Experimental and simulative dissociation of dimeric Cu,Zn superoxide dismutase doubly mutated at the intersubunit surface.

The equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase mutant bearing two negative charges in the amino acid clusters at the association interface has been studied, experimentally and computationally, and compared to those of the native enzyme. Pressure-dependent dissociation is observed for the mutant, as observed by the fluorescence shift of the unique tryptophan residue located at the intersubunit surface. The spectral shift occurs slowly, reaching a plateau after 15-20 min, and is fully reversible. Measurement of the degree of dissociation allows us to calculate the standard volume variation upon association and the dissociation constant at atmospheric pressure. On the other hand the native protein is undissociable at any pressure. In the simulative approach, the dissociation free energy has been calculated through the blue moon calculation method for the case of a multidimensional reaction coordinate, corrected for the rotational contribution within the semiclassical approximation for a free rigid-body rotor. The scheme permits to define a definite path for the rupture of the dimer and to calculate the effective force involved in the process. The calculated free energy difference is close to the experimental one, and the value obtained for the mutant is well below that obtained for the native protein, indicating that the theoretical reaction scheme is able to reproduce the experimental trend. Moreover, we find that, when the separation distance increases, the protein structure of the monomer is stable in line with the fast recovery of the original fluorescence properties after decompression, which excludes the presence of partly unfolded intermediates during the dimer-monomer transition.

Active-site copper and zinc ions modulate the quaternary structure of prokaryotic Cu,Zn superoxide dismutase.

The influence of the constitutive metal ions on the equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase has been studied for the wild-type and for two mutant protein forms bearing a negative charge in the amino acid clusters at the dimer association interface. Depletion of copper and zinc dissociates the two mutant proteins into monomers, which reassemble toward the dimeric state upon addition of stoichiometric amounts of zinc. Pressure-dependent dissociation is observed for the copper-depleted wild-type and mutated enzymes, as monitored by the fluorescence shift of a unique tryptophan residue located at the subunit association interface. The spectral shift occurs slowly, reaching a plateau after 15-20 minutes, and is fully reversible. The recovery of the original fluorescence properties, after decompression, is fast (less than four minutes), suggesting that the isolated subunit has a relatively stable structure, and excluding the presence of stable intermediates during the dimer-monomer transition. The dimer dissociation process is still incomplete at 6.5 kbar for the copper-depleted wild-type and mutated enzymes, at variance with what is generally observed for oligomeric proteins that dissociate below 3 kbar. Measurement of the degree of dissociation, at two different protein concentrations, allows us to calculate the standard volume variation upon association, Delta V, and the dissociation constant K(d0), at atmospheric pressure, (25 ml/mol and 3 x 10(-7)M, respectively). The holoprotein is fully dimeric even at 6.5 kbar, which allows us to evaluate a lower Delta G degrees limit of 11.5 kcal/mol, corresponding to a dissociation constant K(d0)<10(-9)M.

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.

Dynamics-function correlation in Cu, Zn superoxide dismutase: a spectroscopic and molecular dynamics simulation study.

A single mutation (Val29-->Gly) at the subunit interface of a Cu, Zn superoxide dismutase dimer leads to a twofold increase in the second order catalytic rate, when compared to the native enzyme, without causing any modification of the structure or the electric field distribution. To check the role of dynamic processes in this catalytic enhancement, the flexibility of the dimeric protein at the subunit interface region has been probed by the phosphorescence and fluorescence properties of the unique tryptophan residue. Multiple spectroscopic data indicate that Trp83 experiences a very similar, and relatively hydrophobic, environment in both wild-type and mutant protein, whereas its mobility is distinctly more restrained in the latter. Molecular dynamics simulation confirms this result, and provides, at the molecular level, details of the dynamic change felt by tryptophan. Moreover, the simulation shows that the loops surrounding the active site are more flexible in the mutant than in the native enzyme, making the copper more accessible to the incoming substrate, and being thus responsible for the catalytic rate enhancement. Evidence for increased, dynamic copper accessibility also comes from faster copper removal in the mutant by a metal chelator. These results indicate that differences in dynamic, rather than structural, features of the two enzymes are responsible for the observed functional change.

Structural perturbations of azurin deposited on solid matrices as revealed by trp phosphorescence.

The phosphorescence emission of Cd-azurin from Pseudomonas aeruginosa was used as a probe of possible perturbations in the dynamical structure of the protein core that may be induced by protein-sorbent and protein-protein interactions occurring when the macromolecule is deposited into amorphous, thin solid films. Relative to the protein in aqueous solution, the spectrum is unrelaxed and the phosphorescence decay becomes highly heterogeneous, the average lifetime increasing sharply with film thickness and upon its dehydration. According to the lifetime parameter, adsorption of the protein to the substrate is found to produce a multiplicity of partially unfolded structures, an influence that propagates for several protein layers from the surface. Among the substrates used for film deposition, hydrophilic silica, dextran, DEAE-dextran, dextran sulfate, and hydrophobic octodecylamine, the perturbation is smallest with dextran sulfate and largest with octodecylamine. The destabilizing effect of protein-protein interactions, as monitored on 50-layer-thick films, is most evident at a relative humidity of 75%. Stabilizing agents were incorporated to attenuate the deleterious effects of protein aggregation. Among them, the most effective in preserving a more native-like structure are the disaccharides sucrose and trehalose in dry films and the polymer dextran in wet films. Interestingly, the polymer was found to achieve maximum efficacy at sensibly lower additive/protein ratios than the sugars.

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.

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.

Tryptophan phosphorescence as a monitor of protein conformation in molecular films.

This report enquires on the potentiality of Trp phosphorescence for probing the conformational state of proteins deposited on solid dry films. Thin, amorphous protein films were fabricated with Apoazurin, alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and glutamate dehydrogenase the protein being incorporated into a DEAE-dextran matrix and deposited on quartz slides. The results, obtained with appositely constructed instrumentation, demonstrate that thanks to the low background radiation associated with long-lived, delayed emission phosphorescence can be readily detected down to single protein layer matrices and that both spectrum and lifetime are important indicators of the integrity of the protein globular fold. In fact, denaturation of the proteins by guanidinium hydrochloride or heat treatment points out that disruption of the native fold leads to a red shift and broadening of the spectrum with loss of vibronic structure, accompanied to considerably shorter-lived and more heterogeneous decay kinetics. It is also shown that the sensitivity of the phosphorescence lifetime towards the detection of altered, looser conformations of the polypeptide are remarkably enhanced on partial hydration of the sample.

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 (

Phosphorescence emission of 7-azatryptophan and 5-hydroxytryptophan in fluid solutions and in alpha2 RNA polymerase.

The tryptophan analogues 7-azaindole (7-Aza W) and 5-hydroxytryptophan (5-OH W) have a significant absorbance between 310-320 nm, which allows them to act as selective luminescence probes in protein mixtures containing a large number of tryptophan residues. To assess the potential of their phosphorescence emission in probing the nature of protein environments the delayed emission was examined as a function of temperature and solvent viscosity. Whereas in low temperature (135 K) propylene glycol/buffer glasses the phosphorescence of both 7-aza W and 5-OH W is structured, intense and exhibit a lifetime of a few seconds, above the glass transition temperature (180 K) the delayed emission is considerably quenched. Temperature profiles show that as the solvent is more fluid the phosphorescence of 5-OH W becomes red shifted, poorly structured and the triplet lifetime drops steeply reaching 29 micro(s) in buffer at 274 K. For 7-aza W the alterations are more drastic and no phosphorescence could be detected above 193 K. This implies that in fluid aqueous media the excited triplet state of these analogues is deactivated by vary efficient nonradiative processes. The quenching of 5-OH phosphorescence is not prevented even when the chromophore is inserted in a solvent protected protein environment. Indeed, substitution of the single Trp of a2 RNA Polymerase, which according to its relatively low phosphorescence lifetime at ambient temperature is substantially shielded from the solvent, with 5-OH did not inhibit the quenchability of the latter. Knowledge of the quenching mechanisms is therefore needed for this emission to report on the nature of the protein environment.

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.

Pressure-induced dissociation of yeast glyceraldehyde-3-phosphate dehydrogenase: heterogeneous kinetics and perturbations of subunit structure.

In studies of pressure-induced subunit dissociation of oligomeric proteins, the thermodynamic dissociation constant and the dissociation volume change are derived by assuming that high pressure itself does not significantly perturb the structure of both oligomer and isolated subunit. In this report, the intrinsic phosphorescence emission of Trp reveals that high-pressure dissociation of tetrameric yeast glyceraldehyde-3-phosphate dehydrogenase results in a dramatic shortening of the phosphorescence lifetime, from 300 to less than 2 ms, that is consistent with a profound loosening of the polypeptide structure about the phosphorescence probe. On pressure release, subunit reassociation occurs readily whereas recovery of the native phosphorescence properties is a very slow, thermally activated, process which goes hand in hand with the recovery of the catalytic activity. Further, the comparison between the kinetic traces that describe the degree of dissociation and the change in phosphorescence lifetime, at various applied pressures, has established the following: (1) that high pressure plays a direct role on the structural rearrangement, the extent of which increases with pressure; (2) that the conformational change in the monomer is concomitant with, or follows closely after, the break up of the tetramer, in any case long before an apparent tetramer-monomer equilibrium is established; (3) that native tetramers are highly heterogeneous with regard to their rate of dissociation. The influence of temperature, of protein concentration, of binding of NAD+, and of the addition of 2 M urea on the dissociation/phosphorescence kinetic profiles was also examined. The complications arising from these conformational changes for the derivation of the dissociation free energy change as well as their relevance for understanding the lack of concentration dependence of the degree of dissociation are discussed.

Structural mapping of the epsilon-subunit of mitochondrial H(+)-ATPase complex (F1).

Phosphorescence and fluorescence energy transfer measurements have been used to locate the epsilon-subunit within the know structural frame of the mitochondrial soluble part of F-type H(+)-ATPase complex (F1). The fluorescence probe 2'-O-(trinitrophenyl)adenosine-5'-triphosphate was bound to the nucleotide binding sites of the enzyme, whereas the probe 7-diethylamino-3'-(4'-maleimidylphenyl)-4-methylcoumarin was attached to the single sulfhydryl residue of isolated oligomycin sensitivity-conferring protein (OSCP), which was then reconstituted with F1. Fluorescence and phosphorescence resonance energy transfer yields from the lone tryptophan residue of F1 present in the epsilon-polypeptide and the fluorescence labels attached to the F1 complex established that tryptophan is separated by 3.7 nm from Cys-118 of OSCP in the reconstituted OSCP-F1 complex, by 4.9 nm from its closest catalytic site and by more than 6.4 nm from the two other catalytic sites, including the lowest affinity ATP site. These separations together with the crystallographic coordinates of the F1 complex (Abrahams, J.P., A. G. W. Leslie, R. Lutter, and J.E. Walker. 1994. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370:621-628) place the epsilon-subunit in the stem region of the F1 molecule in a unique asymmetrical position relative to the catalytic sites of the enzyme.

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.

Pressure effects on the structure of oligomeric proteins prior to subunit dissociation.

In studies of pressure-induced subunit dissociation of protein aggregates, now widely used to evaluate the association free energy, entropy and enthalpy of very stable complexes, it is assumed that high pressure does not influence their structure/thermodynamic parameters and that some peculiarities of these equilibria, such as the decrease in subunit affinity at larger degrees of dissociation (alpha) and hysteresis in alpha/pressure diagrams are imputable to the slow conformational drift of isolated subunits. To test this premise, the conformation of dimeric alcohol dehydrogenase from horse liver and alkaline phosphatase from Escherichia coli was monitored as a function of pressure (up to 3 kbar) and temperature (0 to 50 degrees C) by means of the intrinsic Trp fluorescence and phosphorescence emission and binding of the 1-anilinonaphatalene-8-sulphonic acid (ANS) fluorophore. The results show a distinct influence of high pressure on the native dimers whose changes in conformation may, depending on whether or not these alterations are promptly reversed, be distinguished in elastic and inelastic changes. Elastic changes are ubiquitous and refer to pronounced modulations of the phosphorescence lifetime which is a monitor of the internal flexibility of the macromolecules. They attest to a tightening of the globular structure in the lower pressure range (below 1.5 kbar) as opposed to an increased fluidity in the higher range. The trend is similar between the two proteins and the tightening/loosening effect is fully consistent with the role that internal cavities and hydration of polypeptide is expected to play in determining the compressibility of these biopolymers. Inelastic perturbations reveal a more profound loosening of the globular fold and were observed only with alcohol dehydrogenase under conditions (low temperature (t < 10 degrees C) and high pressure (p > 2.5 kbar)) that favour protein hydration. They involve slow consecutive reactions that produce drastic reductions in phosphorescence lifetime, spectral red shifts, quenching of fluorescence and phosphorescence emission and modulation of ANS binding. Judging from the full protection afforded by glycerol as cosolvent, or the remarkable enhancement caused by modest concentrations of urea, the driving force of these perturbations appears to be pressure-induced hydration of the protein. Inelastic conformational changes are accompanied by a slow and often incomplete recovery of enzymatic activity. The characteristic times of these processes, their pressure dependence and the slow, thermally activated, reversibility are discussed in the light of hysteresis phenomena and changes of subunit affinity in dissociation equilibria.

Effects of NAD+ binding on the luminescence of tryptophans 84 and 310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

The individual fluorescence and phosphorescence properties of W84 and W310 in Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase were identified through the construction of a single tryptophan mutant (W84F) and by comparison of the emission between mutant and wild-type enzymes. The results show that the luminescence of W310 is red-shifted and substantially quenched relative to that of W84. It displays an average subnanosecond fluorescence lifetime (tau F) and a very short, 50 microseconds, room-temperature phosphorescence (RTP) lifetime (tau P). The perturbation of W310 luminescence is believed to arise from a stacking interaction with Y283. In contrast, W84 exhibits a fluorescence lifetime tau F of several nanoseconds and a long-lived phosphorescence lifetime tau P, typical of buried, unperturbed TrP residues. NAD+ binding to the tetrameric enzyme causes a 55% reduction of W310 fluorescence intensity together with a nearly complete quenching of its low-temperature phosphorescence. W84, which is located far from the nicotinamide moiety of NAD+, is much less affected by the binding of the coenzyme; the reduction in fluorescence intensity is 35%, and its phosphorescence intensity is unchanged. Another consequence of NAD+ binding is a significant decrease of the RTP lifetime tau P of W84, manifesting thereby a conformational change in the region of the coenzyme-binding domain. However, no change is observed in the RTP lifetime tau P of W310 located in the catalytic domain. These findings and those obtained at partial coenzyme saturation support the conclusions derived from high-resolution crystallographic structures [Skarzynski, T., & Wonacott, A. J., (1988) J. Mol. Biol. 203, 1097-1118] that the NAD(+)-induced conformational change is sequential and that subtle rearrangement in the structure of unligated subunits might be responsible for the negative cooperative behavior of NAD+ binding.

Tryptophan luminescence as a probe of enzyme conformation along the O-acetylserine sulfhydrylase reaction pathway.

O-Acetylserine sulfhydrylase A (OASS-A) is a pyridoxal 5'-phosphate- (PLP-) dependent enzyme that catalyzes the last step in the synthesis of L-cysteine, the beta-replacement of acetate in O-acetyl-L-serine (OAS) by sulfide. The phosphorescence properties of the two tryptophans of wild-type OASS-A, W51 and W162, and of W162 in the W51Y mutant protein have been characterized over the temperature range 170-273 K. In glasses at 170 K, the apoenzyme exhibits a phosphorescence spectrum which is the superposition of two spectra with well-resolved 0,0 vibronic bands centered at 405 and 410 nm, the blue lambda max suggesting that one of the two Trp residues in OASS-A is in a polar pocket, while the other is in a relatively hydrophobic pocket. The presence of PLP in the OASS-A holoenzyme reduces the intrinsic fluorescence by 40-45%, but the spectrum is unaltered except for the appearance of the internal Schiff base ketoenamine fluorescence band centered at 484 nm. The phosphorescence is strongly quenched by PLP, with about 70% reduction in intensity and lifetime. Further, the phosphorescence spectrum of the holoprotein exhibits a single and narrow 0,0 vibronic band centered at 405 nm and a broad band in the 450-550-nm range resulting from delayed fluorescence of the ketoenamine tautomer of the internal Schiff base, sensitized by triplet-singlet energy transfer from tryptophan to the ketoenamine tautomer of PLP. Comparison with data obtained for the W51Y mutant strongly suggests that the 405-nm phosphorescence band derives from W162, and that W51 in the wild type is entirely quenched either by singlet or triplet energy transfer to PLP or by some local group in the protein. From the rate of energy transfer, the separation between W162 and PLP is estimated to be about 25 A. Substrates other than OAS affect only the intensity of the coenzyme fluorescence band (484 nm) and the intensity of delayed fluorescence relative to that of phosphorescence, effects that are attributable to changes in fluorescence quantum yield of the ketoenamine chromophore. Addition of OAS, on the other hand, leads to a splitting of the 0,0 vibronic band in the phosphorescence spectrum of W162, yielding poorly resolved peaks at 406 and 408.5 nm, indicating thereby a change in the environment of the tryptophan residue and therefore in the conformation of the macromolecule as the internal Schiff base is converted to the alpha-aminoacrylate Schiff base. In buffer at 273 K, both the fluorescence and phosphorescence spectra relax to longer wavelengths and the phosphorescence lifetime is reduced to a few milliseconds, all indications that W162 is in a flexible region of the macromolecule, probably in close proximity to the aqueous interface. The phosphorescence lifetime in fluid medium reveals conformational heterogeneity in OASS-A and unveils important structure modulating effects of cofactor, substrates, and pH. Binding of PLP to the apoprotein increases the rigidity of the polypeptide in the region of W162 (in agreement with the greater thermal stability of the holoprotein), while OAS and L-serine have an opposite effect. Increasing the pH from 6.5 to 9 results in a 1.7-fold increase in tau av and a change in the relative amplitudes of the two lifetime components. Since the phosphorescence originates from a single tryptophan residue, the two tau components reflect distinct conformations of the subunit. In this case the conformational equilibrium (slow on the phosphorescence time scale) is governed by one or more groups in the protein with a pK around 8.

Proteins in frozen solutions: evidence of ice-induced partial unfolding.

From a drastic decrease in the phosphorescence lifetime of tryptophan residues buried in compact rigid cores of globular proteins, it was possible to demonstrate that freezing of aqueous solutions is invariably accompanied by a marked loosening of the native fold, an alteration that entails considerable loss of secondary and tertiary structure. The phenomenon is largely reversible on ice melting although, in some cases, a small fraction of macromolecules recovers neither the initial phosphorescence properties nor the catalytic activity. The variation in the lifetime parameter was found to be a smooth function of the residual volume of liquid water in equilibrium with ice and to depend on the morphology of ice. The addition of cryoprotectants such as glycerol and sucrose profoundly attenuates or even eliminates the perturbation. These results are interpreted in terms of adsorption of protein molecules onto the surface of ice.

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.

Pressure effects on protein flexibility monomeric proteins.

Alterations in flexibility of monomeric proteins induced by hydrostatic pressure in the predenaturational range (< or = 3 kbar) were probed through the decay kinetics of tryptophan phosphorescence. With apoazurin, ribonuclease T1, wild-type and V67G mutant and phosphoglycerate kinase, pressure effects on the triplet lifetime (tau) and the amplitudes of multicomponent decays emphasize that subtle changes in conformation are ubiquitous. With apoazurin the increase in tau attests to a tightening of the protein core that is enhanced at high temperature. On the contrary, tau decreases with ribonuclease T1, wild-type and mutant, and with phosphoglycerate kinase, indicating that pressure induces a greater flexibility to protein regions in proximity to the surface of the macromolecule. For phosphoglycerate kinase the decrease in tau and the parallel increase in fluorescence intensity and red-shift of the fluorescence spectrum unveil an "unfolding" like transition with midpoint pressures of 1.1 kbar at 5 degrees C and 1.6 kbar at 25 degrees C. Evidence that unfolding of the C-domain of this protein is, however, less than complete is provided by a delta G zero that is about half of that obtained by denaturation in guanidine hydrochloride and also by the ability of this structure to undergo conformational drift. In 70% glycerol, pressure effects on tau of apoazurin are attenuated while for ribonuclease T1 there is a reversal of the tendency with a pronounced increase in tau. With phosphoglycerate kinase glycerol abolishes entirely the "unfolding" transition and all hysteresis effects. A consistent picture of these findings is provided in terms of the location of the probe and of the opposing effects that pressure exerts on protein flexibility by reducing internal cavities and increasing the hydration of the polypeptide.