Studies on the C. rugosa lipase have indicated iPrOH to induce the opening of the lid (Chamorro et al. 1998). Accordingly, it was of interest to study the effects of this solvent on the structural dynamics of the closely related lipase from Thermomyces lanuginosa.
Wild-type TLL contains four Trps, two of which, W89 and W117, are located in two different α-helices and the other two, W221 and W260, in two different β-pleated sheets. Changes in the intrinsic fluorescence of the wild-type TLL thus report on the global conformational changes of this protein. Accordingly, we were particularly interested in the single Trp mutant W89m, in which the fluorescent residue is contained in the lid, an α-helical structure undergoing a major reorientation upon the lid opening.
Thus the CD data suggest partial unfolding of the protein tertiary structure in 50% iPrOH. Time resolved fluorescence of the single Trp in W89m demonstrated decreased residual anisotropy on increasing iPrOH concentration from 25% to 50%. Increase in Trp mobility together with the changes in near-UV CD suggest enhanced overall conformational fluctuations. This is in keeping with our results from the esterase activity measurements with decreased enzymatic activity for the W89m in 50% iPrOH. Our results thus demonstrate loosening of the tertiary structure while secondary structure is preserved, indicating a molten globule state (Dolgikh et al. 1985).
This is in keeping with the suggestion that the function of interfacially active enzymes can be significantly influenced by interactions with apolar molecules which may help to maintain and modulate rather than to destroy their three-dimensional structures (Ransac et al. 1995b; Graupner et al. 1999). Yet, fluorescence spectroscopy did reveal major alterations in the conformational dynamics of TLL to be induced by iPrOH, which had significant effects also on the catalytic activity of the lipase. The major conclusion is the opening of the lid in the presence of iPrOH. A complex pattern of alterations in the different parameters measured was evident as a function of iPrOH concentration.
Notably, the lack of coinciding changes with respect to [iPrOH] is not unexpected. This stems from the very nature of fluorescence, which allows to distinguish between changes in fluorophore mobility (r, r∞, and the derived rotational correlation times) and subtle changes in its electronic transitions (reflected by λmax, intensity, and τ1 and τ2) which are further determined by different constraints imposed by the environment of the fluorophore. Accordingly, whereas the recorded fluorescence parameters obviously are connected, they also differ in their responses to the solvent-induced changes in the structure of the protein. Moreover, on increasing [iPrOH], it is expected that the solvent can induce more subtle changes in protein conformational dynamics, preceeding the closed open conformational transition. Also the presence of the soluble substrate is likely to influence the conformational equilibrium of the lipase, most likely shifting it towards the open conformation, thus representing another complication affecting the concentration dependence of the effects of iPrOH.
Steady-state conformational changes in TLLs
At [iPrOH] = 50%, there was a blue shift by 1 nm in the λmax of the wild-type TLL. The opposite was true for W89m, with a small red shift (from 345 to 346.5 nm), suggesting an increment in the polarity of the microenvironment of the Trp in the lid. Fluorescence intensities changed dramatically on increasing [iPrOH] (Fig. 4). The quantum yield of Trp fluorescence increases when the polarity of microenvironment decreases (She et al. 1998). Accordingly, this indicates that iPrOH caused the Trps in wild-type TLL to move into less polar average microenvironments. Fluorescence intensity of W89m increased only slightly, by ∼3% in 30% iPrOH where after it decreased when 40% iPrOH was exceeded, thus indicating the microenvironment of Trp89 to become more polar. The observed red shift revealed by an increase in I350/I330 for W89m in 50% iPrOH (Fig. 5) supports this conclusion (Zhao and London 1986; Wang et al. 1997). The initial, minor increase in emission intensity at [iPrOH] ≤ 30% suggests that Trp89 in the absence of iPrOH, (i.e., in the closed conformation) contacts a vicinal residue slightly quenching its fluorescence. As Trp89 contributes ∼61% to the fluorescence of the wild-type TLL (Stobiecka et al. 1998), the change in the quantum yield of this residue has a significant effect on the Trp fluorescence of this protein.
Interestingly, the fluorescence intensity of the wild-type TLL started to decrease when [iPrOH] exceeded 25%. Accordingly, this can be concluded to result from the exposure of Trp89 to a more polar environment. Our stopped-flow measurements on the mixing of W89m with iPrOH revealed fluorescence intensity to reach a maximum in <2 ms (data not shown), indicating the opening of the lid to be rapid. Compared to the ∼3% increase in fluorescence intensity by 25% iPrOH in steady-state measurements, the emission increased ≈2.5-fold in the stopped-flow chamber under these conditions. A reason for this difference is likely to be a slow solvation of iPrOH into the buffer, resulting in incomplete solvent mixing within 2 ms. Accordingly, when Trp89 moves into the incompletely solvated iPrOH upon opening of the lid, its quantum yield should increase.
Fluorescence lifetimes provide further insight into the changes in conformational dynamics induced by iPrOH. Intensity decay of W89m is one-exponential in ≤10% iPrOH, whereas, two-exponential fitting was required in ≥15% iPrOH (Fig. 8). The two populations of Trp(s) with different fluorescence lifetimes have been ascribed to different Trp rotamer conformations (Chen et al. 1991).
In proteins, the shorter lifetime component τ1 has been further suggested to reflect interactions of the surface-exposed Trp(s) with the solvent (Graupner et al. 1999). The crystal structure of the open conformation of TLL revealed Trp89 to be exposed on the protein surface (A.M. Brzozowski, pers. comm.), in accordance with the pronounced increase in τ1 on increasing [iPrOH]. Notably, the fractional intensity of τ1 increased from 3.5% to 23.2% with [iPrOH], indicating a considerable shift in the conformational equilibrium of W89m. For the wild-type TLL with four Trps a similar change in the fractional intensity of τ1 could not be resolved.
Changes in conformational dynamics of TLL induced by isopropanol
Decrease in the steady-state anisotropy r of a fluorophore corresponds to its augmented mobility (Lakowicz 1999).
Judged from these data on W89m, the movements of Trp89 became more restricted in ≤20% iPrOH, whereas the opposite is true above this iPrOH concentration. The same overall tendency, yet less pronounced, is observed for the wild-type TLL. It should be noted that steady-state anisotropy measurements as such do not allow one to distinguish between local segmental motions of Trp from changes in the rate of Brownian tumbling of the protein in solution. Accordingly, the increase in r may also report oligomerization of TLL, further supported by the time-resolved fluorescence anisotropy measurements.
More specifically, Trp fluorescence anisotropy decays reflect rapid local motions of the Trp side chains as well as the overall rotational diffusion of the entire protein (Steiner 1991). Two rotational correlation times were measured for W89m. The shorter correlation time ϕ1 can be assigned to the local segmental motions of Trp89 (Davidson et al. 1999). Movement of Trp89 became faster when [iPrOH] approaches 20%, with ϕ1 reaching a minimum of 0.08 ns in 25% iPrOH (Fig 10A). Residual anisotropy r∞ has been interpreted as resulting from an energy barrier that prevents rotational diffusion of the fluorophore beyond a certain angle. Therefore r∞ does not decay to zero when the angular range of the rotational motion of the fluorophore is restricted within the time regime of the fluorophore lifetime (Lakowicz 1999). The values for r∞ decreased steeply when [iPrOH] exceeded 10%, indicating less restriction for the motion of Trp89 (Fig 10B), i.e., diminished constraints for its mobility. These data would be compatible with the location of Trp89 on the protein surface in the open conformation.
The longer correlation time ϕ2 reflects the motions of the entire lipase molecule, relating to the hydrodynamic volume by the Einstein-Stoke's equation
where η is the viscosity of the medium at temperature T, M is the molecular mass of protein, v is the specific volume of protein, h is the hydration factor, and R is the gas constant. Our results show ϕ2 and thus also the hydrodynamic volume of the lipase to increase with increasing [iPrOH]. Moreover, values of ϕ2 for W89m increased in a stepwise manner and in multiples of the original value upon increasing [iPrOH] between 5% and 25% iPrOH. The value of ϕ2 in the absence of iPrOH, 20 ns, is consistent with that expected for a spherical 30 kD protein with a typical hydration of 0.2 g of H2O/g (Lakowicz 1983). Using a value of 0.89 cp for the viscosity of water at 25°C, this ϕ2 corresponds to a hydrodynamic radius of 27.8 Å. This radius is in excellent agreement with further value the radius of 28 Å (= the maximum distance from the center of mass) obtained from crystal structure (Swaminathan et al. 1996), and thus confirms W89m to be a monomer in the absence of iPrOH. Accordingly, it is possible that the stepwise increments in ϕ2 reflect the formation of dimers and trimers by W89m in the presence of iPrOH. Notably, steady-state anisotropies for wild-type and W89m TLL increased abruptly between 5% and 10% iPrOH. In the light of the ϕ1 values, the frequency of the movement of the lid did not change in this concentration range. Therefore, the decrease in the rotation of Trp89 should result from a decreased tumbling of the entire TLL molecule in solution, in keeping with protein oligomerization. TLL can be aggregated by an electrostatic attraction in crystals in the presence of the detergent pentaoxyethylene octylether (C8E5) and trioxyethylene decylether (C10E3) (A.M. Brzozowski, pers. comm.).
The reason that iPrOH can induce the aggregation of TLL is likely to be a partially open structure of lipase when hydrophobic interactions are attenuated. Aggregates are thus likely to form when the structure is partially opened so that the exposed hydrophobic regions from different molecules interact (Swaminathan et al. 1996). Another possibility is that the organic solvent promotes the formation of intermolecular hydrogen bonds.
Above 20% iPrOH however, the Trp89 rotates at a high frequency, evidenced by a decrease in ϕ1, while the oligomeric state of the protein is maintained, as evidenced by ϕ2. We interpret the decrease in steady-state anisotropy at iPrOH>15% to result from conformational changes accompanying the formation of higher oligomers. Changes in the conformational dynamics induced by iPrOH are further reflected in the measurements of the catalytic activity of the wild-type and W89m TLL as a function of [iPrOH]. For W89m, the catalytic activity towards the water-soluble substrate carboxy-DCFDA increased up to 15% iPrOH, whereas exceeding this concentration caused an inhibition. Activation of TLL by iPrOH is in accordance with the opening of the lid. Inhibition of the hydrolytic activity at iPrOH concentrations above 20%–30 % could result from a number of reasons. Accordingly, iPrOH could occupy the active site after the opening of the lid, thus impeding the access of the substrate as well as possibly also the mandatory water to the catalytic residues. Increasing the concentration of substrate should offset this effect, as observed (Fig. 1). Another mechanism is diminished water activity in general.
Conformational changes induced by high [iPrOH] leading to an unfavorable active site geometry cannot be excluded. The above mechanism is in alignment with the strongly correlating changes in steady-state anisotropy (Fig. 6) and catalytic activity (Fig. 1) for both the wild-type and W89m TLL.
This correlation is very clear when the relative activities for the lipases are illustrated as a function of r (Fig. 11). Accordingly, on increasing [iPrOH] up to 15% and 10% respectively, a change in the conformation of lipase caused the steady-state anisotropy r to increase for both the wild-type and W89m TLL. This change in protein conformation also resulted in an ∼2- to 6-fold enhancement in the catalytic activity and is likely to represent the opening of the lid. On increasing [iPrOH] up to 50%, a further change in the conformational dynamics occurs, accompanied by a decrease in r and inhibition of the catalytic activity. The latter would be compatible with formation of higher oligomers by TLL in the open conformation at high concentrations of iPrOH, perhaps involving the burying of the active site in the protein–protein contact site.