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G Irace

Publications and source records attributed to G Irace.

At least 37 records · Page 2Linked to original sources
PubMed1991

Conformational dynamics of unfolded peptides as a function of chain length: a frequency domain fluorescence approach.

The fluorescence emission decays of single-tryptophan-containing peptides of different chain lengths in their unfolded state were investigated in the frequency domain. The data were analyzed using different functions, i.e., exponential fit and probability-density functions of different shape. We found that unimodal Lorentzian distributions best describe the fluorescence decays. This finding agrees with the point of view, now broadly accepted, that rapid motions exist in polypeptides. As a consequence of this flexibility, a large variety of conformations, with an unequal perturbation of tryptophan in its excited state, is generated. The lifetime distribution center was independent of the length of the polypeptide chain but strongly related to the nature of the amino acid residues located in the proximity of the tryptophan in the primary structure. The full width at half maximum, W, of the lifetime distribution was found to be related to the length of unfolded polypeptide by the empirical logarithmic relationship W = 0.83 log n, where n indicates the number of residues. For short peptides, a single lifetime or a narrow range of lifetimes is observed because of the fast relaxation of the tryptophanyl environment. On peptide lengthening, the spectrum of conformations, which the peptide can assume, increases; this causes a complex fluorescence decay represented by a lifetime distribution. For long polypeptide chains, the motions of the regions far from tryptophan do not significantly perturb the chromophore environment.

Amino Acid Sequence↗
PubMed1991

Molecular organization and dynamics of the outer membrane of Salmonella thyphimurium mutant strains detected by frequency domain fluorometry.

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the outer membrane bilayer of two mutant strains of Salmonella thyphimurium, i.e., SH 5014 and SH 6261, at different temperatures was analyzed in terms of continuous Lorentzian lifetime distributions. The results were compared with those obtained for the free fluorophore in an isotropic nonviscous solvent. The incorporation of DPH in the outer membrane fragments resulted in a broadening of the lifetime distribution which was attributed to the microenvironmental heterogeneity of the membrane bilayer for the extrinsic fluorophore. The differences observed between the two types of membrane bilayers were interpreted in terms of a different molecular organization and, to a lesser extent, in terms of a different fluidity. The comparison between the DPH lifetime distributions obtained using two different excitation wavelengths, i.e., 280 and 350 nm, suggested that the structural organization of the membrane domains, which are richest in proteins, is almost identical in the two examined mutant strains. This observation indicates that the different susceptibility of the two mutant strains toward phagocytosis and complement-mediated lytic action may depend on the molecular organization and dynamics of the lipid regions far from those containing proteins.

Bacterial Outer Membrane Proteins↗
PubMed1989

Conformational substates of myoglobin detected by extrinsic dynamic fluorescence studies.

The extent of conformational substates of two apomyoglobins, i.e., sperm whale and tuna apomyoglobin, was investigated by examining the fluorescence decay in the frequency domain of the extrinsic fluorophore TNS [6-(p-toluidino)-2-naphthalenesulfonic acid] bound to the heme binding site. Data analysis was performed in terms of a continuous, unimodal lifetime distribution having a Lorentzian shape. The results were compared with those for the free fluorophore in an isotropic nonviscous solvent. The incorporation of TNS into the protein matrix resulted in a broadening of the lifetime distribution due to the microenvironmental heterogeneity generated by structural fluctuations. The larger width of lifetime distribution observed for TNS bound to tuna apomyoglobin was related to a more extended conformational space accessible to the fluorophore in this protein compared to sperm whale myoglobin. A temperature increase from 15 to 40 degrees C produced a further broadening of the lifetime distributions of TNS bound to both proteins. This result can be explained by assuming the existence of conformational substates at high energy content or separated by high energy barriers, which are not populated at low temperature. The overall picture emerging from the reported data is that the lifetime distributions of TNS bound to apomyoglobins are determined largely by the number of conformational substates accessible to the protein matrix and, to a lesser extent, by the interconversion rates among these states.

Animals↗
PubMed1989

Multiple conformational states in myoglobin revealed by frequency domain fluorometry.

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the chi 2 obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Effect of unfolding on the tryptophanyl fluorescence lifetime distribution in apomyoglobin.

Proteins exhibit, even in their native state, a large number of conformations differing in small details (substates). The fluorescence lifetime of tryptophanyl residues can reflect the microenvironmental characteristics of these subconformations. We have analyzed the lifetime distribution of the unique indole residue of tuna apomyoglobin (Trp A-12) during the unfolding induced by temperature or guanidine hydrochloride. The results show that the increase of the temperature from 10 to 30 degrees C causes a sharpening of the lifetime distribution. This is mainly due to the higher rate of interconversion among the conformational substates in the native state. A further temperature increase produces partially or fully unfolded states, resulting in a broadening of the tryptophanyl lifetime distribution. The data relative to the guanidine-induced unfolding show a sigmoidal increase of the distribution width, which is due to the transition of the protein structure from the native to the random-coiled state. The broadening of the lifetime distribution indicates that, even in the fully unfolded protein, the lifetime of the tryptophanyl residues is influenced by the protein matrix, which generates very heterogeneous microenvironments.

Animals↗
PubMed1988

Protein conformational changes induced by guanidine at predenaturational concentrations.

The nature of the molecular event that apomyoglobin undergoes at predenaturational concentrations of guanidine has been investigated by means of steady-state and multifrequency phase and modulation fluorometry. The results have been compared to those observed for liver alcohol dehydrogenase. From these studies has been hypothesized a different susceptibility of the distinct elements of secondary, super-secondary, and tertiary structure towards the denaturing action of guanidine at predenaturational concentrations.

Alcohol Dehydrogenase↗
PubMed1987

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins.

Dynamic aspects of the heme-binding site of myoglobins derived from two phylogenetically distant species, namely sperm whale and bluefin tuna, have been investigated by studying steady-state and time-resolved emission properties of 2-p-toluidinyl-6-naphthalene sulfonic acid (TNS) apomyoglobin conjugates. Multi-frequency phase and modulation fluorometry data indicate that charge movements occur in the fluorophore environment during the excited state lifetime in the sperm whale myoglobin system. In the case of the bluefin tuna myoglobin TNS adduct these movements were not detected, indicating that the relaxation processes differ in the two types of myoglobins.

Animals↗
PubMed1987

Unfolding pathway of myoglobin: effect of denaturants on solvent accessibility to tyrosyl residues detected by second-derivative spectroscopy.

The effects of denaturants on the solvent accessibility to tyrosyl residues of apomyoglobin have been examined by means of second-derivative spectroscopy in the near-ultraviolet. Three apomyoglobins, i.e., sperm whale, horse, and tuna, were selected because of the different distribution of tyrosyl residues in their primary structure. The results are consistent with the occurrence of two independent consecutive events in the guanidine-induced denaturation pattern of apomyoglobin. The first event, which is responsible for the lack of the ability to bind the heme, has been proved to involve conformational changes in both the domains, i.e., segments 1-79 and 80-153, identified in the myoglobin molecule. However, the conformational changes are not of the same type. In fact, the solvent accessibility to tyrosine HC2 is increased probably because of a partial unfolding of the 80-153 domain. Conversely, the solvent accessibility to tyrosine B2 is decreased, thus indicating that a refolding occurs in some region of the N-terminal moiety (1-79 domain) of the molecule.

Animals↗
PubMed1986

Unfolding pathway of myoglobin: molecular properties of intermediate forms.

The guanidine-induced unfolding of myoglobin as well as apomyoglobin has been found to involve the occurrence of at least a molecular intermediate observed at low denaturant concentrations, the molecular properties of which resemble those possessed by the acid-denatured form of the protein. The two partially unfolded forms show the same secondary structure and similar tryptophanyl fluorescence emission and polarization but exhibit marked differences in the tyrosine contributions to the near-ultraviolet circular dichroism and in the degree of solvent accessibility to tyrosyl residues. The molecular characterization of the two structural forms indicates that acids disorganize the 80-146 molecular domain identified in the myoglobin molecule to a great extent with respect to that induced by low guanidine concentration, whereas the structure of the 1-79 domain appears to be quite similar in the two molecular forms.

Anilino Naphthalenesulfonates↗
PubMed1985

Myoglobin structure and regulation of solvent accessibility of heme pocket.

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.

Animals↗
PubMed1984

Conformational stability and basal metabolic rate: reexamination of the case of myoglobin.

The free energy of unfolding of several myoglobins from different animal species has been determined from their denaturation pattern by using the ligand binding model. The results indicate that no simple correlation exists between the free energy of unfolding of myoglobin and the basal metabolic rate of the animal species from which the myoglobin was isolated.

Animals↗
PubMed1984

Determination of tyrosine exposure in proteins by second-derivative spectroscopy.

The mutual interference between the second-derivative bands of tyrosine and tryptophan in proteins has been evaluated in terms of the ratio r between two peak to peak distances. The r values have been found to be not only related to the tyrosine/tryptophan ratio but also dependent on the polarity of the medium in which tyrosyl residues are embedded. The results obtained on purified proteins have been found consistent with the available X-ray information and with the existing solvent perturbation data.

Acetylation↗
PubMed1984

Solvent accessibility of the heme pocket in tuna myoglobin.

The accessibility of the heme binding site of two apomyoglobins, i.e. tuna and sperm whale apomyoglobin, has been evaluated by quenching the fluorescence of their ANS-conjugates. The quenching pattern obtained by using charged and uncharged quenchers revealed that the heme pocket of tuna apomyoglobin is more accessible than that of sperm whale. Moreover, a larger number of positively charged groups is present in the heme pocket of tuna apomyoglobin as indicated by comparing the extent of quenching produced by iodide and cesium ion. The relaxation time of ANS bound to tuna apomyoglobin is lower than that of the same chromophore bound to sperm whale globin thus indicating that there is some localized flexibility in the tuna globin.

Animals↗
PubMed1983

The effect of evolution on the structure of tuna myoglobin.

The circular dichroic activity of tuna myoglobin in the far ultraviolet has been found to be lower than that of mammalian myoglobin, thus indicating a lower content of alpha-helix. Fluorescence and absorption studies have indicated that the structure of the N-terminal region of the molecule is essentially the same in all the examined apomyoglobins, whereas differences have been observed in the heme microenvironment. The prediction of secondary structure has revealed that the alpha-helical segments of tuna myoglobin, especially those involved in the formation of the heme pocket, are shorter than those of sperm whale myoglobin.

Animals↗
PubMed1983

Unfolding pathway of myoglobin. Evidence for a multistate process.

The free energy of unfolding of horse myoglobin has been calculated from the denaturation pattern induced by guanidine hydrochloride as well as by acid. The delta GH2O, i.e., the value in the absence of denaturant obtained by using the two-state transition model, was found to be 25% lower than that determined from the acid denaturation pattern, i.e., 12.0 kcal/mol, although the extent of protein denaturation produced by acid was much lower. The amount of helical structure surviving the acid-induced conformational change was estimated to be 50% of that present in the native protein, and it could be destroyed only after exposure of myoglobin samples kept at pH 3.0 to concentrated guanidine. From the guanidine denaturation pattern at acidic pH, a further variation of free energy of unfolding of 5.5 kcal/mol could be calculated, thus indicating that the overall free energy of unfolding determined from the two consecutive processes corresponds to 17.5 kcal/mol. The discrepancy between the two sets of data, i.e., guanidine unfolding at neutral pH and acid unfolding followed by addition of denaturant, has been considered to depend on the general assumption that the guanidine unfolding of myoglobin is a two-state process in the transition region. According to the recent experimental evidence showing the occurrence of at least two molecular events during the guanidine unfolding of apomyoglobin [Colonna, G., Balestrieri, C., Bismuto, E., Servillo, L., & Irace, G. (1982) Biochemistry 21, 212-215], the guanidine denaturation pattern of myoglobin was analyzed in terms of two independent steps.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Structural and functional aspects of the heart ventricle myoglobin of bluefin tuna.

The heart ventricle myoglobin of bluefin tuna has been purified to an apparent homogeneity. The amino acid analysis has revealed only a limited number of substitutions between the myoglobins of yellowfin and bluefin tuna. The alpha-helix content of tuna myoglobin has been found considerably lower than that of mammalian myoglobin. No correlation has been discovered between the conformational stability and alpha-helix content. Denaturation experiments have shown that the whole structure of tuna myoglobin results from the interaction of two structural units which represent the product of independent folding processes. The structure of tuna myoglobin has been found more open and disorganized than that of sperm whale. This result has been related to the low content of electrostatic interactions and explained in terms of evolutive adaptations.

Amino Acid Sequence↗
PubMed1982

Properties of clathrin coat structures.

Clathrin polymerizes to form characteristic coat structures (baskets) closely resembling those found on coated vesicles. Two sizes of baskets are formed from clathrin, depending on the purity of the preparation and on other factors. A protein of Mr 110 000 has been separated from clathrin by lysine-Sepharose chromatography which is needed for the formation of 150S baskets. In its absence, polymerization results in the larger size baskets, i.e., 300S. Addition of Ca2+ or Mg2+ stimulates 300S formation in the presence of the 110 000 protein.

Calcium↗