Biomedical subjects

G B Strambini

Publications and source records attributed to G B Strambini.

At least 37 records · Page 2Linked to original sources

PubMed1994

Heterogeneity of protein conformation in solution from the lifetime of tryptophan phosphorescence.

The decay of Trp phosphorescence of proteins in fluid solutions was shown to provide a sensitive tool for probing the conformational homogeneity of these macromolecules in the millisecond to second time scale. Upon examination of 15 single Trp emitting proteins multiexponential decays were observed in 12 cases, a demonstration that the presence of slowly interconverting conformers in solution is more the norm rather than an exception. The amplitude of preexponential terms, from which the conformer equilibrium is derived, was found to be a sensitive function of solvent composition (buffer, pH, ionic strength and glycerol cosolvent), temperature, and complex formation with substrates and cofactors. In many cases, raising the temperature, a point is reached at which the decay becomes practically monoexponential, meaning that conformer interconversion rates have become commensurate with the triplet lifetime. Estimation of activation free energy barriers to interconversion shows that the large values of DeltaG* are rather similar among polypeptides and that the protein substates involved are sufficiently long-lived to display individual binding/catalytic properties.

Journal Article↗

PubMed1993

Tryptophan phosphorescence as a structural probe of mitochondrial F1-ATPase epsilon-subunit.

We report the detection of tryptophan phosphorescence emission from the sole residue in the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex. The phosphorescence spectrum, intensity and decay kinetics have been measured over the temperature range 160-273 K. The fine structure in the phosphorescence spectrum at low temperature, with the 0-0 vibrational band centered at 411 nm, reveals the hydrophobic nature of the chromophore's environment. Both the large width of the 0-0 vibrational band and the heterogeneous decay kinetics in fluid solution emphasize the existence of multiple conformations of the epsilon-subunit, structures which are rather stable as they do not interconvert in the millisecond time scale. Further, from the relatively long triplet lifetime at 273 K, it is possible to infer the existence of a tight, rigid core in the structure of the epsilon-subunit. Under subunit-dissociating conditions (6 M urea), the spectrum at 160 K undergoes a slight blue shift but since the phosphorescence lifetime, at all temperatures, is similar or longer than in the absence of dissociant, we conclude that dissociation does not lead to solvent exposure of the tryptophanyl side-chain. This conclusion is supported by the results obtained at 273 K by dissociating F1 in the presence of 0.3 M guanidine hydrochloride. Phosphorescence lifetimes indicate that 6 M urea leads to a more compact structure of the epsilon-subunit, whereas the opposite occurs when Mg-ATP is added to nucleotide-depleted F1. These spectroscopic changes establish unequivocally that the binding of the adenine nucleotide to the enzyme is accompanied by conformational changes involving the epsilon-subunit.

Adenosine Triphosphate↗

PubMed1993

Tryptophan phosphorescence as a monitor of the solution structure of phosphoglycerate kinase from yeast.

The enzyme phosphoglycerate kinase from yeast possesses two tryptophan residues whose phosphorescence spectrum in low-temperature glasses is resolved into two distinct components with 0-0 vibronic bands centered at 408 and 412.5 nm. The thermal profile of the phosphorescence intensity and lifetime shows that the red (longer wavelength) component is quenched in fluid solutions so that the long-lived phosphorescence observed at ambient temperature in buffer is due entirely to the blue (shorter wavelength) component. The remarkable heterogeneity in flexibility of the two chromophores' sites inferred from the thermal behaviour, when analyzed in terms of the crystallographic structure, allows to make a straightforward assignment of the long-lived emission to internal Trp-333. Because in buffer the phosphorescence is due to only one Trp residue the biphasic nature of the decay reveals the presence of stable, slowly interconverting, conformers with profound differences in the internal fluidity of the C-domain. Further, according to the triplet lifetime, complex formation with substrates affect the protein structure in a very selective way. Thus, while 3-phosphoglycerate has practically no influence on the average lifetime, Mg ATP and Mg ADP increases tau by a factor of 1.9 and 5.3, respectively. The change in lifetime implies a remarkable stiffening of the C-domain which is partly relaxed in ternary complexes with 3-phosphoglycerate. These findings are discussed in terms of ligand-induced "closed" conformations of the protein.

Buffers↗

PubMed1993

Glycerol effects on protein flexibility: a tryptophan phosphorescence study.

In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C. Temperature effects were interpreted by proposing that solvent damping of internal protein motions is particularly effective for low frequency, large amplitude, structural fluctuations yielding highly flexible conformers of the macromolecule.

Alcohol Dehydrogenase↗

PubMed1992

Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli.

The Trp phosphorescence spectrum, intensity and decay kinetics of apo-aspartate aminotransferase, pyridoxamine-5P-aspartate-aminotransferase and pyridoxal-5P-aspartate aminotransferase were measured over a temperature range 160-273 K. The fine structure of the phosphorescence spectra in low-temperature glasses, with 0-0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine-5P-enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal-5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0-0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp-sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer. All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal-5P enzyme shows a decrease in lifetime-normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal-5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.

Apoenzymes↗

PubMed1992

Characterization of tryptophan and coenzyme luminescence in tryptophan synthase from Salmonella typhimurium.

Tryptophan synthase from Salmonella typhimurium is a bifunctional alpha 2 beta 2 complex that catalyzes the formation of L-tryptophan. We have characterized over the temperature range from 160 to 293 K the fluorescence and phosphorescence properties of the single tryptophan present at position 177 of the beta-subunit and of the pyridoxal 5'-phosphate bound through a Schiff's base in the beta-active site. The comparison between the fluorescence of the pyridoxal phosphate bound either to the protein or to valine free in solution indicates substantial protection for the coenzyme against thermal quenching and a greater intensity of the ketoenamine tautomer band. Trp-177 is highly luminescent, and its proximity to the pyridoxal moiety leads to an over 50% quenching of its fluorescence with both reduced and native coenzyme. The Trp phosphorescence spectrum possesses a narrow, well-defined, 0-0 vibrational band centered at 418.5 nm, a wavelength that indicates strong polar interactions with neighboring charges. The observation of delayed fluorescence in the native complex implies that the excited triplet state is involved in a process of triplet-singlet energy transfer to the ketoenamine tautomer. The rate of energy transfer, heterogeneous in low-temperature glasses with rate constants of 2.26 and 0.07 s-1, becomes homogeneous in fluid solutions as the coenzyme tautomer interconversion is likely faster than the phosphorescence decay. In both apo- and holo-alpha 2 beta 2, the phosphorescence from Trp-177 is long-lived even at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1992

Conformational changes and subunit communication in tryptophan synthase: effect of substrates and substrate analogs.

The transmission of regulatory signals between the alpha- and beta-subunits of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium has been investigated by monitoring the luminescence properties of the enzyme in the presence and in the absence of the alpha-subunit ligand DL-alpha-glycerol 3-phosphate, the alpha- and beta-subunit substrate indole, and the beta-subunit substrate analog L-histidine. The beta-subunit contains as intrinsic probes Trp-177 and pyridoxal 5'-phosphate, whereas the alpha-subunit has been mutagenized by replacing Ala-129 with a Trp residue. In contrast to the inertness of L-histidine, DL-alpha-glycerol 3-phosphate was found (i) to alter the phosphorescence spectrum of Trp-129, (ii) to shift the fluorescence thermal quenching profile of both Trp-177 and coenzyme to higher temperature, (iii) to slow down the triplet decay kinetics of Trp-177 in fluid solution, and (iv) to affect the equilibrium between different conformations of the enzyme. These findings provide direct evidence that DL-alpha-glycerol 3-phosphate binding affects the structure of the alpha-subunit and, in the presence of coenzyme, induces a conformational change in the beta-subunit that leads to a considerably more rigid structure. As opposed to DL-alpha-glycerol 3-phosphate, the shortening of the phosphorescence lifetime upon indole binding suggests that this substrate increases structural fluctuations in the beta-subunit. Implications for the mechanism of the allosteric regulation between alpha- and beta-subunits are discussed.

Amino Acid Sequence↗

PubMed1991

Tryptophan phosphorescence of G-actin and F-actin.

The tryptophan phosphorescence spectrum, intensity and decay kinetics of G-actin and F-actin were measured over a temperature range of 140-293 K. The fine structure in the phosphorescence spectra at low temperature, with O,O vibrational bands centered at 405 nm and 415.5 nm for both species, reveals a marked heterogeneity of the chromophore environment. The thermal quenching profile distinguishes these sites in terms of their flexibility, and shows that probably only one of the four tryptophan residues is still phosphorescent at ambient temperature due to its location in a relatively rigid buried core. Although some differences are demonstrated between G-actin and F-actin at low temperature, the identity of the triplet lifetime at ambient temperature strongly supports the notion that the conformation of the macromolecule is largely unaffected by polymerization. Preliminary phosphorescence anisotropy measurements demonstrate both the occurrence of singlet-singlet energy transfer among tryptophan residues and a strong immobilization of actin in the polymerized state.

Actins↗

PubMed1990

Tryptophan luminescence from liver alcohol dehydrogenase in its complexes with coenzyme. A comparative study of protein conformation in solution.

The extent of fluorescence quenching and that of phosphorescence quenching of Trp-15 and Trp-314 in alcohol dehydrogenase from horse liver as well as the intrinsic phosphorescence lifetime of Trp-314 in fluid solution have been utilized as structural probes of the macromolecule in binary and ternary complexes formed with coenzyme, analogous, and various substrate/inhibitors. Luminescence quenching by the coenzyme reveals that (1) while the reduced form quenches Trp emission exclusively from the fluorescent state, the oxidized form is very effective on the phosphorescent state as well and that (2) among the series of NADH binary and ternary complexes known by crystallographic studies to attain the closed form, distinct nicotinamide/indole geometrical arrangements are inferred from a variable degree of fluorescence quenching. Information of the dynamic structure of the coenzyme-binding domain derived from the phosphorescence lifetime of Trp-314 points out that within the series of closed NADH complexes there is considerable conformational heterogeneity. In solution, the variability in dynamical structure among the various protein complexes emphasizes that the closed/open forms identified by crystallographic studies are not two well-defined macrostates of the enzyme.

Alcohol Dehydrogenase↗

PubMed1990

Room temperature phosphorescence of Trp-314 as a monitor of subunit communications in alcohol dehydrogenase from horse liver.

The phosphorescence properties of liver alcohol dehydrogenase from horse were characterized at limiting concentrations of coenzyme and coenzyme analogues. The emission decay kinetics of Trp-314 in strong, slowly exchanging, ternary complexes with NADH/isobutyramide, NAD/pyrazole, and NADH/dimethyl sulfoxide displays a markedly nonexponential character. The analysis of decay components over the saturation curve reveals that the phosphorescence from singly bound protein molecules has a lifetime from 1 to 1.3 s, which is 2-3 times larger than observed with fully bound and unliganded enzyme. The remarkably tighter configuration reported by the triplet probe for the coenzyme-binding domain in half-saturated macromolecules is not exclusive of strongly inhibited ternary complexes. Measurements on binary complexes with NADH, ADPR, and the inactive coenzyme analogue 1,4,5,6-tetrahydronicotinamide adenine dinucleotide confirm that binding of the ligand to one subunit has qualitatively the same influence on protein structure. If the lifetime of Trp-314 provides clear evidence for an appreciable change in conformation at half-binding that is apparently triggered by the ADPR fragment of the coenzyme, such communication between subunits does not lead to allosteric phenomena in coenzyme binding.

Alcohol Dehydrogenase↗

PubMed1990

Temperature dependence of tryptophan phosphorescence in proteins.

The phosphorescence yield and decay kinetics of tryptophan (Trp) in apoazurin from Pseudomonas aeruginosa, subtilisin Carlsberg, Staphylococcal nuclease and liver alcohol dehydrogenase were determined as a function of temperature from 150 K (glassy matrix) to 300 K (fluid solution). The constancy of the lifetime-normalized phosphorescence yield with apoazurin and with Trp-314 in alcohol dehydrogenase establishes that the intersystem crossing quantum yield is practically unaffected across the temperature range. Consequently, any decrease in phosphorescence intensity not accounted for by lifetime-shortening is a signal either of the selective quenching of specific Trp residues in the same macromolecule or that the protein sample is heterogeneous in its emission properties. From an analysis of the thermal profile it is concluded that subtilisin Carlsberg and S. nuclease, as opposed to apoazurin, are not phosphorescent at ambient temperature, their residual emission probably arising from protein impurities. Criteria for distinguishing conformer emission from a contribution by protein impurities are discussed.

Alcohol Dehydrogenase↗

PubMed1989

Tryptophan phosphorescence as a monitor of the structural role of metal ions in alkaline phosphatase.

The phosphorescence properties of Trp109 in alkaline phosphatase from Escherichia coli have been utilized to probe the conformation of the polypeptide following the removal of metal ions, reconstitution with Zn2+ and Cd2+ and phosphorylation. The complete removal of metal ions induces a drastic loosening of the protein structure that extends to the inner core of the macromolecule. While binding of a single metal ion/subunit (A-site occupancy) restores the holoconformer, practically no structuring effect is observed upon B-site occupancy by the second incoming metal ion. An exception to this rule occurs at alkaline pH and when the adjacent subunit in the dimer is metal-free. Under these circumstances a conformation of the subunit more compact than that of the fully saturated dimer manifests some degree of communication across the subunit interface. The binding of more than two metal ions/monomer generally destabilizes the protein, the effect being more pronounced at acid pH. Finally, the binding of inorganic phosphate restores the native-like configuration abolishing any destabilization induced by excess metal ions and acid pH. If the negative cooperativity towards metal binding to A sites in doubly metalated forms at pH 8 is in substantial agreement with 113Cd-NMR data, the equivalence in conformation between Zn2+- and Cd2+-reconstituted alkaline phosphatase emphasizes that no serious structural changes are introduced by the metal replacement.

Alkaline Phosphatase↗

PubMed1989

Dynamical structure of glutamate dehydrogenase as monitored by tryptophan phosphorescence. Signal transmission following binding of allosteric effectors.

Changes in conformation of glutamate dehydrogenase from beef liver as a result of interactions with allosteric effectors have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas activators ADP and L-leucine enhance considerably the rigidity of the protein structure surrounding the chromophore, inhibitors GTP, Zn2+ and Ag+ act in an opposite manner increasing the flexibility of this region of the macromolecule. Such changes in dynamical structure of the protein are confirmed independently for the ADP and GTP complexes by oxygen diffusion studies. Phosphorescence lifetime measurements at various protein concentrations and with the enzyme crosslinked by glutaraldehyde demonstrate that ADP and GTP exert the same effect on the structure of the protein regardless of its degree of polymerization. The connection between changes in protein structure and regulatory function is strengthened by the finding that (1) ligands with no regulatory function (Eu3+) do not affect protein structure; (2) pairs of opposite effectors which neutralize each other's influence on catalytic activity do restore an apparent native-like structure in the enzyme. Mutual neutralization and the observation that ADP and GTP display maximum activity at partial saturation of the binding sites has been interpreted in terms of a model which assumes asymmetry in the hexameric enzyme at the trimer level. Evidence for the existence of conformational heterogeneity among the subunits of the enzyme has been provided.

Adenosine Diphosphate↗

PubMed1989

Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function.

The fluorescence and phosphorescence properties of the tryptophan residues in glutamate dehydrogenase were utilized to probe the conformation of the macromolecule at various states of aggregation of its subunits (hexamer, trimer, and monomer) in guanidine hydrochloride. According to the phosphorescence lifetime no gross alteration in the conformation of the protein follows from complete dissociation of the hexamer into native monomer, implying that the native fold is stabilized exclusively by intrasubunit bonding. Although modest concentrations of denaturant induce a change in configuration in the enzyme, a comparison with the macromolecule cross-linked into the hexameric form by glutaraldehyde confirms that this alteration in structure is not the result of subunit dissociation. Inhibition of catalysis by the denaturant is found to be considerably smaller than anticipated from the extent of hexamer dissociation. Furthermore, this inhibition is in no way prevented by cross-linking the enzyme in its hexameric form. This finding together with the ability of the trimer to bind the coenzyme and to undergo the characteristic structural changes induced by the effectors ADP and GTP suggests that, contrary to what is generally believed, the smallest functional unit of glutamate dehydrogenase is not the hexameric form.

Animals↗

PubMed1989

Phosphorescence properties and protein structure surrounding tryptophan residues in yeast, pig, and rabbit glyceraldehyde-3-phosphate dehydrogenase.

An investigation of the phosphorescence emission properties of tryptophan (Trp) was carried out in glyceraldehyde-3-phosphate dehydrogenase from yeast and from pig and rabbit muscle. Aided by the external heavy-atom effect of iodide, the dependence on excitation wavelength, and thermal quenching profiles, it was established that the 0,0 vibronic band peaked at 406 nm in the pig and rabbit proteins is made up of overlapping contributions from two Trp residues. In contrast to a previous report [Davis, J.M., & Maki, A.H. (1984) Biochemistry 23, 6249-6256], this implies that even in the muscle enzymes all three aromatic side chains are phosphorescent. Further, when the nature of the local environment of each residue is compared to the crystallographic structure of lobster GPDH, it leads to a complete new assignment of the individual phosphorescence spectra. With each protein, a single Trp, identified as Trp-310, was found to display long-lived phosphorescence at room temperature. The decay of this emission gives evidence of conformational homogeneity among the subunits of the tetrameric molecule.

Animals↗

PubMed1989

Phosphorescence properties of Trp-84 and Trp-310 in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

The phosphorescence spectra of Trp-84 and Trp-310 in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus in an aqueous glass show distinct 0,0 vibrational bands with peaks at 406.5 and 410.5 nm. With the aid of external heavy-atom perturbation of iodide and the thermal quenching profile, it is concluded that although both chromophores are effectively buried, only one, viz., the 406.5 nm component, is embedded in a sufficiently rigid core of the protein to phosphoresce in fluid solutions at room temperature. From inspection of the crystallographic structure is it evident that only Trp-310 embedded in the beta-sheet of the catalytic domain may satisfy the requirements of a long triplet-state lifetime and slow migration of O2 to its site. This identification confirms previous analysis of the phosphorescence properties of the enzymes from yeast, pig and rabbit muscle.

Geobacillus stearothermophilus↗

PubMed1988

Tryptophan phosphorescence and the conformation of liver alcohol dehydrogenase in solution and in the crystalline state.

Information on the effects of crystallization upon the structure of liver alcohol dehydrogenase from horse is obtained from a comparison of the phosphorescence properties of its tryptophan residues in solution and in the crystalline state. In the crystalline state the red shift in the phosphorescence spectrum of the solvent-exposed Trp-15 attests to a decreased polarity of its environment consistent with its shielding away from the aqueous solvent probably through its involvement in an intermolecular contact. On the other hand, the triplet-state lifetime of Trp-314 which is buried deeply in the coenzyme-binding domain demonstrates that the flexibility of this region of the macromolecule is unaffected by crystallization; a conclusion supported also by the similarity in the rate of oxygen quenching of its phosphorescence. Given that lattice constraints strongly inhibit large-scale conformational changes these results allow us to identify the average solution structure with the 'open' conformer determined crystallographically.

Alcohol Dehydrogenase↗

PubMed1987

Phosphorescence anisotropy of liver alcohol dehydrogenase in the crystalline state. Apparent glasslike rigidity of the coenzyme-binding domain.

Phosphorescence anisotropy from internal tryptophan (Trp) residues in proteins which are in the crystalline state may provide an experimental approach suitable to study the flexibility of rather rigid segments of protein structure. The phosphorescence anisotropy of Trp-314 in liver alcohol dehydrogenase, which is enclosed within the beta-sheet forming the coenzyme-binding domain, was measured with the protein free in solution and in the crystalline state. In contrast to the free protein, where the rotational correlation time reflects the tumbling rate of the whole macromolecule, there is effectively no loss in anisotropy in the crystalline state. At room temperature, the triplet lifetime of 0.5 s implies that the rotational correlation time of the indole side chain must be larger than 1 s. Anisotropy data show that fluctuations of the indole ring about the average position can only be of limited amplitude (cone of semiangle less than 15 degrees) and that the resistance opposed by the beta-sheet to out-of-plane rotational motions is equivalent to a viscosity larger than 2.5 X 10(8) P, a value which confirms the particular rigidity anticipated for such an assembly of secondary structure.

Alcohol Dehydrogenase↗