Biomedical subjects

G Colotti

Publications and source records attributed to G Colotti.

11 recordsLinked to original sources

PubMed2001

Two different crystal forms of sorcin, a penta-EF-hand Ca2+-binding protein.

Sorcin is a 198 amino-acid Ca(2+)-binding protein that belongs to the penta-EF-hand family. Its Ca(2+)-binding domain (residues 33-198) has been crystallized in the absence of Ca(2+) in two different crystal forms. Two complete data sets have been collected on a synchrotron source under cryocooling conditions from crystals grown using ammonium sulfate as precipitant: monoclinic crystals in space group C2, with unit-cell parameters a = 130.93, b = 103.85, c = 78.55 A, beta = 118.0 degrees, diffracting to 2.1 A, and tetragonal crystals in space group P42(1)2, with unit-cell parameters a = b = 103.33, c = 79.15, diffracting to 2.7 A. Crystals were also grown using PEG 6000 as precipitating agent. They also belong to space group C2, diffract to 2.8 A and their unit-cell parameters are very similar to the first form. Structure determination by molecular replacement has been initiated. Structural information should be useful for elucidating the interaction of sorcin with membrane targets.

Amino Acid Sequence↗

PubMed2000

Structure-function relationships in sorcin, a member of the penta EF-hand family. Interaction of sorcin fragments with the ryanodine receptor and an Escherichia coli model system.

Sorcin, a 21.6 kDa cytosolic EF-hand protein which undergoes a Ca(2+)-induced translocation from cytoplasm to membranes, has been assigned to the newly defined penta EF-hand family. A molecular model of the C-terminal Ca(2+)-binding domain has been generated using as a template the X-ray coordinates of the corresponding domain in the calpain light subunit, the family prototype [Lin, G., et al. (1997) Nat. Struct. Biol. 4, 539-546]. The model indicates that in sorcin the three-dimensional structure is conserved and in particular that of EF1, the novel EF-hand motif characteristic of the family. On this basis, two stable fragments have been obtained and characterized. Just like the native protein, the sorcin Ca(2+)-binding domain (residues 33-198) is largely dimeric, interacts with the ryanodine receptor at physiological calcium concentrations, and undergoes a reversible, Ca(2+)-dependent translocation from cytosol to target proteins on Escherichia coli membranes. In contrast, the 90-198 fragment (residues 90-198), which lacks EF1 and EF2, does not bind Ca(2+) with high affinity and is unable to translocate. Binding of calcium to the EF1-EF2 pair is therefore required for the activation of sorcin which uses the C-terminal calcium-binding domain for interaction with the ryanodine receptor, a physiological target in muscle cells.

Amino Acid Sequence↗

PubMed1999

Ligand-linked changes at the subunit interfaces in Scapharca hemoglobins probed through the sulfhydryl infrared absorption.

FTIR spectra of native Scapharca homodimeric hemoglobin (HbI) and of the Phe97-->Ile mutant have been measured in the region 2400-2700 cm(-1) where the absorption of the sulfhydryl groups can be observed. In native HbI, the two Cys92 residues give rise to a relatively intense band centered at 2559 cm(-1) that is shifted to 2568 cm(-1) and strongly quenched upon ligand binding. In the Phe97-->Leu mutant, such ligand-linked changes are not observed and the strong peak at around 2560 cm(-1) persists in the liganded derivatives. In native HbI, the observed changes have been attributed to the decrease in polarity of the interface due to the ligand-induced extrusion of the Phe97 phenyl ring from the heme pocket to the interface and the subsequent release of several water molecules that are clustered in the vicinity of Cys92. In contrast, in the Phe97-->Leu mutant, the Leu residue does not leave the heme pocket upon ligand binding and the interface is unaltered. The Cys92/S-H infrared band, therefore, represents a sensitive probe of the structural rearrangements that take place in the intersubunit interface upon ligand binding to HbI. The heterotetrameric Scapharca hemoglobin HbII contains, in addition to the Cys92 residues in the interfaces, two extra sulfhydryl groups per tetramer (Cys9 in the B chain) that are exposed to solvent in the A helix. The frequency of the Cys9/S-H stretching vibration occurs at 2582 cm(-1) in the unliganded and at 2586 cm(-1) in the liganded derivative, pointing to the involvement of the A helix in the ligand-linked polymerization characteristic of HbII.

Animals↗

PubMed1998

The apolar distal histidine mutant (His69-->Val) of the homodimeric Scapharca hemoglobin is in an R-like conformation.

The effect of the apolar mutation of the distal histidine (His69-->Val) has been studied in the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis. Absorption, circular dichroism, and resonance Raman spectroscopy point to a more symmetric heme structure of the deoxy derivative, which is indicative of an R-like conformation of the deoxy heme. Resonance Raman spectroscopy also brings out alterations in the geometry and interactions of the bound CO molecule. The iron-carbon stretching frequency is decreased by about 30 cm-1 with respect to the native protein, while the diatomic ligand stretching frequency is increased by about the same degree. Consistent with the structural changes, the ligand binding properties are significantly altered. In the mutant the overall rate and the affinity for CO binding are increased about 100-fold with respect to the native protein, and cooperativity is abolished. In addition, the amplitude and the rate of the geminate rebinding process increase significantly. This finding may be correlated to the longer average residence time of the photolyzed CO molecule within the heme pocket of the H69V mutant, as indicated by molecular dynamics simulations.

Amino Acid Substitution↗

PubMed1997

Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin.

Residue Phe97, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding. This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity. Kinetic measurements of ligand binding to the Leu97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states. Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure. Increased oxygen affinity results from the absence of the Phe97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation. The absence of the Phe97 side chain is also associated with diminished cooperativity, since Leu97 packs in the heme pocket in both states. Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin.

Allosteric Regulation↗

PubMed1996

Cooperative oxygen binding to scapharca inaequivalvis hemoglobin in the crystal.

Oxygen binding to homodimeric Scapharca inaequivalvis hemoglobin (HbI) crystals has been investigated by single-crystal polarized absorption microspectrophotometry. The saturation curve, characterized by a Hill coefficient nH = 1.45 and an oxygen pressure at half saturation p50 = 4.8 torr, at 15 degrees C, shows that HbI in the crystalline state retains positive cooperativity in ligand binding. This finding will permit the correlation of the oxygen-linked conformational changes in the crystal with the expression of cooperativity. Polarized absorption spectra of deoxy-HbI, oxy-HbI, and oxidized HbI crystals indicate that oxygenation does not induce heme reorientation, whereas oxidation does. Lattice interactions prevent the dissociation of oxidized dimers that occurs in solution and stabilize an equilibrium distribution of pentacoordinate and hexacoordinate high spin species.

Animals↗

PubMed1995

Bacterial expression of Scapharca dimeric hemoglobin: a simple model system for investigating protein cooperatively.

Recombinant Scapharca homodimeric hemoglobin has been expressed at high levels from a synthetic gene in Escherichia coli. Addition of the heme precursor delta-aminolevulinic acid to the expression culture results in a considerable increase in the yield of soluble hemoglobin. The recombinant hemoglobin exhibits cooperative oxygen binding properties indistinguishable from native protein. Crystals of the recombinant protein, like those of native hemoglobin, diffract to high resolution which will allow functional studies of site-directed mutants to be correlated with detailed structural analyses.

Amino Acid Sequence↗

PubMed1994

Identification of the site of ferrocyanide binding involved in the intramolecular electron transfer process to oxidized heme in Scapharca dimeric hemoglobin.

The cooperative homodimeric hemoglobin (HbI)2 from the mollusc Scapharca inaequivalvis is characterized by unusual properties of the ferric derivative. The dimeric aquomet form undergoes a pH-dependent reversible dissociation into a monomeric low-spin hemichrome. Moreover, in HbI oxidized with ferricyanide the ferrocyanide anion produced in the reaction remains bound to the oxidized protein with high affinity and forms an intramolecular redox couple with the heme iron. Thus, the reduced HbI-CO adduct is obtained readily in the presence of carbon monoxide. The ferrocyanide binding site of HbI has been identified by modifying the only cysteine residue of the polypeptide chain, Cys 92 (F2), which is located at the subunit interface near the proximal histidine (His 101, F11). In HbI modified with organomercurials the rate of oxidation by ferricyanide depends on the presence and position of a negatively charged group on the aromatic ring, indicating that the binding site of the ferrocyanide anion is located near Cys 92. The tendency to dissociate into the monomeric hemichrome of the various Cys 92-reacted proteins and the study of the intramolecular electron transfer reaction between bound ferrocyanide and the heme iron confirmed this location. The proposed binding site of the ferrocyanide anion comprises a cluster of positive charges at the subunit interface formed by Lys 96, Arg 53', Lys 65', and Arg 67' where apices indicate residues of the contralateral subunit.

Animals↗

PubMed1992

The homodimeric hemoglobin from Scapharca can be locked into new cooperative structures upon reaction of Cys92, located at the subunit interface, with organomercurials.

In the cooperative, homodimeric hemoglobin from Scapharca inaequivalvis, HbI, the subunit interface is formed by the heme-carrying E and F helices and contains the only cysteine residue of the globin chain (Cys92, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to HbI is cooperative and entails major quaternary rearrangements. The reaction of Cys92 with p-chloromercuri-benzoate (PMB) and p-nitro-o-chloromercuriphenol (PN), a sensitive reporter of the cysteine microenvironment at neutral pH values, has been followed in stopped flow experiments. Kinetic evidence for the cooperativity of mercurial binding has been obtained and the rate of the corresponding conformational transition has been estimated. As expected PN, but not PMB, is able to monitor the oxygen-linked change of the cysteine microenvironment. The modification of Cys92 with PN has unique functional effects. In PN-reacted HbI cooperativity is maintained, albeit to a different extent, depending on the ligation state of the protein during mercaptide formation. It may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophenol moiety and the contralateral subunit.

Animals↗

PubMed1992

Halothane does not alter Ca2+ affinity of troponin C.

Troponin C has been suggested as a possible target for the negative inotropic action of volatile anesthetics. This study has examined the effect of halothane on the structure and response of isolated cardiac troponin C to Ca2+ and the response of skinned soleus and cardiac muscle fibers to Ca2+. The high-affinity Ca(2+)-binding sites of cardiac troponin C were assessed by measurement of the change in intrinsic tyrosine fluorescence and ultraviolet circular dichroism in response to Ca2+ in the presence and absence of halothane. Halothane (0.9 mM, 1.4%) did not alter the 45% enhancement in intrinsic tyrosine fluorescence that occurs with saturation of the high-affinity sites or change the Ca2+ concentration at which half-maximal enhancement occurred. The molar ellipticity in the far ultraviolet region, a measure of the secondary structure, increased to a similar extent with addition of 10(-6) M Ca2+ in the absence and presence of 1.0 mM (1.6%) halothane. The binding rate of the sulfhydryl reagent, 5,5'-dithiobis (2-nitrobenzoic acid), to troponin C in response to Ca2+ titration was used as a measure of the integrity of the low-affinity Ca(2+)-binding site in troponin C in the presence and absence of 1.0 mM (1.6%) halothane. The rate of reaction was stimulated twofold, and the half maximal effect was observed at pCa 4.8 +/- 0.2 in both control and halothane-treated samples. Halothane (5 mM; 7.8%) did not change the pCa/tension response of skinned soleus fibers; the data were fit to the Hill equation and yielded dissociation constants of 6.2 x 10(-7) M for control and halothane-treated specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes.

Pseudomonas aeruginosa synthesizes two siderophores, pyochelin and pyoverdin, characterized by widely different structures, physicochemical properties, and affinities for Fe(III). Titration experiments showed that pyochelin, which is endowed with a relatively low affinity for Fe(III), binds other transition metals, such as Cu(II), Co(II), Mo(VI), and Ni(II), with appreciable affinity. In line with these observations, Fe(III) and Co(II) at 10 microM or Mo(VI), Ni(II), and Cu(II) at 100 microM repressed pyochelin synthesis and reduced expression of iron-regulated outer membrane proteins of 75, 68, and 14 kDa. In contrast, pyoverdin synthesis and expression of the 80-kDa receptor protein were affected only by Fe(III). All of the metals tested, except Mo(VI), significantly promoted P. aeruginosa growth in metal-poor medium; Mo(VI), Ni(II), and Co(II) were more efficient as pyochelin complexes than the free metal ions and the siderophore. The observed correlation between the affinity of pyochelin for Fe(III), Co(II), and Mo(VI) and the functional effects of these metals indicates that pyochelin may play a role in their delivery to P. aeruginosa.

Bacterial Outer Membrane Proteins↗