ABSTRACT
The effects of two single point cavity forming mutations, F110S and I7S, on the unfolding volume change (ΔV^sup 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 ΔV^sup 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 �^sup 3^ in I7S and 100 �^sup 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 (τ^sub 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 ~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.
INTRODUCTION
Close atomic packing in protein structures is an important determinant of the stability of native folds ( 1.2). The presence of internal cavities may be crucial, however, for conferring the conformational flexibility needed for their biological function. The extent to which naturally occurring or engineered cavities, sufficiently large to accommodate one or more water molecules, are empty or hydrated may depend on several factors (size, hydrophobicity. etc.) and is currently the subject of active debate (3-8). Unfortunately, internal mobile water molecules are not resolved by x-ray crystallography and neither by NMR spectroscopy, unless they are rotationally restrained (9). Some weakly polar cavities created inside proteins by mutations were indeed found to be empty (10) and studies of binding of noble gases within engineered cavities (7) have indicated that the occupancy of water in an apolar cavity is expected to be vanishingly small. Eriksson et al. (10) estimated that the probability of a single water molecule being in a purely hydrophobic cavity is extremely small, a conclusion strengthening the belief that absence of electron density in a crystallographic map is evidence for the absence of matter (9). However, free-energy considerations point out that empty cavities are very costly on stability, and recent x-ray, NMR, and simulation studies show that water molecules can be present, at least transiently (4,8,10-15).
One of the most sensitive approaches to the detection of internal cavities in macromolecules is the response of the system to applied pressure (16-19). When hydrostatic pressure is applied, the protein-solvent system evolves toward the global conliguration that occupies the least volume (20). According to compressibility data (21-23). a decrease in volume can be achieved by both the reduction of internal cavities, voids that result from imperfect packing of amino acids, and greater hydration of the polypeptide, including the penetration of water molecules into the globular fold. (21,22,24). Since the major contribution to the change of volume on unfolding. ΔV^sup 0^, is the elimination of internal voids upon disruption of the folded structure, the introduction of an additional cavity of volume V^sub c^ should increase the value of ΔV^sup 0^ by roughly the same amount, were the cavity empty. Further, at predenaturating pressure reduction of cavity size and hydration exerts opposing influences on protein dynamics. From the correlation between compressibility, volume fluctuations, and flexibility (25) compression of cavities is expected to restrict the mobility of the polypeptide mainly of internal regions. Hydration, on the other hand, as it substitutes intrapeptide bonds with water bonds, will exert a lubricating action on segmentai flexibility.
This works applies high pressure to a homogeneous series of proteins to enquire specifically on the extent to which relatively large internal cavities, created by the replacement of bulky side chains with smaller ones, may be empty or filled with water as well as on what is the influence of these cavities on pressure modulation of protein dynamics. Azurin from Pseudomonas aeruginosa was chosen as a model system for the wealth of structural (crystallographic, spectroscopic, and theoretical), thermodynamic, and kinetic data available on both native and mutated forms. Azurin is a 14 kDa copper-binding protein with an eight-stranded β-sandwich structure arranged in a double-wound Greek-key topology (26). The β-sandwich is closely packed and forms a highly hydrophobic core about the unique Trp residue (Trp-48) of the polypeptide (26,27), which serves as a natural probe of the local structure. The introduction of Ser in place of a bulky Phe (F110) or Ile (17) creates in the neighborhood of Trp-48 a cavity of ~40 �^sup 3^ in I7S and of 100 �^sub 3^ in F110S, whereas for the rest the structure remains essentially native (28). The two single-point mutations destabilize the globular fold by 3-4 kcal/mole relative to the wild-type (WT) (29) and change the internal dynamics of the protein (30). A similar destabilisation of azurin is observed upon severing the superficial C3-C26 disulfide link in the double mutant C3A/ C26A (31), and for this reason the latter azurin species was taken to represent an example of compact but destabilized azurin fold.
The pressure unfolding equilibrium was monitored by the accompanying large change in fluorescence spectrum and yield of Trp-48, whereas the influence of pressure on the dynamics of the protein core was probed by phosphorescence lifetime of Trp-148 (32). For WT and the above azurin mutants, the phosphorescence emission of Trp-48 in copper free azurin is strong and long-lived even in buffer at ambient temperature (33), an emission that has proved to be remarkably sensitive to the flexibility of the structure surrounding the chromophore induced by a wide range of experimental conditions: metal binding (33), freezing (34), dehydration (35), high pressure (32), sugars (36), and pH (37).
The results of both stability and flexibility response to pressure demonstrate that water molecules do fill the nonpolar cavities of azurin and suggest that these hydrated cavities open the way to further internal hydration of the macromolecule, thus acting as nucleation sites for the unfolding process. The change in internal protein flexibility under pressure shows that with well compact protein folds, WT and C3A/ C26A, the response is an initial enhancement of structural rigidity followed by a progressive loosening of the macro-molecule in the high pressure range. The pressure at which there is an inversion between the two trends is apparently not related to the thermodynamic stability of the native state, hut was found to be signilicantly lowered with the introduction of internal cavities effective in breaking up the compactness of the protein core.
MATERIALS AND METHODS
All chemicals were of the highest purity grade available from commercial sources. Water, doubly distilled over quartz, was purified by Milli-Q Plus system (Millipore, Bedford, MA). All glassware used for sample preparation was conditioned in advance by standing for 24 h in 10% HCl suprapur (Merck, Darmstadt, Germany).
Azurin WT and mutants I7S, F110S, and CJA/C26A were prepared following published protocols. The plasmid carrying the WT sequence was a generous gift from Prof. A. Desideri (Universit� di Roma, "Tor Vergata", Italy). The procedure of isolation and publication of WT has been described by van de Kamp et al. (38). Details about site-directed mutagenesis, protein expression, isolation, and purification of I7S. F100S, and C3A/C26A mutants have been described elsewhere (39,40). Copper free azurins (apoazurins) were prepared from holo-azurins by adding potassium cyanide and EDTA to final concentrations of 0.1 M potassium cyanide and 1 mM EDTA in 0.15 M Tris-HCl, pH 8. followed by column chromalography (38). The proteins were dialyzed and stored in Tris HCl, 10 mM, pH 7.5.
Sample preparation for phosphorescence measurements
Before luminescence measurements, all proteins were extensively dialyzed in Tris-HCl (2 mM, pH 7.5) whose pH is one of the least sensitive to pressure. For phosphorescence measurements, it is paramount to rid the solution of all O2 traces. Deoxygenation of protein samples was obtained by adding an enzymatic system composed of SO nM glucose oxidase, 16 nM catalase, and 0.3% (w/v) glucose. No emission from these proteins could he detected at the amplification levels of phosphorescence measurements. The protein concentration in all phosphorescence experiments ranged between 2 and 4 �M.
Luminescence measurements
Fluorescence spectra and phosphorescence spectra and decays were measured with pulsed excitation (λ^sub ex^ = 292 nm) on a homemade apparatus (41), modified to implement spectral measurements by means of a charge-coupled device (CCD) camera. The main advantages of CCD detection over the traditional scanning monochromator-photomultiplier assembly are enhanced sensitivity to low light levels (>100-fold) and simultaneous acquisition of the entire spectrum.
Pulsed excitation was provided by a frequency-doubled Nd/Yag-pumped dye laser (Quanta Systems, Milan, Italy) with pulse duration of 5 ns. pulse frequency up to 10 Hz, and energy per pulse varying from 10 to 1000 �J. For spectra measurements, the emission was collected at 90� from the excitation and dispersed by a 0.3 m focal length triple grating imaging spectrograph (SpectraPro-2300i, Acton Research, Acton, MA) with a band pass ranging from 1.0 to 0.2 nm. The emission was monitored by a back-illuminated 1340 � 400 pixels CCD camera (Princeton Instruments Spec-10:400B (XTE). Roper Scientific, Trenton. NJ) cooled to -80�C. Phosphorescence decays were monitored by collecting the emission at 90� from vertical excitation through a filter combination with a transmission window of 405-445 nm (WG405, Lot-Oriel, Milan Italy; plus interference filter DT-Blau, Balzer, Milan, Italy). A gating circuit that inverts the polarity of dynodes 1 and 3, for up to 1.5 ms after the laser pulse, protects the photomultiplier (Hamamatsu R928, Hamamatsu City, Japan) from the intense fluorescence light pulse.
The photocurrent was amplified by a current-to-voltage converter (SR570, Stanford Research Systems, Stanford, CA) and digitized by a 16 bits high speed (1.25 MHz) multi-function data acquisition board (NI 6250 PCI, National Instrument Italy, Milan, Italy) supported by LabVIEW software capable of averaging multiple sweeps. Prompt fluorescence was collected through a 310-375 hand pass filter combination (WG305 nm plus Schott UG11) and detected by an ultraviolet-enhanced photodiode (OSD100-7, Centronics, Newbury Park, CA).
An analog circuit was used to integrate the pholocurrent, and its output was digitized and averaged by a multifunctional board (PCI-20428, Intelligent Instrumentation, Tucson, AZ) utilizing LabVIEW software. The prompt fluorescence intensity was used to account for possible variations in the laser output between measurements as well as to obtain fluorescence-normalized phosphorescence intensities. All phosphorescence decays were analyzed in terms of a sum of exponential components by a nonlinear least-squares fitting algorithm (DAS6, Fluorescence decay analysis software, Horiba Jobin Yvon, Milan, Italy).
Each spectral and lifetime determination was repeated at least three times. Luminescence measurements under pressure were carried out by placing the sample cuvette in a pressure cell (SITEC, Zurich, Switzerland) provided with sapphire windows and employing water as pressurizing fluid. Details of the sample cuvette and procedure to avoid O2 inlet during pressure cycles have been reported before (32). The reversibility of luminescence measurements was checked at the end of each pressure cycle. Particular care was taken Io assure temperature equilibration of the sample after each pressure variation, which required at least 5 min.
RESULT AND DISCUSSION
Effects of internal cavities on the unfolding ΔV^sup 0^ The volume change of azurin denaturation was obtained from the linear relationship describing the pressure dependence of the unfolding free energy, ΔG(p) = ΔG^sup o^+ ΔV^sup o where pis the applied pressure(42). At any p, the fraction of unfolded azurin, f^sub U^, was determined from the change in the fluorescence emission, spectrum and quantum yield, of the single Trp residue (W48) buried in the central hydrophobic core of the macromolecule. The nonpolar rigid environment confers to W48 the bluest and most structured spectrum reported to date in proteins, a feature particularly suitable for distinguishing native from unfolded states of the macromolecule. Fig. 1 shows the large red shift, λ^sub max^, increasing from 308 to 355 nm, and the decrease in fluorescence yield accompanying pressure denaturation of WT azurin. The spectral change is similar to that found with chemical denaturation by GdnHCl (29.36) and is typical for the full exposure of the aromatic residue to the solvent. Note that relative to WT and the mutant C3A/C26A, the fluorescence spectrum of mutants I7S and F110S is partially red-shifted and less resolved (39), an indication that the environment of W48 has been modified by the amino acid substitution. For each protein, f^sub U^ as function of applied pressure was evaluated from the change in the center of mass of the spectrum, λg, as described in Materials and Methods.
Pressure denaturation of azurin was found to be a reversible process, although for the cavity mutants recovery of the native fold is not immediate nor complete on decompression, particularly if the sample is maintained for a long time, over 20-30 min, at denaturing pressures. Evidently, for these proteins, slow, partly irreversible processes set in with the formation of denatured species. The phenomenon is not concentration-dependent, indicating that aggregation of protein is not responsible of it. To minimize the interference of these side reactions on the unfolding equilibrium, the time of equilibration after each pressure change was reduced to 5 min and the equilibration kinetics accelerated by raising the temperature to 40�C. Specific tests confirmed that under these conditions, recovery of native azurin at the end of a complete pressure cycle was better than 90%.
The main conclusion from the pressure denaturation profile is that the volume change on azurin unfolding is not significantly different between compact globular folds, WT, and C3A/C26A mutant, species in which a sizable internal cavity was introduced by the amino acid replacement.
The crystallographic structure of the two azurin cavity mutants I7S and F1100S shows that by replacing 17 or F110 with the smaller and polar Ser, a predominantly hydrophobic cavity of -24 and 60 mL/mole, respectively, has been engineered in proximity of Trp-48 (28), with important consequences on the stability and internal dynamics of azurin (29.30). In contrast to I7S, where no water molecules have been detected in the cavity, one water molecule sufficiently structured to give a diffraction pattern was indeed detected in the cavity of F110S (28). However, since the larger cavity of F110S can in principle accommodate 7-8 water molecules, x-ray data could be interpreted to mean that in both mutants the cavities are largely empty.
The increments to the total volume change measured for the mutants by x-ray measurements are significantly larger than the experimental error of ΔV^sup o^ (~10 mL/mole) and therefore were the cavities empty would show a 40% and 100% increase of ΔV^sup o^, respectively. We must infer that the cavities are largely filled with water.
Effects of internal cavities on the response of protein dynamics to applied pressure
The application of high pressure will promote any structural rearrangement of the macromolecule/solvent system that accommodates a reduction in its volume. At predenaturating pressures, the decrease in volume can involve both the reduction of internal cavities and greater hydration of the polypeptide. including the penetration of water molecules into the globular fold. Cavity size reduction and hydration exert opposing influences on protein dynamics, and the net effect will manifest which of the two trends is the prevailing structural adaptation to applied pressure.
The internal dynamics of azurin is assessed by the phosphorescence lifetime of W48 (τ^sub 0^). a parameter whose magnitude depends directly on the flexibility of the protein matrix around the chromophore (43,44). In buffer (2 mM Tris, pH 7.5) at 40�C. the phosphorescence decay of W48 in WT azurin and in the C3A/C26A double mutant is uniform with a lifetime of 0.12 and 0.10 s, respectively. For the cavity-forming mutants F110S and I7S. the decay is heterogeneous, reflecting the presence of more than one stable conformation of the macromolecule in the millisecond-second timescale, each with its own τ (30) (Table 2). Throughout, heterogeneous decays were adequately fitted in terms of two lifetime components (see as an example F110S in Fig. 3). This analysis yielded an averaged lifetime (τ^sub av^ = α^sub 1^τ^sub 1^ + α^sub 2^τ^sub 2^) of 0.076 s for F110S and 0.022 s for I7S . Thus, according to the triplet lifetime, both cavity mutants are more flexible in the central core of the protein harboring the phosphorescent probe.
The application of high pressure affects both the phosphorescence intensity and lifetime of each azurin species. It should he pointed out that when the protein unfolds, the phosphorescence lifetime falls to below detection limit and consequently the denatured fraction does not contribute to the phosphorescence emission of the sample. Thus, the intensity of phosphorescence extrapolated to time zero, P^sub 0^, when normalized by the flourence intensity F, P^sub 0^/F, is a direct measure of the native fraction of the protein population. For every protein, the pressure profile of P^sub 0^/F was found to be practically identical, within the ~5% precision ol these measurements, to the fraction of azurin in the native state, f^sub N^ = f^sub U^, as determined from the fluorescence f^sub U^ profiles of Fig. 2. This good correspondence between the native fraction of azurin determined by P^sub 0^/F and fluorescence indicates that the macromolecule is either fully native, with long-lived phosphorescence, or completely unfolded. Hence, even at denaturing pressures, there is no evidence of intermediate, partially structured conformations of the protein, that could be too flexible to be phosphorescent but with W48 still shielded from the solvent to fluoresce to the blue. The above correspondence between independent monitors of azurin denuturation supports the validity of a two-state unfolding equilibrium, meaning that in general unfolding proceeds from fully compact states.
The pressure dependence τ^sub 0^ is reported for each protein in Fig. 4. Throughout, the pressure modulation of τ^sub 0^ was found to be a totally elastic process, as on decompression the change is promptly reversed. For the WT protein, we observe a biphasic τ profile characterized by an initial lengthening of the lifetime up to 3 kbar followed by its progressive reduction to values below τ^sub 0^. From the correlation between τ and the fluidity of the chromophore's environment (45), the pressure profile of τ attests to an initial lightening of the protein core, presumably owed to the preeminence of cavity reduction, followed by its progressive loosening in high pressure range, reflecting enhanced internal hydration.
The C3A/C26A azurin mutant behaves similarly to the WT protein. Although the much lower stability of the mutant relative to the WT limits the observation range to 3 khar, τ is also found to increase, the compaction of the inner fold reaching its highest point on the same pressure range of the WT. This finding points out that despite the much smaller stability (6 kcal/mole) of C3A/C26A relative to WT, caused by the removal of the superficial disulphide link, hydration of the inner core and consequent loss of structural rigidity is as hindered as in the WT protein.
The introduction of a large cavity in proximity of W48 is expected to abate the rigidity of the local structure. The smaller To of F110S and I7S and the 3-4 orders of magnitude increase of the permeability of acrylamidc to the protein core (30) confirm that cavity mutants are considerable more flexible than the WT. The response of protein dynamics to applied pressure will tell if the gain in flexibility of the cavity mutants is due to the presence of free voids or to the lubricating action of an internal water pools. Only in the former case would the macromolecules be highly compressible and pressure exert a drastic reduction of structural fluctuations. The flexibility changes inferred from τ (Fig. 4) show that for F110S, the initial compaction reaches a maximum at much lower pressure (1.5 kbar) than WT and C3A/C26A azurins, the trend reverting thereafter with a sizable twofold reduction of 7 before reaching 3 kbar. For the more flexible cavity mutant, I7S, there is even no sign of compaction as the lifetime decreases monotonically above 0.5 kbar. In the latter, the triplet probe reports a sharp enhancement of protein flexibility at relatively low pressure, consistent with an overwhelming effect of internal protein hydration over any compaction of the structure. A similar behavior was also observed for the WT when approaching freezing temperatures where protein hydration is the dominant reaction to pressurization (46). In either case, we do not observe the sharp tightening of conformational flexibility anticipated tor empty cavities. Instead, the main response to pressure is consistent with the promotion of protein internal hydration. Hence, it appears that the new cavities of I7S and F110S are largely hydrated and that such a configuration confers high plasticity to the native state of azurin even at relatively low pressure. The lack of or decreased compaction of the inner core of cavity mutants with applied pressure points out that cavities are already filled with water, a rinding that is consistent the invariance of ΔV� of the among the four azurin species.
The main conclusions to draw from the limited but homogeneous set of proteins examined is that the creation of an internal cavity will enhance the plasticity and lower the stability of the globular structure.
The possibility to discriminate between empty and water-oiled cavities is also important for testing the validity of the relationship predicting the decrease in protein stability resulting from the creation of internal empty cavities. Based on a series of mutations in the core of phage T4 lysozyme, a correlation has been proposed (10,47) between ΔΔG and the size (ΔV) of the cavity created. According to this relationship, the energetic cost of making a cavity is linearly dependent on the volume of the created cavity plus an additional free-energy term determined by the difference in hydrophobicity of the two amino acid residues involved in the substitution. The cost of creating a cavity derives mostly from the missing van der Waals interactions between the side chain to be replaced and the surrounding atoms in the native structure and is estimated at ~0.022 kcal mole^sup -1^ [Angstrom]^sup 3^ (10,47), a value frequently used in studies predicting protein stability (3,48). This ΔΔG versus ΔV relationship was found to hold in some cases (8,49) but data is also available that contrasts with it (3,50-52). In the case of I7S and F110S, the measured ΔΔG is ~6 kcal mole^sup -1^ for both mutants, and this value is fairly close to that predicted theoretically considering the substitution of Ile and Phe with a Ser plus the creation of cavity of the dimension calculated by x ray. However, we note that the agreement is totally fortuitous and misleading because the cavities are not empty but largely filled with water, and consequently ΔΔG should be much smaller. Both the difficulty in establishing the actual volume of a cavity, which depends on the shape of the cavity and the size of the probe used, and its variable extent of hydration suggest that ΔV is not a useful parameter for predicting free-energy changes in response to mutations. Moreover, buried water molecules in the interior of proteins can form a variety of hydrogen-bonded networks that can have a significant if unpredictable impact on protein stability (53,54).
The author is grateful to Dr. Giovanni Strambini for helpful discussions and for critical reading of the manuscript.
[Reference]
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[Author Affiliation]
Patrizia Cioni
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Pisa 56100, Italy
[Author Affiliation]
Submitted March 27, 2006, and accepted for publication June 12, 2006
Address reprint requests to Patrizia Cioni, E-mail: patrizia.cioni@pi.ibf.cnr.it.
� 2006 by the Biophysical Society
0006-3495/06/11/3390/07 $2.00
