including ¹¹⁹Sn NMR and X-ray crystallography were used in the characterization of the compounds.     

Analytical expressions for PLM(t), CL(t) and CLM(t) without use of Laplace transforms

Based on the age density functions for each phase of the cell life cycle (G₁, S, G₂ and M) in an exponentially growing steady state population derived by Trucco &; Brockwell (1968), the expressions for the percentage labeled mitoses curve [PLM(t)], the continuous labeling curve [CL(t)] and the continuous labeled mitotic curve [CLM(t)] are obtained explicitly without use of Laplace transforms. This approach is useful in describing the cell population when the steady state is disturbed due to, for example, irradiation. The mitotic index [MI(t)] for this case is considered.     

Reaction of [Pt3(μ-CO)3(PBu3)3] with activated olefins and alkynes: the X-ray-structure of [Pt(CO)(PBu3)(CF3CCCF3)]

The reactions of the triangulo-cluster [Pt₃(μ-CO)₃(P^tBu₃)₃] with activated olefins and alkynes have been examined under various conditions. At low temperature, cluster fragmentation occurs yielding the Pt(0) complexes [Pt(CO)(P^tBu₃)(olefin)] (olefin = maleic anhydride and maleimide), while di(tert-butyl)acetylenedicarboxilate reacts quantitatively giving the dinuclear Pt(0) complex [Pt₂(CO)₂(P^tBu₃)₂(μ-η²:η²-^tBuO₂CCCCO₂^tBu)]. At higher temperature and in the presence of alkyne in large excess, the latter dimer converts quantitatively to the monomers [Pt(CO)(P^tBu₃)(alkyne)] (alkyne = CF₃CCCF₃ and ^tBuO₂CCCCO₂^tBu). The stereochemistry of these complexes has been established by NMR and IR measurements. The structure of [Pt(CO)(P^tBu₃)(CF₃CCCF₃)] was confirmed by X-ray diffraction analysis.     

Site-Directed Mutagenesis in Basic Amino Acid Residues of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V _max/K _mADP. These results indicate that Arg76 is not involved in CO₂ binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207–210], does not perform an essential function for the enzyme activity.     

A two-state tension-displacement model for hemoglobin-ligand binding

Based on the Perutz view of hemoglobin co-operativity and the methodology of statistical physics, a two-state (t → r) model for the co-operative response is presented. The motion of the iron atom with respect to the heme plane is assumed to be the important feature of the binding process, and results in an expression for hemoglobin saturation as an explicit function of the internal tension of the hemoglobin molecule. Closure of the equation is achieved with the assumption of linearity between the internal tension and the displacement of the iron atom above the heme plane. The result is a linear dependence of log_e [(ψ/(1?ψ)/(1/X_L)] on the fractional saturation, ψ, the slope and intercept being expressed in terms of physically realizable parameters characteristic of the hemoglobin-ligand reaction. Agreement with experimental data for hemoglobin-oxygen and hemoglobin-carbon monoxide is obtained using parameter values that are reasonable in terms of the interactions they represent.     

Investigation of DNA Binding Modes for a Symmetrical Cyanine Dye Trication: Effect of DNA Sequence and Structure

Abstract

The DNA binding behavior of a tricationic cyanine dye (DiSC₃₊(5)) was studied using the [Poly(dA-dT)]₂, [Poly(dI-dC)]₂ and Poly(dA)?Poly(dT) duplex sequences and the Poly(dA) ?2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA)?Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]₂ duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]₂ sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987–2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.     

Enzymatic binding of aminoacyl-tRNA and peptide chain elongation by ribosomes and ribosome subunits in pea

In vitro aminoacyl transfer from aminoacyl-tRNA to elongating peptide chains and binding of aminoacyl-tRNA to ribosomes were studied with n     

Biomolecule Association Rates Do Not Provide a Complete Description of Bond Formation

The efficiency of many cell-surface receptors is dependent on the rate of binding soluble or surface-attached ligands. Much effort was exerted to measure association rates between soluble molecules (three-dimensional k_on) and, more recently, between surface-attached molecules (two-dimensional [2D] k_on). According to a generally accepted assumption, the probability of bond formation between receptors and ligands is proportional to the first power of encounter duration. Here we provide new experimental evidence and review published data demonstrating that this simple assumption is not always warranted. Using as a model system the (2D) interaction between ICAM-1-coated surfaces and flowing microspheres coated with specific anti-ICAM-1 antibodies, we show that the probability of bond formation may scale as a power of encounter duration that is significantly higher than 1. Further, we show that experimental data may be accounted for by modeling ligand-receptor interaction as a displacement along a single path of a rough energy landscape. Under a wide range of conditions, the probability that an encounter of duration t resulted in bond formation varied as erfc[(t₀/t)^1/2], where t₀ was on the order of 10 ms. We conclude that the minimum contact time for bond formation may be a useful parameter to describe a ligand-receptor interaction, in addition to conventional association rates.     

A chemical mapping technique for exploring the location of proteins along the ribosome-bound peptide chain

A series of peptidyl-tRNA analogs with varying peptide chain length, BrAc(Gly) _nPhe-tRNA^phe, n = 0 to 16 has been prepared. When bound to Escherichia coli 70 S ribosomes these all react covalently with certain ribosomal proteins. The overwhelming majority of the reaction is with 50 S ribosomal proteins L2, L16, L24, L26–L27 and L32–L33. The extent of reaction with each protein is a function of peptide chain length, making it possible to estimate the relative proximity of these proteins to the 3′-terminus of tRNA bound in the ribosomal P site. This fact, coupled with the findings of others about the length dependence of the binding and peptide donor activity of peptidyl-tRNAs suggests that there is actually a binding site for the growing peptide chain. If this is true, the results presented here permit the ordering of the proteins in this site: L2 is closest to the 3′-end of tRNA followed by L26–L27, L32–L33 and last L24. Evidence is also given that the direction of the growing peptide chain must point away from the A site.     

An affinity scale for the interaction of amino acid residues with nucleic acids

The affinity of amino acid residues to nucleic acids is probed by measurements of melting temperatures t_m for the helix–coil transition at various concentrations of amino acid amides. The increase of t_m on addition of ligand is described by the equation t_m = t*_m + αlog(1+K_tc_λ), where t*_m is the melting temperature in the absence of ligand, c_λ the ligand concentration, and K_t the “t_m-onset” constant, which is analogous to an equilibrium constant. It is shown that K_t is closely related to the affinity of the ligands to the double helix, whereas the slope α mainly reflects the preference of the ligand binding to the helix versus the coil form. In the case of the amino acid amides, α is found to be virtually independent of the nature of the side chain with few exceptions, e. g., aromatic amides. The t_m-onset constant, however, strongly depends on the nature of the amino acid side chain. For simple aliphatic amino acids, the relative free energy of binding decreases with increasing hydrophobic free energy, e.g., a high affinity is found for Gly-amide and a low affinity for Leu-amide. This relation is modified by functional groups like OH in Ser-amide. The helices poly[d(A-T)], ploy[d(I-C)]. and poly[d(A-C)]·poly[d(G-T)] exhibit similar affinity scales with relatively small variations. Our results demonstrate that the hydrophilic character of double helices at their surface disfavors binding of hydrophobic ligands unless special contacts can be formed. From our results we establish an affinity scale for the binding of amino acids to double helices.     

Structure of chain d of the gigantic hemoglobin of the earthworm

The extracellular hemoglobin of the earthworm has four major O₂-binding chains, a, b, c and d, together with additional non-heme structural chains that are required for assembly. Although the abc trimer self-associates extensively at least to (abc)₁₀, addition of chain d results in the formation of a discrete 280 kDa complex corresponding to (abcd)₄. Thus a primary function of chain d is to cap the abc association and convert an abc trimer that binds O₂ with weak cooperativity to a highly cooperative (abcd)₄ complex. Amino-acid sequences of the major globin chains a, b, c have been determined previously by peptide and cDNA analysis. However, the peptide sequence reported for the major chain d (Shishikura, F., Snow, J.W., Gotoh, T., Vinogradov, S.N. and Walz, D.A. (1987) J. Biol. Chem., 262, 3123–3131), has a calculated molecular mass 134–167 Da higher than masses for components of chain d determined by mass spectrometry (Ownby, D.W., Zhu, H., Schneider, K., Beavis, R.C., Chait, B.T. and Riggs, A.F. (1993) J. Biol. Chem. 268, 13539–13547). Reverse-phase HPLC confirms the presence of two distinct polypeptides, d₁ and d₂, together with d₁′, a variant of d₁. cDNA-derived amino-acid sequences have been determined for chains d₁′ and d₂ by application of the polymerase chain reaction with primers based on the NH₂-terminal sequences and oligo-dT. Each of the two cDNA-derived sequences has 140 residues and they differ by 28 substitutions. The data show that the sequence originally reported had been derived from peptides generated from both polypeptides.     

Rh and Ir dibenzo[a,e]cyclooctatetraene complexes: Chloro, hydroxo, and mixed Au oxo complexes

New and improved procedures are reported for the synthesis of [M(DBCOT)(μ-Cl)]₂ (M = Rh, Ir; DBCOT = dibenzo[a,e]cyclooctatetraene) from MCl₃(H₂O)_x or [M(COD)(μ-Cl)]₂ and DBCOT. Treatment of [M(DBCOT)(μ-Cl)]₂ with [(LAu)₃(μ-O)]BF₄(L = PPh₃, P^tBu₃) yields the mixed-metal oxo complexes [M(DBCOT)(μ⁴-O)(AuL)₂]₂(BF₄)₂. Dimeric [Rh(DBCOT)(μ-OH)]₂ is obtained from the reaction of [M(DBCOT)(μ-Cl)]₂ with KOH in EtOH/H₂O. All complexes except [Rh(DBCOT)(μ-Cl)]₂ have been structurally characterized by single crystal X-ray diffraction.