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.     

Role of Protein Cavities on Unfolding Volume Change and on Internal Dynamics under Pressure

The effects of two single point cavity forming mutations, F110S and I7S, on the unfolding volume change (DeltaV(0)) of azurin from Pseudomonas aeruginosa and on the internal dynamics of the protein fold under pressure were probed by the fluorescence and phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Pressure-induced unfolding, monitored by the shift of the center of mass of the fluorescence spectrum, showed that DeltaV(0) is in the range of 60-70 mL/mol, not significantly different between cavity mutants and compact azurin species such as the wild-type and the mutant C3A/C26A, in which the superficial disulphide has been removed. The lack of extra volume in F110S and I7S proves that the engineered cavities, 40 A(3) in I7S and 100 A(3) in F110S, are filled with water molecules. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)). The application of pressure in the predenaturation range initially decreases the internal flexibility of azurin, the trend eventually reverting on approaching unfolding. The main difference between compact folds, wild-type and C3A/C26A, and cavity mutants is that the inversion point is powered from approximately 3 kbar to 1.5 kbar for F110S and <0.1 kbar for I7S, meaning that in the latter species pressure-induced internal hydration dominates very early over any compaction of the globular fold resulting from the reduction of internal free volume. The similar response between wild-type and the significantly less-stable C3A/C26A mutant suggests that thermodynamic stability per se is not the dominant factor regulating pressure-induced internal hydration of proteins.     

Protein dynamics and pressure: what can high pressure tell us about protein structural flexibility?

After a brief overview of NMR and X-ray crystallography studies on protein flexibility under pressure, we summarize the effects of hydrostatic pressure on the native fold of azurin from Pseudomonas aeruginosa as inferred from the variation of the intrinsic phosphorescence lifetime and the acrylamide bimolecular quenching rate constants of the buried Trp residue. 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 the hydration of the polypeptide. The study of the effect of two single point cavity forming mutations, F110S and I7S, on the unfolding volume change (ΔV(0)) of azurin and on the internal dynamics of the protein fold under pressure demonstrate that the creation of an internal cavity will enhance the plasticity and lower the stability of the globular structure. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Amplitude spectrum of structural fluctuations in proteins from the internal diffusion of solutes of increasing molecular size: a Trp phosphorescence quenching study

The accessibility of O(2), acrylamide, and four acrylamide derivatives of increasing molecular size {N-(hydroxymethyl)acrylamide, N,N'-methylene-bisacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid} to buried Trp residues in four proteins, as determined by dynamic quenching of their phosphorescence emission, was utilized for probing the amplitude range of structural fluctuations in these macromolecules. The quenching rate constant of each solute, k(q), was determined (at 25 and -5 °C) for liver alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, azurin, and alkaline phosphatase. The results show that high-frequency small amplitude motions pervade the protein globular fold, permitting relatively unhindered diffusion of small diatomic molecules all the way to compact cores of the macromolecule. For larger solutes, the access to deep regions drops sharply with molecular size, with acrylamide probably representing a threshold for diffusion of a solute through homogeneous compact domains, on the long second time scale. The results emphasize the variability in the amplitude of protein motions between deep cores and more superficial regions of the globular fold and unveil the existence of unexpectedly large amplitude low-activation barrier fluctuations permitting the penetration of solutes with comparatively large M(w) values.     

Cavity-creating mutations in Pseudomonas aeruginosa azurin: effects on protein dynamics and stability

Changes in flexibility and structural stability of Pseudomonas aeruginosa azurin in response to cavity-creating mutations were probed by the phosphorescence emission of Trp-48, which was deeply buried in the compact hydrophobic core of the macromolecule, and by measurements of guanidinum hydrochloride unfolding, respectively. Replacement of the bulky side chains Phe-110, Phe-29, and Tyr-108 with the smaller Ala introduced cavities at different distances from the hydrophobic core. The phosphorescence lifetime (τ₀) of Trp-48, buried inside the protein core, and the acrylamide quenching rate constant (k_q) were used to monitor local and global flexibility changes induced by the introduction of the cavity. The results of this work demonstrate the following: 1), the effect on core flexibility of the insertion of cavities is not correlated readily to the distance of the cavity from the core; 2), the protein global flexibility results are related to the cavity distance from the packed core of the macromolecule; and 3), the increase in protein flexibility does not correspond necessarily to a comparable destabilizing effect of some mutations.     

Pressure/temperature effects on protein flexibilty from acrylamide quenching of protein phosphorescence.

Pressure is an effective modulator of protein structure and biological function. The influence of hydrostatic pressure (相似文献   

Oxygen and acrylamide quenching of protein phosphorescence: correlation with protein dynamics

Oxygen quenching of protein phosphorescence and activation enthalpies for the structural fluctuations underlying O2 and acrylamide diffusion were determined for RNase T1, glyceraldehyde-3-phosphate dehydrogenase and beta-lactoglobulin, which have the phosphorescing residues located in relatively solvent-exposed and flexible regions of the polypeptide. The results, compared with those obtained for proteins characterised by a very rigid environment, established that kqO2 was directly correlated to the flexibility of the protein matrix surrounding the chromophore. While the migration of acrylamide was characterised by delta H(double dagger), which was strongly dependent on the fluidity of the structure about the Trp residue, the values of the activation enthalpies for the oxygen migration of all the proteins studied were rather similar, approximately 10 kcal mol(-1), in spite of the depth of the chromophore and the rigidity of its environment. The implications of these findings for the migration of small solutes inside proteins have been discussed.     

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.     

Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

The fine structure of luminescence spectra of azurin.

The spectra of azurin absorption, fluorescence, phosphorescence and fluorescence excitation have been measured in aqueous solutions at ordinary and liquid nitrogen temperatures. The fluorescence spectra of azurin even at ordinary temperatures have a well resolved fine vibrational structure. The frequency analysis reveals practically the same wave number distances between the main structure peaks in fluorescence spectra at room and low temperatures and in phosphorescence spectra. The comparison of the protein absorption and excitation spectra shows that all the energy absorbed by tyrosine residues is transferred onto indole chromophore. These data suggest an unusual tryptophan environment in this protein, which is characterized by the absence of any hydrogen bonding or other polar interaction of tryptophan with its environment. The problem of the possibility of contributions of two electronic transitions (1La in equilibrium A and 1Lb in equilibrium A) in absorption and emission spectra of azurin tryptophan arising from their mirror symmetry is discussed.     

Dynamic features of the subunit interface of Cu,Zn superoxide dismutase as probed by tryptophan phosphorescence.

As part of the more general inquiry on the molecular basis of specific recognition between macromolecules, the subunit-subunit interface structure of dimeric superoxide dismutase from Photobacterium leiognathi has been probed selectively by the phosphorescence emission of Trp-73, located at the subunit contact region. Copper at the catalytic site was found to quench completely the delayed emission and therefore all studies were conducted with the copper-free or Cd(2+)-substituted protein. The spectrum at 140 K is diagnostic for an indole ring located in a hydrophobic environment whereas a degree of spectral broadening indicates that the local structure is not unique. Environmental heterogeneity is confirmed by the nonuniform phosphorescence decay in buffer, at 274 K, with lifetime components of 44 and 20 ms of practically equal amplitude. Information on the flexibility of the interface region was gathered from both the intrinsic lifetime and the accessibility of acrylamide to the site of the chromophore. The magnitude of the intrinsic lifetime, its temperature dependence, and the accessibility to solutes like acrylamide describe a tight dimeric structure in which hydrophobic interactions seem to play an important role. In particular the acrylamide bimolecular rate constant is 1.4 x 10(4) M(-1) s(-1) and indicates highly hindered diffusion of the solute through the interface region. Cd(2+) complexation to the apoprotein caused no detectable changes in protein conformation although the metal was able to influence the flexibility of the Trp-73 environment, indicating the occurrence of a long-range communication between the intersubunit surface and the active site, which is more than 16 A away.     

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.     

Conformational changes in azurin from Pseudomona aeruginosa induced through chemical and physical protocols

Azurin from Pseudomona aeruginosa is a small copper protein with a single tryptophan (Trp) buried in the structure. The Gibbs free energies associated with the folding of holo azurin, calculated monitoring Trp fluorescence and changes in absorbance on the ligand-to-metal band, are different because these techniques probe their local environments, thereby being able to probe different conformational changes. The presence of an intermediate state was observed during the chemical denaturation of the protein. Upon denaturation, a 30-fold increase is observed in the magnitude of the quenching constant of the tryptophan fluorescence by acrylamide, because this residue becomes more accessible to the quencher. Entrapping the protein in sol-gel materials lowers its stability possibly because the solvation properties of the macromolecule are changed. The thermal denaturation of azurin immobilized in a sol-gel monolith is irreversible, which tends to rule out an aggregation mechanism to account for the irreversibility of the denaturation of the protein free in solution. Unlike the Cu(II) ion, the Gd(III) ion accommodates in site B of azurin with high affinity and the folding free energy of Gd-azurin is larger than that of apo azurin.     

Quenching of room temperature protein phosphorescence by added small molecules

A number of molecular agents that can efficiently quench the room temperature phosphorescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequence, namely, N-acetyltryptophanamide (NATA) greater than parvalbumin approximately lactoglobulin approximately ribonuclease T1 greater than liver alcohol dehydrogenase greater than aldolase greater than Pronase approximately edestin greater than azurin greater than alkaline phosphatase. Quenching rate constants for O2 and CO are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to 10(5) lower for H2S and CS2. Larger and more polar quenchers--including organic thiols, conjugated ketones and amides, and anionic species--were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structural dynamics of myoglobin

Conformational fluctuations in proteins were initially invoked to explain the observation that diffusion of small ligands through the matrix is a global phenomenon. Small globular proteins contain internal cavities that play a role not only in matrix dynamics but also in controlling function, tracing a pathway for the diffusion of the ligand to and from the active site. This is the main point addressed in this Review, which presents pertinent information obtained on myoglobin (Mb). Mb, a simple globular heme protein which binds reversibly oxygen and other ligands. The bond between the heme Fe(II) and gaseous ligands can be photodissociated by a laser pulse, generating a non-equilibrium population of protein structures that relaxes on a picosecond to millisecond time range. This process is associated with migration of the ligand to internal cavities of the protein, which are known to bind xenon. Some of the results obtained by laser photolysis, molecular dynamics simulations, and X-ray diffraction of intermediate states of wild-type and mutant myoglobins are summarized. The extended relaxation of the globin moiety directly observed by Laue crystallography reflects re-equilibration among conformational substates known to play an essential role in controlling protein function.     

Extended molecular dynamics simulation of the carbon monoxide migration in sperm whale myoglobin

We report the results of an extended molecular dynamics simulation on the migration of photodissociated carbon monoxide in wild-type sperm whale myoglobin. Our results allow following one possible ligand migration dynamics from the distal pocket to the Xe1 cavity via a path involving the other xenon binding cavities and momentarily two additional packing defects along the pathway. Comparison with recent time resolved structural data obtained by Laue crystallography with subnanosecond to millisecond resolution shows a more than satisfactory agreement. In fact, according to time resolved crystallography, CO, after photolysis, can occupy the Xe1 and Xe4 cavities. However, no information on the trajectory of the ligand from the distal pocket to the Xe1 is available. Our results clearly show one possible path within the protein. In addition, although our data refer to a single trajectory, the local dynamics of the ligand in each cavity is sufficiently equilibrated to obtain local structural and thermodynamic information not accessible to crystallography. In particular, we show that the CO motion and the protein fluctuations are strictly correlated: free energy calculations of the migration between adjacent cavities show that the migration is not a simple diffusion but is kinetically or thermodynamically driven by the collective motions of the protein; conversely, the protein fluctuations are influenced by the ligand in such a way that the opening/closure of the passage between adjacent cavities is strictly correlated to the presence of CO in its proximity. The compatibility between time resolved crystallographic experiments and molecular dynamics simulations paves the way to a deeper understanding of the role of internal dynamics and packing defects in the control of ligand binding in heme proteins.     

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.