Replacement of a conserved proline and the alkaline conformational change in iso-2-cytochrome c

Although point mutations usually lead to minor localized changes in protein structure, replacement of conserved Pro-76 with Gly in iso-2-cytochrome c induces a major conformational change. The change in structure results from mutation-induced depression of the pK for transition to an alkaline conformation with altered heme ligation. To assess the importance of position 76 in stabilizing the native versus the alkaline structure, the equilibrium and kinetic properties of the pH-induced conformational change have been compared for normal and mutant iso-2-cytochrome c. The pKapp for the conformational change is reduced from 8.45 (normal iso-2) to 6.71 in the mutant protein (Gly-76 iso-2), suggesting that conservation of Pro-76 may be required to stabilize the native conformation at physiological pH. The kinetics of the conformational change for both the normal and mutant proteins are well-described by a single kinetic phase throughout most of the pH-induced transition zone. Over this pH range, a minimal mechanism proposed for horse cytochrome c [Davis, L. A., Schejter, A., & Hess, G. P. (1974) J. Biol. Chem. 249, 2624-2632] is consistent with the data for normal and mutant yeast iso-2-cytochromes c: NH KH----N + H+ kcf in equilibrium kcb A NH and N are native forms of cytochrome c with a 695-nm absorbance band, A is an alkaline form that lacks the 695-nm band, KH is a proton dissociation constant, and kcf and kcb are microscopic rate constants for the conformational change. The Gly-76 mutation increases kcf by almost 70-fold, but kcb and KH are unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

EPR study of Cu(II) complexes of tridentate amino acids

The Cu(II) complexes of tridentate amino acids and related amines in alkaline solution were studied by EPR spectroscopy. Line shapes, g∥ and A∥ of each amino acid complex were compared with those of the corresponding amine complex. The results indicate that aromatic amino acids, monoaminodicarboxylic amino acids, arginine, methionine, and lysine bind to Cu(II) via the amino and carboxyl α groups. On the other hand cysteine, 2-3-diaminopropionic acid and hydroxy amino acids appear to be coordinated through the α-amino group and the third potentially binding group. Evidence is presented for the formation of mixed complexes in the cases of histidine and 2-4-diaminobutyric acid, whereas a glycine-like complex with apical coordination of the δ-amino groups is proposed for the ornithine-Cu(II) complex.     

EPR study of the electronic properties and weak exchange interactions in bis(L-phenylalaninamidato)Cu(II)

We performed EPR measurements in powder and single crystal samples of bis(L-phenylalaninamidato)Cu(II) (C18H22CuN4O2). We evaluated the crystal and molecular g-factors, and estimate an exchange interaction of 0.15 K< or = J(AB)/k < or =0.32 K between coppers spaced apart 6.24 A, transmitted through an amidate bridge Cu-O-C-N-Cu. An unusual line shape is observed in powder samples arising from the relative orientation of symmetry-related molecules in the structure. The dipolar interaction and the layered structure of the copper ions produce a strong temperature variation of the spectral shape when the copper spins are polarized by the applied magnetic field.     

Methylamine-induced conformational change of alpha 2-macroglobulin and its zinc (II) binding capacity. An X-ray scattering study

Methylamine induces a conformational change of alpha 2-macroglobulin which is very similar to that obtained by proteinase reaction and binding. This was shown by small-angle X-ray scattering at 21 degrees C in 0.03 M Hepes buffer of pH 8.0 containing 0.15 M NaCl and 0.3 mM EDTA. When alpha 2-macroglobulin reacts with methylamine the side maximum virtually disappears from the X-ray scattering curve and the radius of gyration decreases from 7.8 nm to 7.2 nm. The X-ray data of alpha 2-macroglobulin are consistent with an open shape model similar to that deduced via electron micrographs [Schramm, H. J. and Schramm, W. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 803-812]; one projection of the model resembles the letter H; the four subunits are mainly represented as elliptical cylinders which are connected via a central, quite flat cylinder. Zinc(II) ions cause aggregation of alpha 2-macroglobulin even at such a low total zinc concentration as 12.5 microM; for 25 microM zinc(II) concentration, the average molecular mass indicates that the aggregation goes beyond the dimeric stage. Monomeric species of alpha 2-macroglobulin appear to have the capacity specifically to bind 8.0 zinc(II) ions per molecule, which corresponds to two zinc(II) ions per subunit.     

DNA-fiber EPR study of the orientation of Cu(II) complexes of 1,10-phenanthroline and its derivatives bound to DNA: mono(phenanthroline)-copper(II) and its ternary complexes with amino acids

The orientation of mono(1,10-phenanthroline)copper(II), [Cu(phen)]2+, and the ternary complexes with amino acids, [Cu(phen)X(aa)]n+, where X(aa) stands for an alpha-amino acid, has been investigated by electron paramagnetic resonance (EPR) spectra of the complexes on DNA fibers. It has been revealed that these complexes bind to DNA with several different binding modes. The observation of a species whose g axis is almost parallel to the DNA double helical axis has suggested that the phenanthroline moiety intercalates to DNA. An absence of the intercalated species for the corresponding 2,2'-bipyridine complex has shown that the three-fused aromatic rings in phenanthroline are critical for the intercalative binding of the complexes. The intercalative binding was promoted by 5,6-dimethyl groups on the phenanthroline ring, whereas it was disturbed by 2,9-dimethyl groups, indicating that the planarity of the coordination sphere is important for the intercalative binding. In all cases, the amount of the non-intercalated species was larger than that of the intercalated one. The amino acids in the ternary complexes of glycine, leucine, serine, threonine, cysteine, methionine, and asparagine were partly substituted with some coordinating groups in DNA, whereas the ternary complexes of lysine, arginine, and glutamine remained intact on DNA.     

Picosecond spectroscopy of Cu(II) cytochrome c.

We have observed a strong pH dependence in the relaxation rate of Cu(II) cytochrome c following excitation at 532 nm. At pH 8.0 the excited state relaxes with a lifetime of 10 +/- 5 ps while at pH extremes of 2.5 and 13.0 we find that the lifetime becomes longer than 1 ns. This change of more than two orders of magnitude in the lifetime may be due to the Cu coordination number, which is six at neutral pH but five at pH extremes.     

The octapeptide repeat region of prion protein binds Cu(II) in the redox-inactive state

The octapeptide repeat region of human prion protein is known to bind four Cu(II) ions per molecule. A peptide, Octa(4), representing this region was tested for inhibitory effects on copper-catalyzed oxidation of l-ascorbate or glutathione and on generation of OH(*) during the former reaction. The result indicated that the catalytic activity of the first Cu(II) ion bound to an Octa(4) molecule was completely suppressed. The valence state of the copper under reducing conditions was Cu(II), as determined by a newly developed method using bathocuproinedisulfonate under acidic conditions. Furthermore, it was shown that Escherichia coli cells expressing the octapeptide repeat region were significantly resistant to copper treatment compared with control cells. The results taken together indicate that prion protein can function to sequester copper ions in the redox-inactive state, rendering copper-induced generation of reactive oxygen species impossible.     

Raman spectroscopic study on the copper(II) binding mode of prion octapeptide and its pH dependence.

The cellular form of prion protein is a precursor of the infectious isoform, which causes fatal neurodegenerative diseases through intermolecular association. One of the characteristics of the prion protein is a high affinity for Cu(II) ions. The site of Cu(II) binding is considered to be the N-terminal region, where the octapeptide sequence PHGGGWGQ repeats 4 times in tandem. We have examined the Cu(II) binding mode of the octapeptide motif and its pH dependence by Raman and absorption spectroscopy. At neutral and basic pH, the single octapeptide PHGGGWGQ forms a 1:1 complex with Cu(II) by coordinating via the imidazole N pi atom of histidine together with two deprotonated main-chain amide nitrogens in the triglycine segment. A similar 1:1 complex is formed by each octapeptide unit in (PHGGGWGQ)2 and (PHGGGWGQ)4. Under weakly acidic conditions (pH approximately 6), however, the Cu(II)-amide- linkages are broken and the metal binding site of histidine switches from N pi to N tau to share a Cu(II) ion between two histidine residues of different peptide chains. The drastic change of the Cu(II) binding mode on going from neutral to weakly acidic conditions suggests that the micro-environmental pH in the brain cell regulates the Cu(II) affinity of the prion protein, which is supposed to undergo pH changes in the pathway from the cell surface to endosomes. The intermolecular His(N tau)-Cu(II)-His(N tau) bridge may be related to the aggregation of prion protein in the pathogenic form.     

DNA-fiber EPR spectroscopy as a tool to study DNA-metal complex interactions: DNA binding of hydrated Cu(II) ions and Cu(II) complexes of amino acids and peptides

DNA-fiber EPR spectroscopy and its application to studies of the DNA binding orientation and dynamic properties of Cu(II) ions and their complexes with amino acids and peptides are reviewed. Cu(II) ions bind in at least two different binding modes; one mode was mobile while the other mode fixed the orientation of the coordination plane. The hydroxyl groups of L-Ser and L-Thr fixed the coordination plane of their respective Cu(II) complexes parallel to the DNA base pair plane, whereas Cu(II) complexes of Lys and Arg induced several binding modes, depending on the tertiary structure of the DNA and the chirality of the amino acids. Unusually broadened signals observed for the His complex were assigned to a mono-L-His complex stacked stereospecifically along the DNA double helix. In comparison, Cu(II). Xaa-Xaa' -His type complexes oriented in the minor groove with different affinities and extents of randomness depending on the Xaa-Xaa' sequence and the chirality of Xaa or Xaa' while the C-terminal Xaa residues in Cu(II).Arg-Gly-His-Xaa (Xaa=L-Leu or L-Glu) decreased the stereospecificity and the stability of the complexes bound to DNA. In contrast to Xaa-Xaa'- His complexes, the coordination planes of Cu(II).Gly-L-His-Gly and Cu(II).Gly-L-His-L-Lys complexes were found to lie parallel to the DNA-fiber axis. Dinuclear Cu(II).carnosine complexes were also shown to bind to DNA stereospecifically.     

Copper(II)--nucleic acid interactions--a conformational study

The effect of copper complexation upon the conformational angles in mononucleotides and dinucleotides was studied using classical potential energy expressions. The geometrical parameters were taken from crystallographic reports on the base-metal complexes. It was found that the preferred conformations of the complexes were quite different from the preferred conformations of the corresponding pure nucleotides. Notable among the disallowed conformations in case of Cu(II)-dinucleotide complexes were the regions corresponding to RRNA's, RNA's and the A, B and C forms of DNA. It is proposed that the energetics of a single strand is more important than inter-strand interactions in promoting copper-induced denaturation of DNA.     

Interactions between yeast iso-1-cytochrome c and its peroxidase

Isothermal titration calorimetry was used to study the formation of 19 complexes involving yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP). The complexes comprised combinations of the wild-type proteins, six CcP variants, and three Cc variants. Sixteen protein combinations were designed to probe the crystallographically defined interface between Cc and CcP. The data show that the high-affinity sites on Cc and CcP coincide with the crystallographically defined sites. Changing charged residues to alanine increases the enthalpy of complex formation by a constant amount, but the decrease in stability depends on the location of the amino acid substitution. Deleting methyl groups has a small effect on the binding enthalpy and a larger deleterious effect on the binding free energy, consistent with model studies of the hydrophobic effect, and showing that nonpolar interactions also stabilize the complex. Double-mutant cycles were used to determine the coupling energies for nine Cc-CcP residue pairs. Comparing these energies to the crystal structure of the complex leads to the conclusion that many of the substitutions induce a rearrangement of the complex.     

Temperature-dependent conformational transitions and hydrogen-bond dynamics of the elastin-like octapeptide GVG(VPGVG): a molecular-dynamics study

A joint experimental/theoretical investigation of the elastin-like octapeptide GVG(VPGVG) was carried out. In this article a comprehensive molecular-dynamics study of the temperature-dependent folding and unfolding of the octapeptide is presented. The current study, as well as its experimental counterpart (see companion article in this issue) find that this peptide undergoes an inverse temperature transition (ITT), leading to a folding at approximately 40-60 degrees C. In addition, an unfolding transition is identified at unusually high temperatures approaching the normal boiling point of water. Due to the small size of the system, two broad temperature regimes are found: the ITT regime at approximately 10-60 degrees C and the unfolding regime at approximately T > 60 degrees C, where the peptide has a maximum probability of being folded at T approximately 60 degrees C. A detailed molecular picture involving a thermodynamic order parameter, or reaction coordinate, for this process is presented along with a time-correlation function analysis of the hydrogen-bond dynamics within the peptide as well as between the peptide and solvating water molecules. Correlation with experimental evidence and ramifications on the properties of elastin are discussed.     

EPR investigation of Cu(II)-complexes with nitrogen derivatives of dialdehyde starch

Dialdehyde starch obtained by periodate oxidation from potato starch was converted into its disemicarbazone (DSC), dithiosemicarbazone (DTSC), dihydrazone (DHZ) and dioxime (DOX). The Cu(II) complexes of these compounds were prepared and characterized by Raman and EPR spectra, as well as by the measurements of magnetic susceptibility. EPR investigations showed that two types of complexes with different surroundings of copper centres existed in each starch derivative. Besides nitrogen atoms of the CN moiety and sulphur atoms of the CS moiety, also oxygen atoms from starch hydroxyl groups and/or water molecule were proposed as the coordination sites for the central copper ions.     

Chaperone-mediated reduction of RepA dimerization is associated with RepA conformational change

RepA, the initiator protein of plasmid P1, binds to multiple sites (iterons) in the origin. The binding normally requires participation of chaperones, DnaJ, DnaK and GrpE. When purified, RepA appears dimeric and is inactive in iteron binding. On reaction with chaperones, a species active in iteron binding is formed and found to be monomeric. To test whether the chaperones can reduce dimerization, RepA was used to replace the dimerization domain of the λ repressor. The hybrid protein repressed the λ operator efficiently, indicating that RepA can dimerize in vivo . A further increase in repressor activity was seen in dnaJ mutant cells. These results are consistent with a chaperone-mediated reduction of RepA dimerization. We also found that RepA mutants defective in dimerization still depend on DnaJ for iteron binding. Conversely, RepA mutants that no longer require chaperones for iteron binding remain dimerization proficient. These results indicate that the chaperone dependence of RepA activity is not solely owing to RepA dimerization. Our results are most simply explained by a chaperone-mediated conformational change in RepA protomer that activates iteron binding. This conformational change also results in reduced RepA dimerization.     

Coordination geometry of Cu-porphyrin in Cu(II)-Fe(II) hybrid hemoglobins studied by Q-band EPR and resonance Raman spectroscopies

Cu(II)-Fe(II) hybrid hemoglobins were investigated by UV-vis, Q-band (35 GHz) EPR and resonance Raman spectroscopies. EPR results indicated that Cu-porphyrin in alpha-subunit within hybrid hemoglobin had either 5- or 4-coordination geometry depending on the pH conditions, while Cu-porphyrin in beta-subunit had only 5-coordination geometry at high and low pH values. These results were consistent with UV-vis absorption results. A new resonance Raman band appeared around 190 cm(-1), which was present whenever 5-coordinated Cu-porphyrin existed in Cu(II)-Fe(II) hybrid hemoglobins irrespective of the coordination number in Fe(II) subunit. This Raman band might be assigned to Cu-N(epsilon) (His) stretching mode. These results are direct demonstration of the existence of coordination changes of Cu-porphyrin in alpha-subunit within hybrid hemoglobin by shifting the molecular conformation from fully unliganded state to intermediately liganded state.     

EPR study of the dynamic behavior of cis,cis-1,3,5-triaminocyclohexane Cu(II) complex on B-form DNA-fibers

The orientation and the dynamic behavior of [Cu(TACH)](2+)(TACH = cis,cis-1,3,5-triaminocyclohexane) on B-form DNA-fiber have been investigated by electron paramagnetic resonance (EPR) spectroscopy. The complex showed novel EPR spectra indicating that a rapidly moving species (X) is in equilibrium with a stereospecifically oriented species (Y) on the DNA-fiber in the range 20 and -20 degrees C. The thermodynamic parameters Delta H and Delta S of the equilibrium X right arrow over left arrow Y are respectively -51.3 kJmol(-1) and -1.9 x 10(2) JK(-1)mol(-1) for the DNA-fibers prepared at pH 7 and -47.1 kJmol(-1) and -1.7 x 10(2) JK(-1)mol(-1) for the DNA-fibers prepared at pH 9. These results suggest that the equilibrium involved a concerted structural change of the coordination sphere and the hydrogen bonding network around the complex on the B-form DNA-fibers. The orientation of the species Y was completely randomized below -20 degrees C, indicating that the freezing of the water molecules in the DNA-fibers breaks down the hydrogen bonding network which regulated the orientation of the complex in the DNA-fibers. The correlation between the observed dynamic behavior and the hydrolytic ability of the complex was also discussed.     

Covalent binding and conformational change of pUC19 DNA by rhodium (II) metal complex

Binding of Rhodium (II) acetate [Rh(2)(O(2)CCH(3))(4)] (Rh1) compound with plasmid pUC19 DNA has been studied using different molar ratio of Rh1. After incubation for 24hr at 37 degrees C, binding of the Rh1 to pUC19 DNA was confirmed by agarose gel electrophoresis. The electrophoretic results indicated the slower migration speed for the linearized pUC19 DNA. Conformation change of the DNA after Rh1 binding was also indicated at higher molar ratio of Rh1. The atomic force microscopy images showed that the Rh1 induced the conformation change to unwind pUC19 DNA. The Rh1-DNA complexes are observed very stable due to covalent bond. This study clearly demonstrates that [Rh(2)(O(2)CCH(3))(4)] reacts with pUC19 DNA and covalently binds to be stable Rh1-pUC19 DNA as interstrand adducts.     

EPR studies of copper(II) and cobalt(II) complexes of adriamycin

Cu2+ and Co2+ complexes of adriamycin (ADM) in aqueous solutions have been examined using EPR spectroscopy. An appreciable amount of Cu2+ and Co2+ complexes formed in the solutions were found to be in the EPR silent associated form, where the metal ions are antiferromagnetically coupled. The associated form of the Cu2+ complex may be neither a simple dimer nor coordination polymer but aggregates of a stacked type. Formation of a complex having Cu2+-ADM stoichiometry of 1:2 was observed for the solutions containing excess of ADM as an EPR observable species. The complex having Cu2+-ADM stoichiometry of 1:1 was not observed directly by EPR, but the presence of the complex is undeniable, especially at low pH range so far as large excessive ADM is not present. The Co2+ complex of ADM observed by EPR is in the high-spin (S = 3/2) state and may have a coordination structure of tetragonal symmetry. The EPR spectra of these complexes apparently show that the Cu2+ and Co2+ ions are bound at the carbonyl and phenolate oxygen in the 1,4-dihydroxyanthraquinone moiety and the amino nitrogen in the sugar part does not seem to participate in the coordination to the metal ions.     

Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin

Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.