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M Gonnelli

Publications and source records attributed to M Gonnelli.

14 recordsLinked to original sources
PubMed2001

No effect of trimethylamine N-oxide on the internal dynamics of the protein native fold.

Trimethylamine N-oxide (TMAO) is a natural osmolyte accumulated in cells of organisms as they adapt to environmental stresses. In vitro, TMAO increases protein stability and forces partially unfolded structures to refold. Its effects on the native fold are unknown. To investigate the interrelationship between protein stability, internal dynamics and function, the influence of TMAO on the flexibility of the native fold was examined with four different proteins by Trp phosphorescence spectroscopy. Its influence on conformational dynamics was assessed by both the intrinsic phosphorescence lifetime, which reports on the local structure about the triplet probe, and the acrylamide bimolecular quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results demonstrate that for apoazurin, alcohol dehydrogenase, alkaline phosphatase and glyceraldehydes-3-phosphate dehydrogenase 1.8 M TMAO does not perturb the flexibility of these macromolecules in a temperature range between - 10 degreesC and up to near the melting temperature. This unexpected finding contrasts with the dampening effect observed with polyols as well as with the expectations based on the preferential exclusion of the osmolyte from the protein surface.

Acrylamide↗
PubMed2000

Sensitive monitoring of the dynamics of a membrane-bound transport protein by tryptophan phosphorescence spectroscopy.

This paper presents a tryptophan phosphorescence spectroscopy study on the membrane-bound mannitol transporter, EII(mtl), from E. coli. The protein contains four tryptophans at positions 30, 42, 109, and 117. Phosphorescence decays in buffer at 1 degrees C revealed large variations of the triplet lifetimes of the wild-type protein and four single-tryptophan-containing mutants. They ranged from <70 microseconds for the tryptophan at position 109 to 55 ms for the residue at position 30, attesting to widely different flexibilities of the tryptophan microenvironments. The decay of all tryptophans is multiexponential, reflecting multiple stable conformations of the protein. Both mannitol binding and enzyme phosphorylation had large effects on the triplet lifetimes. Mannitol binding induces a more ordered structure near the mannitol binding site, and the decay becomes significantly more homogeneous. In contrast, enzyme phosphorylation induces a large relaxation of the protein structure at the reporter sites. The implications of these structural changes on the coupling mechanism between the transport and the phosphorylation activity of EII(mtl) are discussed. Taken as a whole, our data show that tryptophan phosphorescence spectroscopy is a very sensitive technique to explore conformational dynamics in membrane proteins.

Binding Sites↗
PubMed1998

Tyrosine quenching of tryptophan phosphorescence in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

Tyrosine is known to quench the phosphorescence of free tryptophan derivatives in solution, but the interaction between tryptophan residues in proteins and neighboring tyrosine side chains has not yet been demonstrated. This report examines the potential role of Y283 in quenching the phosphorescence emission of W310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by comparing the phosphorescence characteristics of the wild-type enzyme to that of appositely designed mutants in which either the second tryptophan residue, W84, is replaced with phenylalanine or Y283 is replaced by valine. Phosphorescence spectra and lifetimes in polyol/buffer low-temperature glasses demonstrate that W310, in both wild-type and W84F (Trp84-->Phe) mutant proteins, is already quenched in viscous low-temperature solutions, before the onset of major structural fluctuations in the macromolecule, an anomalous quenching that is abolished with the mutation Y283V (Tyr283-->Val). In buffer at ambient temperature, the effect of replacing Y283 with valine on the phosphorescence of W310 is to lengthen its lifetime from 50 micros to 2.5 ms, a 50-fold enhancement that again emphasizes how W310 emission is dominated by the local interaction with Y283. Tyr quenching of W310 exhibits a strong temperature dependence, with a rate constant kq = 0.1 s(-1) at 140 K and 2 x 10(4) s(-1) at 293 K. Comparison between thermal quenching profiles of the W84F mutant in solution and in the dry state, where protein flexibility is drastically reduced, shows that the activation energy of the quenching reaction is rather small, Ea < or = 0.17 kcal mol(-1), and that, on the contrary, structural fluctuations play an important role on the effectiveness of Tyr quenching. Various putative quenching mechanisms are examined, and the conclusion, based on the present results as well as on the phosphorescence characteristics of other protein systems, is that Tyr quenching occurs through the formation of an excited-state triplet exciplex.

Geobacillus stearothermophilus↗
PubMed1998

Alteration of the intramolecular dynamics of glycogen phosphorylase b by allosteric ligands.

Phosphorylase b (E.C. 2.4.1.1), prepared from rabbit skeletal muscle, was used to study whether the binding of allosteric ligands modifies the intramolecular dynamics of the protein matrix. Protein dynamics were monitored through the fluorescence and phosphorescence parameters of the 12 tryptophan (Trp) residues (one monomer) of the enzyme. The phosphorescence lifetime was measured at room temperature both in the absence and the presence of ligands. The addition of an allosteric inhibitor (ATP) decreased the lifetime, while the presence of activator (AMP) and/or substrate (G-1-P) had no detectable effect. The lifetime data allow us to conclude that the environment of the buried tryptophans becomes more flexible upon the binding of ATP, while the other ligands did not induce such change. The ATP-induced perturbation was also examined by the quenching of Trp fluorescence by acrylamide. The quenching parameters did not show any change, suggesting that the effect of ATP is localized to the vicinity of the phosphorescent Trp residues.

Allosteric Site↗
PubMed1997

Time-resolved protein phosphorescence in the stopped-flow: denaturation of horse liver alcohol dehydrogenase by urea and guanidine hydrochloride.

This study reports the implementation of room temperature protein phosphorescence in the stopped-flow technique. Time-resolved Trp phosphorescence can now be detected following rapid mixing of protein solutions with a time resolution of 10 ms and a sensitivity in terms of chromophore concentration down to 0.1 microM. Calibration tests with monomeric and multimeric proteins proved that in all cases the delayed emission is not affected by artefacts that could arise from either enrichment of trace impurities along the flow lines or deformation of the macromolecules by the shear stress of laminar flow. To illustrate the potential of Trp phosphorescence in the stopped-flow to detect the time evolution of protein conformation the interaction of urea and guanidine hydrochloride (GdnHCl) with the native structure of horse liver alcohol dehydrogenase (LADH) has been re-examined under conditions of rapid denaturation. Remarkable differences in the action of the two denaturing agents has been confirmed by the phosphorescence lifetime (tauP) of the internal Trp residue (W314). Whereas in urea, up to 8 M, tauP is not minimally perturbed, in GdnHCl it decreases sharply and progressively from 800 ms down to 23 ms in 6 M solutions. Such reduction of tauP implies that in the region of W314 the polypeptide structure has become highly loose and flexible prior to the major unfolding transition. Therefore, denaturation of LADH in GdnHCl, as opposed to urea, proceeds from a partly unfolded intermediate conformation of the protein. Other characteristics of this intermediate state are a partial loss of tertiary structure, as revealed by the circular dichroism of the aromatics, and an almost complete inhibition of the catalytic activity. Control experiments with equimolar NaCl demonstrate that tauP, the tertiary structure and the catalytic activity are affected to a much smaller extent and that, therefore, salt effects do not account for the difference between urea and GdnHCl. Finally, measurements of the unfolding reaction emphazise that the kinetics of LADH denaturation are heterogeneous with both denaturing agents. From the constancy of tauP during the course of the reaction it is concluded that the multiphasic behavior is a manifestation of multiple unfolding pathways owing to a plurality of stable LADH conformations.

Alcohol Dehydrogenase↗
PubMed1995

Phosphorescence lifetime of tryptophan in proteins.

This investigation enquires into the factors that are responsible for the wide range of room-temperature Trp phosphorescence lifetimes (tau) in proteins. By exploiting the enhanced sensitivity and time resolution of phosphorescence measurements, experiments were conducted to evaluate the triplet quenching potential of each amino acid side chain. From the magnitude of the Stern-Volmer rate constant it is concluded that, among the amino acids, quenching reactions at 20 degrees C are quite effective with His, Tyr, Trp, cysteine, and cystine, with rate enhancements of 20 and 50 times when the side chains of Tyr and His, respectively, are in the ionized form. The distance dependence of the quenching interaction, estimated from the quenching of internal Trp residues in proteins separated from the amino acid in solution by a protein spacer of various thickness, emphasizes the very short-range nature of the process. The importance of these side chains, and to some extent that of the peptide linkage, as intrinsic quenchers of Trp phosphorescence in proteins was also confirmed with short synthetic peptides prepared appositely with only one type of these residues. Finally, very short (microseconds) phosphorescence lifetimes of Trp residues in proteins were shown to be invariably associated with the presence of Tyr or Cys in the immediate neighborhood of the chromophore. From a survey of the amino acids that are nearest neighbors to Trp in proteins and the corresponding value of tau it was established that, in the absence of His, Tyr, Trp, and Cys, tau is > or = 1 ms and appears to reflect mainly the local fluidity of the protein structure. Otherwise, tau can be much shorter, and for bulky His, Tyr, and Trp side chains it seems to depend dramatically on the mutual chromophore-quencher orientation. In these cases the triplet decay kinetics is shown to be a complex function of temperature, pH, and flexibility of the protein site.

Amino Acid Sequence↗
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

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↗
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↗
PubMed1986

Effects of urea and guanidine hydrochloride on the activity and dynamical structure of equine liver alcohol dehydrogenase.

The inactivation of equine liver alcohol dehydrogenase by guanidine hydrochloride and urea has been studied by monitoring the intrinsic tryptophan fluorescence and phosphorescence emission. The use of triplet-state lifetimes to probe the flexibility of protein structure at the site of tryptophan-314 reveals a distinct behavior between the two denaturants. At predenaturational concentrations, the loss of enzyme activity in guanidine hydrochloride is associated with a loosening of intramolecular interactions resulting in a greater fluidity of the interior region of the macromolecule. In contrast, the interaction with urea, even at high concentrations, does not alter the dynamics of the native conformation. Enzyme activity is irreversibly lost as a result of a drastic unfolding of the macromolecule which occurs in a highly cooperative two-stage process.

Alcohol Dehydrogenase↗
PubMed1986

The rate of equine liver alcohol dehydrogenase denaturation by urea. Dependence on temperature and denaturant concentration.

The kinetics of the irreversible urea denaturation of equine liver alcohol dehydrogenase have been studied as a function of temperature and urea concentration. The unfolding of the macromolecule, monitored by means of the phosphorescence properties of a deeply buried tryptophan residue, was found to be strictly a two-state process over the entire temperature range. It is characterized by a steep dependence on urea concentration typical of highly cooperative transitions and below room temperature it possesses large negative activation energies. The reaction is comparatively slow, does not seem to be preceded by a fast phase, and the rate-limiting step does not have the characteristics of proline isomerization. When the data are analyzed in terms of binding equilibria the temperature dependence results from an anomalously large change in heat capacity. Although this is a property of strong hydrophobic interactions in model compounds the slow rates of denaturation are best understood with a model of protein stability which emphasizes the cooperative nature of intramolecular interactions such as hydrogen bonding.

Alcohol Dehydrogenase↗
PubMed1985

Purification and characterization of two leaf polypeptide inhibitors of leaf protease from alfalfa (Medicago sativa).

Two polypeptides with antiproteolytic activities have been isolated from alfalfa leaves. Polypeptide I resembles the previously described plant protease inhibitors in both structural and functional features; it has a molecular weight of 15,000, a random coil secondary structure, and inhibits exogenous protease as well as alfalfa leaf protease. Polypeptide II is a novel type of plant inhibitor with a molecular weight of 6300 and a highly organized structure with a high (40-50%) alpha-helix content. It only inhibits endogenous protease with a molar stoichiometry polypeptide/enzyme protein of 1.

Amino Acids↗