Biochimica et Biophysica Acta 1702 (2004) 181–189 http://www.elsevier.com/locate/bba

Fluorescence spectroscopic characterization of Humicola lanuginosa dissolved in its substrate

Arimatti Jutila*, Keng Zhu, Esa K.J. Tuominen, Paavo K.J. Kinnunen

Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, P.O. Box 63 (Haartmaninkatu 8), FIN-00014 University of Helsinki, Finland

Received 17 February 2004; received in revised form 4 August 2004; accepted 18 August 2004 Available online 8 September 2004

Abstract

The conformational dynamics of Humicola lanuginosa (HLL) and its three mutants were investigated by steady state and time- resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the a-helical surface loop, the so-called dlidT of HLL comprised of residues 86–93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the dopenT conformation in triacetin, with the a-helical surface loop moving so as to expose the . (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue. D 2004 Elsevier B.V. All rights reserved.

Keywords: Humicola lanuginosa lipase; Triacetin; Fluorescence spectroscopy

1. Introduction concentration at the interface [5], a better orientation of the scissile ester bond [6], a reduction in the water shell around Lipases (triacylglycerol , E.C. 3.1.1.3) con- the ester molecule in water [7], as well as conformational stitute a large family of and are widely distributed in changes of the leading to an optimized active site Nature. True lipases are distinguished from by their geometry and enhanced catalytic activity [1,8]. characteristic interfacial activation at lipid–water interfaces The amino acid sequence of Humicola lanuginosa lipase [1,2]. Several mechanisms have been forwarded to explain (HLL) consists of 269 residues, including four tryptophans this property [3,4]. These include an increased substrate [9]. The crystal structure of HLL has been solved at 1.8-2 resolution [10]. Accordingly, HLL consists of a single, roughly spherical domain containing a central eight- Abbreviations: EDTA, ethylenediaminetetraacetic acid; HEPES, N-(2- stranded, predominately parallel h-pleated sheet, with five hydroxyethyl) piperazine-NV-2-ethanesulfonic acid; HLL, Humicola lanu- interconnecting a-helices, compacted to a volume of ginosa lipase; RFI, relative fluorescence intensity; W89m, single Trp HLL 3 3 mutant with substitutions W117F, W221H, and W260H; W89mN33Q, approximately 9.710 2 . The active site contains a single Trp HLL mutant with substitutions W117F, W221H, W260H, and Ser(146)–Asp(201)–His(258) , closely remi- N33Q; wt, wild type; s, fluorescence lifetime; /, rotational correlation niscent to that seen in serine proteases [11]. The active site time; rl, residual anisotropy Ser is additionally involved in maintaining the structure of * Corresponding author. Tel.: +358 9 19125427; fax: +358 9 19125444. HLL and its substitution (S146A) leads to substantial E-mail addresses: [email protected] (A. Jutila)8 conformational alterations as well as different substrate [email protected] (P.K.J. Kinnunen). binding affinities [12]. The a-helical surface loop (amino

1570-9639/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2004.08.011 182 A. Jutila et al. / Biochimica et Biophysica Acta 1702 (2004) 181–189 acids 86–93) constituting the so-called dlidT lies directly (W117F, W221H, W260H, N33Q) were obtained from over the active site S146 [11,13], and is highly mobile in the Novo Nordisk (Bagsv&rd, Denmark). Their concentrations crystals [14]. The lid of HLL contains a single Trp at were determined as described previously [24]. Specific position 89. Site-directed mutagenesis [15,16] showed that activities of wt HLL, W89L, W89m, and W89mN33Q were Trp89 is critical for efficient hydrolysis of tributyrin and that 2158, 1587, 1130 and 1016 Amol/mg/min, respectively [17]. it plays a role in the catalytic steps subsequent to the The buffer used in the experiments was 20 mM HEPES, 0.1 absorption of the lipase to the substrate interface. Trp89 has mM EDTA, pH 7.0 (adjusted with NaOH), prepared in been shown to influence in a qualitative manner the binding freshly deionized (Milli RO/Milli Q, Millipore, Bedford, of substrates into the active site [16]. MA) water. Fluorescence intensity of HLL in triacetin Two distinct conformations, dclosedT and dopenT, inactive increased linearly with the lipase concentration indicating and active, respectively, have been proposed for HLL [17]. ideal solubility. Accordingly, in an aqueous medium, access to the catalytic triad is blocked by the lid and the conformation is closed. 2.2. Stationary fluorescence spectroscopy Martinelle et al. [9] reported that HLL displays a pronounced interfacial activation in the presence of the Steady-state fluorescence measurements were carried out substrate p-nitrophenyl butyrate (PNPB). This would be using a Perkin Elmer LS 50B spectrofluorometer equipped associated with a significant conformational change of the with a magnetically-stirred, thermostated cuvette compart- lid, its opening generating a large hydrophobic surface, ment. The excitation wavelength for Trp was 295 nm which also includes the active site. The ability of HLL to [25,26] with both excitation and emission bandpasses set at efficiently form the acyl-enzyme intermediate at the inter- 5 nm. To improve signal-to-noise ratio in the steady-state face thus requires a number of separate steps: (i) the enzyme anisotropy measurements, excitation and emission band- must adapt the active, lid open conformation, (ii) the lid and passes were 10 nm, and the emission wavelength was varied the hydrophobic surface has to attach to the interface, and between 332 and 346 nm. (iii) the active site has to be saturated with the substrate [15]. Importantly, in complexes with substrate analogues or serine 2.3. Time-resolved fluorescence spectroscopy protease inhibitors the lid helix is displaced and the active site is exposed, yielding the open conformation [18–20].To Commercial laser spectrometer (Photon Technology this end, not only lipids [13,21] but also detergents [22] and International, Ontario, Canada) was used to measure i-propanol, an organic solvent [23], can induce the opening fluorescence lifetimes, rotational correlation times, and of the lid of HLL. residual anisotropies. The minimum lifetime accessible to It is generally difficult to obtain conditions where the the instrument is 200 ps. A train of 500 ps excitation pulses chemical equilibrium for the binding of lipase to the at 337 nm at a repetition rate of 10 Hz was produced by a substrate would be shifted quantitatively to the presence of nitrogen laser, pumping 5 mM solution of rhodamine 6G the lipase–substrate complex. In the present study we (Merck, Darmstadt, Germany) in methanol. Pulses from the utilized fluorescence spectroscopy to compare the confor- lasing dye solution emitting at 590 nm were frequency mations of HLL in an aqueous buffer and dissolved in its doubled for Trp fluorescence measurements at excitation substrate, triacetin. Under the latter conditions we can expect wavelength of 295 nm. Depending on the sample, the the enzyme to be maximally saturated with the substrate. emission wavelength was varied between 332 and 346 nm Moreover, triacetin is optically transparent and does not using a monochromator. impede the use of fluorescence spectroscopy techniques. For the determination of the fluorescence lifetimes, the Both steady state and time-resolved analyses were employed averages of five emission decay curves were analysed using to monitor the changes in the fluorescence of lipases. HLL the software provided by the instrument manufacturer. contains approximately 20 mannose residues, glycosylated Instrument response functions were measured separately to Asn33. The variant with four mutations W89mN33Q using aqueous glycogen solution. The validity of the fit was lacks the glycosylation site and thus also the carbohydrate judged by the value of the reduced chi-square, v2 [27,28] moiety (S.A. Patkar, personal communication). Accordingly, which varied in the range of 0.9–1.2. W89m and W89mN33Q were made to study the impact of Fractional intensities I(t) were calculated according to the carbohydrate moiety on the dynamics of the lid. the equation: XN ItðÞ¼aisi aisi ð1Þ 2. Materials and methods i¼1

where ai is amplitude and si is lifetime. For polarized light, 2.1. Materials the decay of fluorescence intensity, F(t) was calculated from the raw data according to: H. lanuginosa lipase (HLL) and its mutants W89L, W89m (W117F, W221H, W260H), and W89mN33Q FtðÞ¼IjjðÞþt 2 G I8ðÞt ð2Þ A. Jutila et al. / Biochimica et Biophysica Acta 1702 (2004) 181–189 183 where It (t) is the intensity of light detected with both the wild-type HLL contains four Trps residing in positions 89, excitation and emission polarizer being vertical (i.e., parallel 117, 221, and 260, which combined report on more global polarizers) and I8(t) is the intensity of light detected with changes in this lipase. Regarding the interaction of HLL vertical excitation polarizer and horizontal emission polar- with its substrate and the conformational change causing izer (i.e., perpendicular polarizers), and G is the correction enzyme activation, the recently introduced single Trp term for the relative throughput of the respective polarized mutant W89m is of particular interest [22,23,29,30]. light component through the emission optics. Furthermore, mutants W89L and W89mN33Q enable Time-resolved anisotropy r(t) is defined as: studies on the importance of Trp 89 and mannose residues, respectively, for the structure of HLL. Fluorescence spectra rtðÞ¼ IjjðÞÀt G I8ðÞt = IjjðÞþt 2 G I8ðÞt ð3Þ for these lipases are shown in Fig. 1 and the measured photophysical parameters are compiled in Figs. 2–4. where G, It and I8 are as above. The fluorescence and anisotropy decays are described by 3.2. Emission intensity and kmax a sum of exponentials, as follows: X The wavelength of the emission maximum kmax and the Àt=si FtðÞ¼ Aie ð4Þ fluorescence quantum yield for Trp depend on its micro- i environment. In brief, with increase in polarity kmax shifts to X longer wavelength and quantum yield decreases [31,32]. rt r eÀt=/i 5 ðÞ¼ i ð Þ Notably, the fluorescence quantum yield of W89m is high i and the emission of its single Trp is approximately 60% of In the above Ai and ri are the normalized pre-exponential that measured for the wild-type HLL with four Trps [22]. initial fluorescence intensities and initial anisotropies, respectively, and si and /i are the corresponding fluores- cence lifetimes and rotational correlation times. When the angular range of the rotational motion of fluorophore is limited

Àt=/ rtðÞ¼ðr0 À rlÞe þ rl ð6Þ where r0 stands for the anisotropy in the absence of rotational diffusion. Eq. (6) was used only to fit the data so as to obtain the value of the residual anisotropy. Rotational correlation times (/i) and residual anisotro- pies (rl) were derived from the fitted curves. The decays I(t)t and I(t)8 were fitted before calculating the respective r(t) decays from these data. Accordingly, r(t) contains no experimental noise and therefore criteria such as the v2 value do not apply. For the determination of rotational correlation times and residual anisotropies, each emission decay was measured 10 times.

3. Results

3.1. General considerations

Trp residues provide intrinsic fluorescent probes for protein structure, allowing for detailed studies on their conformational dynamics. We used both steady state and time-resolved fluorescence spectroscopy to compare HLL dissolved in an aqueous buffer and in its substrate triacetin. The pH of triacetin is approximately 5.5, whereas that of the buffer used in this study is 7.0. To check the pH effect we recorded steady state fluorescence spectra at these two pHs Fig. 1. Fluorescence spectra of 2 AM wt HLL (.), W89L (4), W89m (n), and W89mN33Q (5) in 20 mM HEPES, 0.1 mM EDTA, pH 7.0 (panel A) in buffer. These spectra were identical verifying that the and in triacetin (panel B) at 25 8C. The concentration of water in the latter differences between the spectra recorded in buffer and medium was approximately 0.5 vol.%. The excitation wavelength was 295 triacetin are due to the conformational changes in HLL. The nm, both excitation and emission bandpasses were 5 nm. 184 A. Jutila et al. / Biochimica et Biophysica Acta 1702 (2004) 181–189

This is likely to be explained by the low quantum yields of the other three Trps, Trp117 and Trp221 in particular. More specifically, the latter two reside in locations with both anionic and cationic residues in their immediate vicinity, which could be sufficient to perturb the electronic config- uration of the Trps so as to cause efficient quenching of their fluorescence [30]. For wt HLL, W89L, W89m, and W89mN33Q in buffer values for kmax are 342, 332, 345, and 346 nm, respectively (Fig. 2A). As expected, in the less polar triacetin a blue shift by 2, 8, and 8 nm in kmax was observed for wt, W89m, and W89mN33Q, respectively. On the other hand, fluorescence intensity was significantly lower in triacetin for these three lipases (Fig. 1), in keeping with reduced quantum yield of Trp89 in triacetin. Accord- ingly, the opposite was true for W89L as a 6-nm red shift and an increase in emission intensity were observed, in keeping with decreased interactions between Trps and their vicinal charged amino acids in triacetin.

3.3. Stationary fluorescence anisotropy

Rotational diffusion of fluorophores is the dominant cause of fluorescence depolarization. In proteins the mobility of fluorescent amino acid residues bears a close relationship with the overall state of a protein, and any Fig. 2. Wavelength of the maximum emission intensity (panel A) and steady factor which affects its size, shape, state, or segmental state fluorescence anisotropy (panel B) for the studied lipases in buffer (open columns) and in triacetin (striped columns). flexibility will also affect the observed anisotropy value

Fig. 3. Fluorescence lifetimes s1 (panel A) and s2 (panel B) and the contribution of the latter (panel C) for the lipases in buffer (open columns) and triacetin (striped columns). Panel D shows the average fluorescence lifetimes in these two media. A. Jutila et al. / Biochimica et Biophysica Acta 1702 (2004) 181–189 185

Fig. 4. Rotational correlation times /1 (panel A), /2 (panel B), and /3 (panel C) and residual anisotropy rl (panel D) for the studied lipases in buffer (open columns) and in triacetin (striped columns).

[33]. Accordingly, anisotropy reports on both conforma- respectively, emerging in the latter medium. For wt, W89m, tional changes and the state of association of a protein. and W89mN33Q the longer fluorescence lifetime (s2) When the medium was changed from the aqueous buffer to component decreased in triacetin (Fig. 3B). Instead, for the more viscous triacetin, the steady state anisotropy (r) W89L the value for s2 increased from 2.15 to 3.76 ns in this values increased for wt, W89m, and W89mN33Q, with solvent. The fractional intensity of the longer fluorescence most pronounced change for the latter, from 0.124 to 0.188 lifetime (s2%) of the wt and W89L increased from 82% and (Fig. 2B). Interestingly, W89L stands again apart from the 58%, respectively, to 91% for both (Fig. 3C). For W89m other studied lipases as the value of r decreased from 0.180 and W89mN33Q, s2% decreased from 100% to 98% and to 0.147 in triacetin. Accordingly, W89L is least compactly 91%, respectively. folded in this solvent with the neighboring amino acids being less restrictive to the motions of the three Trps. 3.5. Rotational correlation times and residual anisotropy

3.4. Fluorescence lifetimes Time-resolved fluorescence anisotropy can provide information on the diffusive motions of a fluorophore during Not only the spectral features but also the fluorescence its excited lifetime. More specifically, these data can reveal lifetimes of Trp depend on its microenvironment, thus whether a fluorophore is free to rotate over all angles, or if allowing to obtain further insight into the conformational the surroundings of the fluorophore restrict its angular changes of a protein. Average fluorescence lifetimes (s¯) Brownian motion. Moreover, these measurements allow to decreased from 3.95, 5.40, and 6.37 to 2.84, 3.58, and 3.51 distinguish between decays of anisotropy due to a single ns for the wt HLL, W89m, and W89mN33Q, respectively, process, and those involving multiple modes of rotations whereas for W89L s¯ increased from 1.58 to 3.43 ns (Fig. [33]. We measured fluorescence anisotropy decays of these 3D). Two-exponential fluorescence decays were measured lipases in the aqueous buffer and triacetin, and calculated the for wt and W89L both in buffer and triacetin. The shorter rotational correlation times (/) and residual anisotropies fluorescence decay (s1) was significantly faster in triacetin (rl) from these data (Fig. 4). In the aqueous medium, two than in the buffer (Fig. 3A). Yet, the measured values (0.12 rotational correlation times were required for all four and 0.09 ns, respectively) are already beyond the resolution proteins for satisfactory fit of the data. The long rotational of the instrument. W89m and W89mN33Q exhibited single- correlation time (/3) varied between 20 and 24 ns (Fig. 4C), exponential decays in buffer and two-exponential in while the short rotational correlation time (/1) was b1ns triacetin, with the shorter component of 0.33 and 0.76 ns, (Fig. 4A). However, in triacetin the anisotropy decays 186 A. Jutila et al. / Biochimica et Biophysica Acta 1702 (2004) 181–189 became three-exponential for wt, W89m, and W89mN33Q, of this lipase to reside in hydrophobic microenvironments whereas for W89L a two-exponential anisotropy decay was [31]. In contrast to the other studied lipases, there is a red measured also in triacetin. The long rotational correlation shift by 6 nm, a threefold¯ increase in fluorescence intensity, times (/3) were increased in triacetin by 3.3, 23.9, 3.4, and and an increased s for the emission of W89L in triacetin. 1.8 ns for wt, W89L, W89m, and W89mN33Q, respectively These effects are probably due to decreased interactions (Fig. 4C). In triacetin the short rotational correlation times between Trps and their vicinal charged amino acids such as (/1) for the wt, W89m, and W89mN33Q were less than 1 His, Lys, Arg, Glu, and Asp [30] upon increase in ns, and the intermediate rotational correlation times (/2) hydrodynamic volume of the lipase. This would result in were in the range of 3.9–6.6 ns (Fig. 4B). For W89L, the decreased quenching and prolonged fluorescence lifetime value for /1 was 3.33 ns. The residual anisotropies (rl) for for this mutant. all these lipases revealed the same behaviour, the measured Time-resolved fluorescence studies provide further values decreasing upon the transfer of the lipases from the insight into the changes in conformational dynamics aqueous buffer into triacetin (Fig. 4D). induced by triacetin. Intensity decay of W89m and W89mN33Q was one-exponential in the aqueous buffer, whereas two-exponential fitting was required in triacetin. 4. Discussion Two populations of Trp(s) with different fluorescence lifetimes have been ascribed to different Trp rotational A major problem in studying substrate induced changes isomers [35]. In proteins, the shorter fluorescence lifetime in an enzyme such as lipase is due to the difficulty in component s1 has been further suggested to reflect obtaining the enzyme to be quantitatively associated with interactions of the surface-exposed Trp(s) with the solvent the substrate. This problem was alleviated in the present [36]. The crystal structure of the open conformation of HLL study by dissolving HLL in its substrate, triacetin. More- revealed Trp89 to be exposed on the protein surface [37],in over, the concentration of water is very low, c0.28 M accordance with the appearance of s1 in triacetin. Similar (approximately 0.5 vol.%), allowing maximally about 6.5% behaviour has been observed for protein tyrosine phospha- of the total triacetin present to be hydrolyzed. Accordingly, tase (PTPase) which exhibits two-exponential emission we can expect most of the enzyme to be in the form of the decay in the dopenT conformation [38]. acyl-enzyme intermediate. The latter state should be Decrease in steady state fluorescence anisotropy of a associated with significant conformational changes espe- fluorophore indicates augmented mobility and vice versa cially in the lid of HLL. Wt HLL contains four Trps two of [33]. Judged from these data on W89m and W89mN33Q, which, Trp89 and Trp117, are located in two different a- the movements of Trp89 became more restricted in triacetin. helices and the other two, Trp221 and Trp260, in two As Trp89 locates on the surface of HLL in the open different h-pleated sheets. Changes in the intrinsic fluo- conformation [29], the increased r is probably due to the rescence of the wt thus include contributions from all four increased viscosity of the medium. An opposite change is Trps and therefore report on the global conformational observed for W89L indicating that the average movements changes of this protein. Accordingly, particular interest was of Trps 117, 221, and 260 became less restricted. It can be in the single Trp mutant W89m, reporting on the changes in concluded that in W89L the interactions of these three Trps the dlidT. In order to survey possible effects of the with their vicinal amino acids are weaker in triacetin. carbohydrate moiety on the conformational dynamics of Time-resolved fluorescence anisotropy yields informa- the lid, the mutant W89mN33Q was employed. tion on the rapid local motions of the Trp side chains as well Tryptophan emission is sensitive to its microenviron- as the overall rotational diffusion of the entire protein [39]. ment. Comparison of kmax in triacetin and aqueous buffer Two rotational correlation times (/1 and /3) were measured revealed blue shifts of 2, 8, and 8 nm for HLL, W89m, and for these four lipases in buffer. However, three rotational W89mN33Q, respectively, in keeping with triacetin being correlation times (/1, /2, and /3) were required for a less polar than water [31]. On the other hand, fluorescence satisfactory fit for wt, W89m, and W89mN33Q in triacetin. intensity was significantly lower in triacetin for these three In buffer, the short correlation time /1 can be assigned to lipases (Fig. 1). This finding is in apparent contradiction the local segmental motions of Trp(s), [40], i.e., movements with the quantum yield of Trp fluorescence being inversely of side chains, a-helices and h-pleated sheets containing proportional to the polarity of microenvironment [32].We Trp(s) [38]. As the lid of HLL is highly mobile in the attribute this decrement to quenching of the Trp89 crystals [14], it can be expected that the rates of motion of fluorescence in triacetin. Fluorescence lifetime of Trp is the Trp89 side chain and the lid are relatively close. related to its microenvironment and reduced polarity of the Consequently, these cannot be distinguished by time- medium is associated with longer lifetimes [34]. Interest- resolved anisotropy measurement, and in the time range ¯ ingly, the average fluorescence lifetimes s of wt, W89m, shorter than 10 ns only one rotational correlation time is and W89mN33Q decreased in triacetin, thus indicating the likely to be present. As indicated by steady state anisotropy quenching of Trp89 in triacetin to be dynamic [33].In measurements the three Trps of W89L in buffer are located buffer, kmax of W89L is at 332 nm suggesting the three Trps inside the protein, in surroundings more restricting than that A. Jutila et al. / Biochimica et Biophysica Acta 1702 (2004) 181–189 187 for Trp89. Accordingly, the local motions of Trps 117, 221, dynamics of the lipase as shown by the emission of Trp89. and 260 would be slower than Trp89, as revealed by time- Comparison of the single Trp mutants W89m and resolved anisotropy measurements showing the rotational W89mN33Q reveals that the absence of the mannose correlation time /1 for W89L to be longer than that for residues causes the microenvironment of Trp89 to become W89m or W89mN33Q. In a medium of higher viscosity, more hydrophilic, indicated by the decrease in emission e.g., triacetin, the motions of the lid can be expected to be intensity (Fig. 1) and a red shift in kmax (Fig. 2A). This slower, and these two different modes of local motion thus could result from more loose packing of HLL and enhanced become separable. Under these conditions it is feasible to access of water to Trp89. This conclusion is supported by attribute the short and the medium correlation times /1 and the lower steady state anisotropy value (Fig. 2B) and shorter /2 to the motion of Trp89 side chain and the lid, rotational correlation time /1 (Fig. 4A) measured for respectively. In triacetin the motions of Trps and a-helices W89mN33Q in aqueous buffer. In the more viscous medium and h-pleated sheets of W89L could not be distinguished, triacetin the observed steady state anisotropy value is likely and only one short rotational correlation time was required to be dominated by the global motions of the lipase. Thus, it for a satisfactory fit. In triacetin the motions of both Trp89 is rational that the glycosylated mutant W89m with tighter and lid are slower for W89mN33Q than for W89m, as packing and smaller radius of hydration has a somewhat indicated by the somewhat larger values for /1 and /2, lower anisotropy value. The long rotational correlation time respectively. This shows that also the carbohydrate moiety /3 (Fig. 4C) obtained from time-resolved anisotropy affects the conformational dynamics of HLL. measurements should measure directly the global motion Non-zero values of residual anisotropy rl have been of the lipase. However, no significant changes in this interpreted to result from an energy barrier preventing parameter were observed in either medium. This contra- rotational diffusion of the fluorophore beyond a certain diction may be apparent only as changes in steady state angle within the fluorophore lifetime [33]. For all the lipases anisotropy are very small, and, as the global motions of in the present study, the value of rl was lower in triacetin HLL are likely to contribute to /2 (Fig. 4B), the value of the than in the aqueous buffer, indicating diminished constraints latter being decreased upon mutation N33Q. In buffer the for mobility of the fluorophores. These data would be average fluorescence lifetime for W89mN33Q is longer than compatible with Trp89 to be located on the protein surface that for W89m (Fig. 3D). As a more hydrophilic micro- in the open conformation [29]. environment of Trp usually decreases its fluorescence The long rotational correlation time /3 reflects the global lifetime, the explanation for this difference remains motions of the entire lipase, and thus it is related to the unknown at present. However, the longer fluorescence hydrodynamic volume of the molecule by the Einstein– lifetime provides a likely reason for the lower residual Stoke’s equation anisotropy value measured for W89mN33Q (Fig. 4C).

/3 ¼ gMvðÞþ h =RT ð7Þ where g is the viscosity of the medium at temperature T, M 5. Conclusions is the molecular mass of protein, v is the specific volume of protein, h is the hydration factor, and R is the gas constant. The data reported here demonstrate open conformation of Using a value of 0.89 cp for the viscosity of water at 25 8C HLL to be induced by its substrate triacetin. In triacetin, [41], /3 of 20.11 ns measured for W89m in buffer W89L increased in volume significantly revealing Trp89 to corresponds to a hydrodynamic radius of 27.8 2. This is play an important role in the structural stability of HLL. In in excellent agreement with the value of 28 2 (=the an aqueous medium, the motions of the W89 side chain and maximum distance from the center of mass) obtained from the lid are not separable by time-resolved fluorescence crystal structure [42], thus excluding the possibility of anisotropy measurements. However, the difference between oligomerization of HLL. the frequencies of these two modes of motion is augmented Viscosity of the medium slows down the rotations of in triacetin, allowing to distinguish between these two molecules, and, as expected, the long rotational correlation modes. Accordingly, the rotational correlation times for times /3 for all the lipases were augmented in triacetin. For Trp89 side chain and the lid, approximately 0.2 and 5 ns, W89L the value for /3 was increased twofold, indicating a respectively, could be resolved by fluorescence anisotropy. very dramatic increase in the hydrodynamic volume of the The carbohydrate moiety attached to Asn33 also contributes rotating unit. This can be attributed to either aggregation or on the conformational dynamics of the lid, although only to increase in volume of single protein. The latter seems more a minor extent. likely, as Trp89 is important for the structural stability of HLL [30]. 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