Biomedical subjects

Giovanni B Strambini

Publications and source records attributed to Giovanni B Strambini.

16 recordsLinked to original sources

PubMed2006

Protein stability in ice.

This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (DeltaG degrees ) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from -8 to -16 degrees C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (V(L)) in equilibrium with the solid phase. The data establish a threshold, around V(L)=1.5%, below which in ice DeltaG degrees decreases progressively relative to liquid state, up to 3 kcal/mole for V(L)=0.285%. The sharp dependence of DeltaG degrees on V(L) is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in DeltaG degrees is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins.

Azurin↗

PubMed2006

Dendrimer-protein interactions studied by tryptophan room temperature phosphorescence.

Dendrimers are a relatively new class of materials with unique molecular architectures, which provide promising opportunities for biological applications as DNA carriers and drug delivery systems. Progress in these fields, however, requires knowledge of their potential interactions with biological components at cellular and molecular level. This study utilizes Trp phosphorescence spectroscopy to examine possible perturbations of the protein native fold in solution by neutral, positively and negatively charged fifth generation polyamidoamine (PAMAM) dendrimers. Phosphorescence lifetime measurements, conducted on model proteins varying in the degree of burial of the triplet probe and in quaternary structure, show that dendrimers interact with proteins in solutions forming stable complexes in which the protein structure may be significantly altered, particularly in superficial, flexible regions of the polypeptide. Both electrostatic and non-electrostatic interactions can give rise to stable complexes, whose affinity and limited number of binding sites distinguish them from mere aspecific molecular associations. Of direct relevance for the application of these polymers in the medical field, structural alterations have also been detected in human plasma proteins such as serum albumin and immunoglobulins. The above results suggest that Trp phosphorescence may provide a useful monitor for working out experimental conditions and protocols that help preserve the structural integrity of proteins in the presence of these polymers.

Dendrimers↗

PubMed2006

ANS fluorescence detects widespread perturbations of protein tertiary structure in ice.

Freeze-induced perturbations of the protein native fold are poorly understood owing to the difficulty of monitoring their structure in ice. Here, we report that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a general monitor of ice-induced alterations of their tertiary structure. Experiments conducted with copper-free azurin from Pseudomonas aeruginosa and mutants I7S, F110S, and C3A/C26A correlate the magnitude of the ice-induced perturbation, as inferred from the extent of ANS binding, to the plasticity of the globular fold, increasing with less stable globular folds as well as when the flexibility of the macromolecule is enhanced. The distortion of the native structure inferred from ANS binding was found to draw a parallel with the extent of irreversible denaturation by freeze-thawing, suggesting that these altered conformations play a direct role on freeze damage. ANS binding experiments, extended to a set of proteins including serum albumin, alpha-amylase, beta-galactosidase, alcohol dehydrogenase from horse liver, alcohol dehydrogenase from yeast, lactic dehydrogenase, and aldolase, confirmed that a stressed condition of the native fold in the frozen state appears to be general to most proteins and pointed out that oligomers tend to be more labile than monomers presumably because the globular fold can be further destabilized by subunit dissociation. The results of this study suggest that the ANS binding method may find practical utility in testing the effectiveness of various additives employed in protein formulations as well as to devise safer freeze-drying protocols of pharmaceutical proteins.

Anilino Naphthalenesulfonates↗

PubMed2005

Substrate-induced conformational changes in the membrane-embedded IIC(mtl)-domain of the mannitol permease from Escherichia coli, EnzymeII(mtl), probed by tryptophan phosphorescence spectroscopy.

Membrane-bound transport proteins are expected to proceed via different conformational states during the translocation of a solute across the membrane. Tryptophan phosphorescence spectroscopy is one of the most sensitive methods used for detecting conformational changes in proteins. We employed this technique to study substrate-induced conformational changes in the mannitol permease, EnzymeII(mtl), of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Ten mutants containing a single tryptophan were engineered in the membrane-embedded IIC(mtl)-domain, harboring the mannitol translocation pathway. The mutants were characterized with respect to steady-state and time-resolved phosphorescence, yielding detailed, site-specific information of the Trp microenvironment and protein conformational homogeneity. The study revealed that the Trp environments vary from apolar, unstructured, and flexible sites to buried, highly homogeneous, rigid peptide cores. The most remarkable example of the latter was observed for position 97, because its long sub-second phosphorescence lifetime and highly structured spectra in both glassy and fluid media imply a well defined and rigid core around the probe that is typical of beta-sheet-rich structural motifs. The addition of mannitol had a large impact on most of the Trp positions studied. In the case of position 97, mannitol binding induced partial unfolding of the rigid protein core. On the contrary, for residue positions 126, 133, and 147, both steady-state and time-resolved data showed that mannitol binding induces a more ordered and homogeneous structure around these residues. The observations are discussed in context of the current mechanistic and structural model of EII(mtl).

Amino Acid Motifs↗

PubMed2005

Effects of sucrose on the internal dynamics of azurin.

Sucrose is a natural osmolyte accumulated in cells of organisms as they adapt to environmental stresses. In vitro, sucrose increases protein stability and forces partially unfolded structures to refold. Its effects on the native fold structure and dynamics are not fully established. This study, utilizing Trp phosphorescence spectroscopy, examined the influence of molar concentrations of sucrose on the flexibility of metal-free azurin from Pseudomonas aeruginosa. In addition, by means of specific mutants of the test protein, namely I7S, F110S, and C3A/C26A, that altered its thermodynamic stability, its intrinsic flexibility, and the extent of internal hydration, this investigation sought to identify possible correlations between these features of protein structure and the influence of the osmolyte on protein dynamics. Alterations of structural fluctuations were assessed by both the intrinsic phosphorescence lifetime (tau), which reports on local structure about the triplet probe, and the acrylamide bimolecular quenching rate constant (k(q)) that is a measure of the average acrylamide diffusion coefficient through the macromolecule. From the modulation of tau and k(q) across a wide temperature range and up to a concentration of 2M sucrose, it is concluded that sucrose attenuates structural fluctuations principally when macromolecules are internally hydrated and thermally expanded. Preliminary tests with trehalose and xylitol suggest that the effects of sucrose are general of the polyol class of osmolytes.

Acrylamide↗

PubMed2004

Effects of cavity-forming mutations on the internal dynamics of azurin.

The effects of two single-point cavity-forming mutations, F110S and I7S, on the internal dynamics of azurin from Pseudomonas aeruginosa were probed by the phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)) whereas more general effects on structural fluctuations were deduced from the phosphorescence acrylamide quenching rate constant (k(q)), which measures the diffusion of the solute through the protein fold. The results show a spectacular, 4-5 orders of magnitude, increase of k(q) emphasizing that large amplitude structural fluctuations permitting acrylamide migration to the protein core have been drastically enhanced in each azurin mutant. The large, 12-15 kcal/mol, decrease in the activation enthalpy associated to k(q) suggests that the rate enhancement is caused, rather than through a generalized increase of protein flexibility, by the elimination of an inner barrier to the diffusion process. According to tau(0) the chromophore environment is more fluid with I7S but strikingly more rigid with F110S, demonstrating that when internal cavities are formed local effects on the mobility at the mutation site are unpredictable. Both tau(0) and k(q) reveal a structure tightening role of bound Cd(2+) that correlates with the increase in stability from apo- to holo-azurin. While these alterations in internal dynamics of azurin do not seem to play a role on electron transfer through the central region, the enhanced migration of acrylamide emphasizes that cavities may be critical for the rapid diffusion of substrates to buried, solvent inaccessible sites of enzymes.

Acrylamide↗

PubMed2004

Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain.

The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.

Actins↗

PubMed2003

Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core.

The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component of Rhodospirillum rubrum transhydrogenase are described. This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer. Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions. Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s). The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50 degrees C. In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime. In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site. It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer. The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.

Acrylamide↗

PubMed2003

Structure and dynamics of proteins encapsulated in silica hydrogels by Trp phosphorescence.

This report establishes the conditions for monitoring the intrinsic Trp phosphorescence of proteins encapsulated in silica hydrogels and demonstrates the usefulness of the delayed emission for examining potential perturbations of protein structure-dynamics by the silica matrix. Phosphorescence measurements were conducted both in low temperature (140 K) glasses and at ambient temperature on the proteins apo- and Cd-azurin, alkaline phosphatase and liver alcohol dehydrogenase together with the complexes of liver alcohol dehydrogenase with coenzyme analogs ADPR and H(2)NADH. While spectral shifts and broadening indicate that alterations of the Trp microenvironment are more marked on superficial regions of the macromolecule the decay kinetics of deeply buried chromophores show that the internal flexibility of the polypeptide in two out of three cases is significantly affected by silica entrapment. Both the intrinsic lifetime and the bimolecular acrylamide quenching constant confirm that, relative to the aqueous solution, in hydrogels the globular fold is more rigid with azurin, looser with alcohol dehydrogenase and substantially unaltered with alkaline phosphatase. It was also noted that large amplitude structural fluctuations, as those involved in coenzyme binding to alcohol dehydrogenase or thermally activated in alkaline phosphatase, were not restricted by gelation. Common features of the three silica entrapped proteins are pronounced conformational heterogeneity and immobilization of rotational motions of the macromolecule in the long time scale of seconds.

Acrylamides↗

PubMed2003

Perturbation of protein tertiary structure in frozen solutions revealed by 1-anilino-8-naphthalene sulfonate fluorescence.

Although freeze-induced perturbations of the protein native fold are common, the underlying mechanism is poorly understood owing to the difficulty of monitoring their structure in ice. In this report we propose that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a useful monitor of ice-induced strains on the native fold. Experiments conducted with copper-free azurin from Pseudomonas aeruginosa, as a model protein system, demonstrate that in frozen solutions the fluorescence of ANS is enhanced several fold and becomes blue shifted relative free ANS. From the enhancement factor it is estimated that, at -13 degrees C, on average at least 1.6 ANS molecules become immobilized within hydrophobic sites of apo-azurin, sites that are destroyed when the structure is largely unfolded by guanidinium hydrochloride. The extent of ANS binding is influenced by temperature of ice as well as by conditions that affect the stability of the globular structure. Lowering the temperature from -4 degrees C to -18 degrees C leads to an apparent increase in the number of binding sites, an indication that low temperature and /or a reduced amount of liquid water augment the strain on the protein tertiary structure. It is significant that ANS binding is practically abolished when the native fold is stabilized upon formation of the Cd(2+) complex or on addition of glycerol to the solution but is further enhanced in the presence of NaSCN, a known destabilizing agent. The results of the present study suggest that the ANS binding method may find practical utility in testing the effectiveness of various additives employed in protein formulations as well as to devise safer freeze-drying protocols of pharmaceutical proteins.

Anilino Naphthalenesulfonates↗

PubMed2003

Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein.

Biosynthetic incorporation of tryptophan (Trp) analogs such as 7-azatryptophan, 5-hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared. Until now, the approach has dealt primarily with soluble proteins. In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EII(mtl)). EII(mtl) overexpression was under control of the lambdaP(R) promoter, and the E. coli Trp auxotroph M5219 was used as host. This strain constitutively expresses the heat labile repressor protein of the lambdaP(R) promoter. Together with the presence of the repressor gene on the EII(mtl) plasmid, this resulted in a tightly controlled promoter system, a prerequisite for high Trp analog incorporation. A new method for determining the analog incorporation efficiency is presented that is suitable for membrane proteins. The procedure involves fitting of the phosphorescence spectrum as a linear combination of the Trp and Trp analog contributions, taking into account the influence of the protein environment on the Trp analog spectrum. The data show that the analog content of EII(mtl) samples is very high (>95%). In addition, we report here that biosynthetic incorporation of Trp analogs can also be effected with less expensive indole analogs, which in vivo are converted to L-Trp analogs.

Cell Membrane↗

PubMed2002

Tryptophan phosphorescence and pressure effects on protein structure.

After a brief introduction of the potentialities of Trp phosphorescence spectroscopy for probing the conformation and flexibility of protein structure, this presentation summarizes the effects of hydrostatic pressure (up to 3 kbar) on the native fold of monomeric and oligomeric proteins as inferred from the variation of the intrinsic phosphorescence lifetime and the oxygen and acrylamide bimolecular quenching rate constants of buried Trp residues. The pressure/temperature response of the globular fold and modulation of its dynamical structure is analyzed both in terms of a reduction of internal cavities and of hydration of the polypeptide. The implications of these findings for the thermodynamic stability of proteins and for the determination of subunit dissociation equilibria under high pressure conditions are also discussed.

Fluorescent Dyes↗

PubMed2002

Effect of heavy water on protein flexibility.

The effects of heavy water (D(2)O) on internal dynamics of proteins were assessed by both the intrinsic phosphorescence lifetime of deeply buried Trp residues, which reports on the local structure about the triplet probe, and the bimolecular acrylamide phosphorescence quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results obtained with several protein systems (ribonuclease T1, superoxide dismutase, beta-lactoglobulin, liver alcohol dehydrogenase, alkaline phosphatase, and apo- and Cd-azurin) demonstrate that in most cases D(2)O does significantly increase the rigidity the native structure. With the exception of alkaline phosphatase, the kinetics of the structure tightening effect of deuteration are rapid compared with the rate of H/D exchange of internal protons, which would then assign the dampening of structural fluctuations in D(2)O to a solvent effect, rather than to stronger intramolecular D bonding. Structure tightening by heavy water is generally amplified at higher temperatures, supporting a mostly hydrophobic nature of the underlying interaction, and under conditions that destabilize the globular fold.

Acrylamide↗

PubMed2002

Interactions between PEG and type I soluble tumor necrosis factor receptor: modulation by pH and by PEGylation at the N terminus.

The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF-RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF-RI bound free PEG in a pH-dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp-107, at the C terminus, with the pH-dependent and PEGylation-dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF-RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp-107. Deprotonation of His-105, which is perpendicular to Trp-107, was integral to the binding mechanism producing a pH-dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.

Etanercept↗

PubMedDate not supplied

The triplet-state lifetime of indole derivatives in aqueous solution.

An important feature of tryptophan phosphorescence, crucial for probing protein structure and dynamics, is the drastic reduction of the lifetime (tau) in fluid solutions. Initial reports of indole and derivatives showed that tau decreases from 6 s in rigid glasses to about 1 ms in aqueous solutions at ambient temperature. Recently a report by Fischer et al. questioned the validity of the millisecond lifetime, claiming that in millimolar electrolyte solutions tau is about 40 micros, similar to the 12-30 micros of earlier determinations based on flash photolysis. Longer lived phosphorescence was detected in pure water but because it exhibited an initial growing phase and an anomalously large triplet yield, the emission was attributed to an artifact arising from the slow, first-order, geminate recombination of the radical cation and electron generated by photochemistry. In this study, we reexamine both the phosphorescence lifetime and the triplet quantum yield of indole, N-acetyl tryptophanamide (NATA), N-methyl tryptophan and the tryptophan-glycine-glycine tripeptide under the same conditions adopted by Fischer et al. as well as over a wider range of electrolyte and buffering salts concentrations, pH, solvent and temperature. Throughout, the results show that the phosphorescence decay is slow and uniform down to the 12 micros resolution of the instrument, with no evidence of short-lived, 40 micros-like components. Most compelling was the similarity between the fluorescence-normalized triplet yield of indole derivatives in water and that of W59 in the protein ribonuclease T1 or of NATA in rigid glasses. Its invariance over experimental conditions that varied the production of photoproducts several fold and the characteristic susceptibility of the triplet lifetime to O2, proton and ground state quenching demonstrated that the triplet state was formed predominantly through normal intersystem crossing and that its unquenched lifetime was at least 9 ms.

Buffers↗

PubMedDate not supplied

Intramolecular quenching of tryptophan phosphorescence in short peptides and proteins.

The phosphorescence lifetime (tau) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of tau in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (k(obs)) in tripeptides of the form acetyl-Trp-Gly-X-CONH2 (WGX), where Trp is joined to X by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (k(obs) > 40 s(-1)), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys >> His+ > Tyr >> Phe approximately His. For the aromatic side chains the corresponding contact rate at 20 degrees C is estimated to be between 3-4 x 10(9) s(-1) for Cys (as determined by Lapidus et al.), 0.8-8 x 10(6) s(-1) for His+, 0.37-3.7 x 10(6) s(-1) for Tyr and 0.2-2 x 10(5) s(-1) for Phe and His. In the cases of His and Tyr, k(obs) drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between -20 degrees C and 20 degrees C, the reaction is characterized by activation energies of about 5 kcal.M(-1) for His+ and Tyr and 8 kcal.M(-1) for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s(-1), showing that these moieties are practically inert with respect to the triplet-state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s(-1), a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

Cysteine↗