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Interdisciplinary Research Centre, K.U. Leuven Campus Kortrijk, B-8500 Kortrijk, Belgium
Reprint requests to: Herman Van Dael, Interdisciplinary Research Centre, K.U. Leuven Campus, Kortrijk, E. Sabbelaan 53, B-8500 KORTRIJK, Belgium; e-mail: Herman.Vandael{at}kulak.ac.be; fax: +32-56-246997.
(RECEIVED October 4, 2002; FINAL REVISION December 6, 2002; ACCEPTED December 11, 2002)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0235303.
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Abstract |
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Keywords: Protein folding; intermediate state; canine lysozyme; stopped-flow fluorescence; kinetics; molten globule
Abbreviations: CL, canine lysozyme • apo-CL, calcium-depleted form of CL • CD, circular dichroism • DSC, differential scanning calorimetry • HLY, human lysozyme • GdnHCl, guanidine hydrochloride • MeU-triNAG, 4-methylumbelliferyl-N,N',N''-triacetyl-ß-chitotriose
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Introduction |
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-lactalbumin/lysozyme family, an analogous correspondence was found using different techniques in hen, human, and equine lysozyme (Kuwajima et al. 1985; Mizuguchi et al. 1998; Haezebrouck et al. 1999) and in bovine, human, and goat -lactalbumin (Balbach et al. 1995; Chaudhuri et al. 2000; Yoda et al. 2001). In an engineered chimeric protein, constructed from parts of human lysozyme and bovine -lactalbumin, on the other hand, an obvious difference between both intermediate states could be detected (Haezebrouck et al. 1999). In most cases, the conclusion that equilibrium and kinetic intermediate states resemble, is based on the observation that both states can be adequately described as molten globules. The molten globule state is characterized by the presence of substantial secondary structure arranged in a native-like overall fold, but its hydrophobic core is exposed to the solvent and it lacks rigid side-chain packing and fixed tertiary interactions. In several studies, however, it has been stressed that a molten globule is not a well-defined, unique structure but that it covers a variety of states, ranging from nearly fully unfolded proteins to highly ordered molten globules with specific and detectable tertiary structure (Dill and Chan 1997; Fink et al. 1998). The exact role played by an intermediate in the folding process is still under debate. Classically, a folding intermediate is seen as an essential element, acting as a collector along the folding pathway and, thus, favoring the transition to the native state. On the other hand, it has also been argued that folding intermediates merely result from the existence of kinetic traps in the folding reaction (Baldwin 1996; Dill and Chan 1997). Possibly, specific non-native interactions stabilize such intermediate states and have to be broken in order to reach the global energy minimum corresponding to the native state. Therefore, a detailed characterization of equilibrium and kinetic intermediate states remains an essential element in elucidating the folding mechanism of globular proteins.
Canine lysozyme belongs to the family of calcium-binding lysozymes (Grobler et al. 1994). Although recent determination of the crystal structure of apo-CL in the native state (Koshiba et al. 2000) revealed that the overall structural motif of CL is not distinguishable from that of conventional noncalcium-binding lysozymes, its physicochemical properties are known to be rather different. Whereas conventional lysozymes unfold in a cooperative two-state manner without the accumulation of an intermediate form, a stable equilibrium intermediate has been shown to accumulate during the GdnHCl—induced unfolding of CL (Kikuchi et al. 1998). In this way, the unfolding behavior of CL is comparable with that observed in -lactalbumin, the equilibrium state of which has extensively been characterized as a molten globule. Further investigations by the Nitta group (Koshiba et al. 1999, 2000; Kobashigawa et al. 2000) have characterized the intermediate of CL as an extraordinarily stable molten globule. This extra stabilization, compared to -lactalbumin and equine lysozyme, is attributed to the strengthened cooperative interaction between secondary structure elements and the stronger protection of the aromatic cluster region around the C and D helices.
In this paper, we fruitfully exploit the exceptional stability of the molten globule intermediate in canine lysozyme in the dissection of the individual steps of its folding process. At pH 7.5 and 25°C in 3 M GdnHCl, the intermediate state of CL is populated for the full 100%. This enabled us to isolate a CL sample in the equilibrium intermediate state (IE) and to study the folding kinetics by stopped-flow fluorescence and circular dichroism spectroscopy in two clearly separated steps, that is, from the unfolded state (U) to IE and from IE to the native state (N). This was not possible in former folding studies of the lysozyme/-lactalbumin family, as the intermediate state in these samples was only partially populated, being in dynamic equilibrium with the U or the N state. The population of the intermediate state maximally amounts to 0.4 in a mutant of human lysozyme (Haezebrouck et al. 1999), to 0.2 and 0.8 in the chimeric molecules Lyla1 and Lyla1B, constituted from parts of human lysozyme and -lactalbumin (Haezebrouck et al. 1999; Joniau et al. 2001), to 0.3 in pigeon lysozyme (Haezebrouck et al. 1998), and to 0.6 in equine lysozyme (Griko et al. 1995). In the present experiments, besides the characterization of the equilibrium intermediate state, we were able to identify a folding intermediate in kinetic experiments. In contrast to what is expected, we showed that the non-native interactions, which occur along the folding pathway in canine lysozyme, are not involved during the refolding from U to IE but that they appear only after the intermediate has been formed and thus during the further evolution to the native state.
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Results |
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). The overall shape and the details of the CD spectra in both spectral regions largely correspond with earlier published spectra of CL at pH 4.5 (Koshiba et al. 1999, 2000) and at pH 2 (Polverino de Laureto et al. 2002).
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max and the important decrease of the far-UV ellipticity are characteristic for gradual further unfolding.
In the holo-form, the two transitions are conserved but, as only the first one is shifted to higher temperatures as a result of the stabilization by Ca2+ binding, they tend to overlap. Nevertheless, from the evolution of max and the ellipticity at 222 nm, also here, a clear distinction between the two transitions remains possible. The first transition starts from about 60°C, while the main unfolding takes place from 80°C on.
The results of the chemical denaturation of apo-canine lysozyme at pH 7.5 can be interpreted in an analogous way (Fig. 2, left panel). A first transition, in which an important decrease in the ellipticity at 287 nm is observed whereas the ellipticity at 222 nm remains nearly constant, occurs in the concentration region from 0 to 2 M GdnHCl. Between 2 and 4 M GdnHCl, the spectral properties do not change, indicating that a stable intermediate is formed. As this intermediate contains reduced tertiary structure and conserved native secondary structure, it can be classified as a classical molten globule. The main transition in which the equilibrium intermediate unfolds, starts at about 4 M and combines a further decrease of the CD signal at 287 nm with a shift of max to higher values and with a drastic decrease of the far-UV CD intensity.
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, right panel), max shifts from 335 nm to 344 nm in a monophasic process situated between 4 M and 7 M GdnHCl. The CD spectral parameters in the near and far-UV region, which monitor changes in the tertiary and secondary structure, behave in the same way. In this case, no evidence remains of the first unfolding step observed for the apo-form.
Refolding kinetics of apo-canine lysozyme
The refolding kinetics of apo-CL were measured at pH 7.5 and 25°C by diluting a solution of unfolded CL in 6 M GdnHCl to yield native CL in 0.54 M GdnHCl. During the dead time of the experiment (2 ms), the total fluorescence intensity drops drastically to a value that is significantly lower than the fluorescence intensity obtained in the final, native state. The overshoot amplitude equals 61% of the intensity change between the U and N state. This final intensity is reached in a monoexponential way with k = 3.6 s-1 (Fig. 3A, Table 1).
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av, defined as
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(Royer et al. 1993). The latter parameter is a good measure for the extent to which the Trp residues are shielded from the solvent. Figure 3B shows that already after 2 ms the Trp residues are in an environment that exhibits nearly the same shielding from solvent as that of the native state.
The development of a functional active site during the refolding of apo-CL was monitored in an additional experiment in which the binding of the fluorescent probe MeU-triNAG to CL was examined. As this experiment has to be done preferentially at pH 6 (Yang and Hamaguchi 1980), the refolding kinetics based on Trp fluorescence have to be determined accordingly at this pH. The overall refolding behavior is similar to that obtained at pH 7.5, but the overshoot phenomenon becomes even more important. It has been shown before (Itzhaki 1994; Haezebrouck et al. 1999) that the presence of MeU-triNAG does not disturb the refolding process of HLY and that the binding of MeUtriNAG to native HLY proceeds in a single exponential process that is considerably faster (k = 145 s-1) than the refolding of HLY. In that way, the signal observed in the refolding experiment (Fig. 3C) can be attributed unambiguously to the refolding process itself. Our present data show that only 5% of the fluorescence change is achieved in the dead time, indicating that, although very important changes were detected in the Trp fluorescence change during the initial collapse, the active site is not yet formed. The substantial increase of the fluorescence intensity reflects the binding of the probe to the active site with k = 4.9 s-1, which is comparable with the rate constant of the increase in Trp fluorescence at this pH.
The near-UV CD signal recorded at 287 nm shows a dead time (8 ms) effect of 53% and the remaining signal proceeds in a monoexponential process with k = 4.5 s-1 (Fig. 3D). It is remarkable that, in contrast to our observations in the Trp fluorescence experiments, no overshoot is detected with CD. In the far-UV spectrum at 225 nm, 81% of the ellipticity change is already achieved in the dead time, indicating that most of the secondary structure is formed in the first 8 ms of the refolding (Fig. 3E). The ellipticity of the fully native protein is reached in an monotone exponential process (k = 3.5 s-1).
Refolding kinetics of holo-canine lysozyme
The refolding kinetics of Ca2+-CL were examined by monitoring the total Trp fluorescence intensity as a function of time at pH 7.5 and pH 6 (Fig. 4A). During the first 2 ms, the fluorescence intensity decreases and reaches a level below that of the native state. In the subsequent phase, the fluorescence intensity achieves the native level in an exponential way with k = 4.8 s-1. Thus, the presence of Ca2+ ions only marginally accelerates the refolding process (k = 3.6 s-1 for apo-CL).
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av as a function of time, it appears that the almost complete shift in av occurs during the dead time (Fig. 4B). In that process, av drops from 353.3 nm to 349.1 nm. Accurate analysis of the earliest observable data points reveals an extremely small rearrangement of the Trp residues reflected in an increase of av of a few hundredths of a nanometer with k = 3.6 s-1. This value corresponds with the slower phase in the fluorescence intensity measurements.
The time course of the organization of the native conformation is followed by the binding of the fluorescent inhibitor MeU-triNAG. Twenty-five percent of the total fluorescence change occurs during the first 2 ms, whereas the remaining 75% of the fluorescence intensity change evolves via a single exponential process with k = 5.9 s-1 (Fig. 4C). The fact that 25% of the fluorescence intensity change occurs during the dead time may refer either to a weak binding of the probe to the whole ensemble of protein molecules or to a strong binding to the completely native state of only a fraction of the protein molecules.
In the far-UV/CD experiments at = 225 nm (Fig. 4D), 84% of the ellipticity change occurs in the dead time (8 ms), indicating that in that period most of the secondary structural elements are formed. The remaining evolution to a complete native structure can be described in a monoexponential way (k = 4.7 s-1 ). As for the apo form, no overshoot is detected in the CD experiments on holo-CL.
Refolding kinetics of apo-CL from the unfolded to the intermediate state
The refolding kinetics of apo-CL from the unfolded state to the equilibrium intermediate state can be followed by diluting a solution of apo-CL in 6 M GdnHCl at pH 7.5 and 25°C with a refolding buffer to a final state in 3 M GdnHCl. From Figure 5 it appears that the total fluorescence intensity change and the complete shift in av take place during the dead time. Also in CD experiments at 287 nm (not shown), the small jump in ellipticity that accompanies the transition is already accomplished in the dead time of the experiment. Thus, the transition from U to IE proceeds very fast and is certainly totally completed during the first milliseconds. The measured intensity change and wavelength shift, however, are not equal to the corresponding quantities observed in the U to N refolding process. This implicates that important additional kinetic phenomena are due to happen in the IE to N unfolding.
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shows an initial, significant drop of the fluorescence intensity. This overshoot phenomenon indicates that in the course of this refolding process, a non-native intermediate is formed in which Trp fluorescence is severely quenched. The native conformation is subsequently restored with k = 4.3 s-1. Thus, starting from IE, the native conformation is not reached in a direct, but in a roundabout way. The overshoot, that is very significant in the intensity measurements is not detected in the measurement of av (Fig. 6B). Starting from 349.3 nm, 62% of the total change of av occurs in the dead time, suggesting that in this kinetic intermediate state a significant number of Trp residues is sequestered from solvent. Further evolution happens monotonically in an exponential way with k = 5.2 s-1. A comparable rate constant (k = 5.3 s-1) is found in the CD experiments at 287 nm (not shown).
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Discussion |
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-lactalbumin in that it is significantly more organized (Van Dael et al. 1993; Griko et al. 1995; Van Dael 1998). Indeed, an ordered core composed of the strongly protected A-, B-, and D-helices favors substantial native-like interactions between hydrophobic residues and stabilizes the intermediate state in equine lysozyme (Morozova et al. 1995,1997). The second transition (70°C–100°C ) in apo-CL represents the further denaturation of the intermediate state to a highly unfolded state. As shown before by differential scanning calorimetry experiments (Koshiba et al. 2000, 2001), the position of the first transition in CL is strongly Ca2+-dependent, resulting in a shift to higher temperature at increasing Ca2+ concentration. In contrast, the second transition is not markedly influenced by the presence of Ca2+ ions. This result indicates that the first transition is associated with the disruption of the calcium binding site and correspondingly results in the loss of the ability to specifically bind Ca2+. A similar behavior was found in a calorimetric study of the heat denaturation of the related equine lysozyme that equally binds Ca2+ (Griko et al. 1995). Nevertheless, the intermediate state of CL has been shown to be 9.0 kJ/mol more stable than that of equine lysozyme at pH 4.5 and 66.4°C (Koshiba et al. 2001).
Our results of the chemical denaturation experiments on natural canine lysozyme largely agree with recent data obtained on apo- and holo-recombinant canine milk lysozyme at pH 4.5 (Kobashigawa et al. 2000). In our experiments, most of the specific tertiary structure is destroyed at 2 M GdnHCl whereas the secondary structure is almost completely retained. Therefore, in a broad concentration range between 2 and 4 M GdnHCl, a very stable equilibrium intermediate is formed. This result is consistent with the DSC studies of CL (Koshiba et al. 2000), which indicated that specific packing interactions in the -domain cause the presence of an extraordinarily stable molten globule. In our case, the population of that intermediate amounts to 100% at 3 M GdnHCl and this enables us to perform refolding experiments from the unfolded to a pure intermediate state.
The U 
N refolding process
Although the evolution of structural organization occurring rapidly after the initiation of refolding cannot be observed directly, interesting information on the early refolding kinetics can be derived from the signals obtained in stopped-flow experiments just after the dead time. These earliest detected events correspond to kinetically unresolved changes in circular dichroism ellipticity or in fluorescence intensity. They are attributed to the rapid formation of a relatively uniform collapsed state, mainly driven by the hydrophobic effect (Dill 1990a,b).
The pronounced overshoot in the total fluorescence intensity of apo-canine lysozyme (Fig. 3A) strongly suggests that non-native interactions are formed that result in quenching of tryptophan fluorescence in the early collapsed state. The evolution of av from fluorescence spectra recorded at discrete emission wavelengths (Fig. 3B) shows that Trp residues are well shielded from solvent. The latter fact, however, does not imply that the protein has adopted its native form. From our fluorescence experiments in the presence of MeU-triNAG (Fig. 3C), it is obvious indeed that the active site has not yet been formed after 2 ms and that the fully native state is only achieved in a further process. The initial value of the far-UV CD signal reveals a rapid stabilization of secondary, particularly helical, structure (Fig. 3E). The development of a native-like, near-UV CD signal indicates that at least some tryptophan residues become immobilized during the hydrophobic collapse (Fig. 3D). Taken together, we can characterize the burst phase as a collapsed state with a substantial amount of secondary structure and with some Trp residues buried in the hydrophobic core, which results in quenching of the total fluorescence intensity. As tertiary contacts are fluctuating and are at least partially non-native, this burst phase does not represent a distinct and fixed folding intermediate state but merely corresponds to a nonspecifically collapsed state with heterogeneous character.
The observable refolding kinetics of apo-canine lysozyme, as they result from either fluorescence or CD measurements, evolve on the same time scale in a monoexponential process with time constants that are nearly independent of the technique used (k = 4.2 ± 0.7 s-1). The assembly of secondary structure and the stabilization of persistent native tertiary contacts thus evolve simultaneously and cooperatively.
The addition of Ca2+ to the sample does not alter the overall kinetics of refolding. The initial collapsed state evolves to the final native state in a monoexponential process with a rate constant that has a value only a little higher than that for the apo-protein (k = 5.3 ± 0.6 s-1). Although the stability of canine lysozyme is drastically affected by the binding of Ca2+ (Koshiba et al. 2000; this work), the binding of this ion has little effect on the folding speed. Previous measurements on the influence of Ca2+ ions on the folding speed of pigeon lysozyme, that is highly comparable with the canine protein, equally reveal that Ca2+ binding does not induce a measurable increase of the refolding rate (Haezebrouck et al. 1998). Conversely, in the case of -lactalbumin, the binding of Ca2+ ions induces a very drastic enhancement of the folding speed by more than two orders of magnitude (Kuwajima et al. 1989; Haezebrouck et al. 1998). Sequential mixing experiments monitored by fluorescence spectroscopy have indicated that, in the case of -lactalbumin, the slower folding in the absence of Ca2+ is not the result of the accumulation of kinetically trapped species (Forge et al. 1999). Rather, Ca2+ stabilizes native-like contacts in the partially folded species and reduces the barriers for the conversion of the protein to its native state (Forge et al. 1999). Also, Ca2+-binding mutants of human lysozyme show a significant increase of the refolding rate upon Ca2+ binding (Haezebrouck et al. 1999).
Characterization of the kinetic intermediate state
In a kinetic experiment, the population of intermediates, which are formed transiently during the folding of the protein, is followed as a function of time. For many proteins, the intermediates appear very native-like, suggesting a direct hierarchical mechanism of folding. In this case, the intermediates could increase the rate of folding by reducing the total number of possible conformations in the search process toward the final native state. An intermediate is then seen as a transient structure that favors the further evolution to the folded state (Kuwajima 1989; Kim and Baldwin 1990). However, the kinetic phenomena can also be explained arguing that intermediates simply represent off-pathway misfolded structures from which the protein must unfold before refolding to the native conformation (Baldwin 1996; Dill and Chan 1997). In this case, intermediates result from the formation of misfolded, trapped species and play a nonproductive role in the folding process. Several proteins are known to form non-native structure in their transient folding intermediates and reorganize this nonnative structure to yield the final native structure. Whether the reorganization step becomes a kinetic trap is determined by the kinetic stability of the non-native structure. Accumulation of a kinetic intermediate always requires that the free-energy barrier between a conformation and the next state on the folding pathway is higher than the one before it.
In hen lysozyme, non-native tertiary structure involving Trp62, Trp108, and disulfide bond 6–127 is formed in the folding intermediate that occurs along the slow folding path (Denton et al. 1994; Rothwarf and Sheraga 1996). This conformation is characterized by a substantial quenching of tryptophan fluorescence, resulting in a total fluorescence intensity even lower than that observed in the native state. Replacement of Trp62 or Trp108 by Tyr or cleavage of the 6–127 disulfide bridge eliminates the intermediate non-native structure and consequently the overshoot in the fluorescence intensity (Denton et al. 1994; Rothwarf and Sheraga 1996). In accordance with the latter observations, natural pigeon lysozyme, which possesses Tyr at position 62, also shows no overshoot (Haezebrouck et al. 1998). Conversely, canine lysozyme, in fact, possesses both Trp residues corresponding to Trp62 and Trp108 in hen lysozyme, giving rise to the overshoot in fluorescence intensity as shown in our kinetic traces.
An important problem to solve is to determine the stage of the folding process in which these non-native interactions take place and to decide if they are rate determining. It is generally accepted that the various proteins from the lysozyme/-lactalbumin family possess two structural domains that act also as distinct folding domains (Radford et al. 1992). The -domain, which contains the four helices, folds faster than the ß-domain, in which the tertiary contacts remain more flexible and nonspecific until the final matching of the two domains into the native conformation. Our experiments on the refolding kinetics from the unfolded to the equilibrium intermediate state show that the decrease of both the fluorescence intensity and the av is completed in less than 2 ms. No further changes of these observables are detected in the subsequent seconds. This implies that a collapsed state with Trp residues sequestered from solvent is formed. The observed value for av (350 nm), however, is clearly higher than that obtained in the native state (348 nm). In the equilibrium intermediate state, the helices and sheets are formed but they are associated with flexible side-chain interactions and they lack persistent native structure. The evolution from this equilibrium intermediate state to the folded native state with fixed side-chain interactions is typified by the presence of a non-native conformation characterized by buried Trp residues that show low fluorescence intensity. The present article shows that this kinetic trap is present only in the last stage of folding.
This can be understood in the framework of the hierarchical protein-folding model as described by Arai and Kuwajima (2000). They make use of two contiguous folding funnels with different shape. The first funnel corresponds to the first stage of folding in which the protein partially folds to a compact but structurally diverse state. This protein compaction brings about a large decrease in conformational entropy suggesting that this funnel is much more widened at its top than funnel two. The first stage of folding from N to Ie in canine lysozyme implies a protein compaction accompanied by a large decrease in conformational entropy and corresponding with a wide and smooth surface of the energy landscape. The specific tertiary packing interactions occurring in stage two bring a number of energetic barriers along funnel two, leading to a more rugged funnel surface. The search for specific tertiary interactions during the Ie to N transition, therefore, takes place on a narrow funnel with rugged surface. To rise from the trough associated with the kinetic trap, a free energy barrier has to be overcome. The location of this energy barrier suggests that the rate-determining step involves the final association of the two domains into their final native conformation.
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We preferred to use authentic canine milk lysozyme rather than the recombinant protein expressed in Escherichia coli as the latter possesses an extra methionine residue at the N terminus (Koshiba et al. 1999). It is very likely that the observed decrease in thermal stability of the recombinant protein compared to the authentic protein arises from this extra methionine residue. Recently, it was demonstrated by different techniques that in bovine, goat, and human -lactalbumins, although the overall structures of the authentic and recombinant proteins are the same, the presence of the extra methionine residue remarkably destabilized the native state. This destabilization was entirely ascribed to an increase of the conformational entropy in the unfolded state (Chaudhuri et al. 1999, 2000).
Equilibrium fluorescence spectra were recorded with an SLM Aminco series 2 fluorometer with automatic correction for lamp and detector characteristics. The excitation wavelength for the tryptophan residues was set at 280 nm and the fluorescence intensity of the emitted light was integrated between 290 and 400 nm. Equilibrium circular dichroism measurements were carried out on a Jasco J-600A spectropolarimeter using cuvettes of 1 cm pathlength in the near-UV and 1 mm in the far-UV region. The temperature was kept constant with circulating thermostated water through the cell holder and was measured with a calibrated thermocouple inside the cell. Protein concentration was about 0.35 mg/mL. The data were expressed as residual ellipticity [
], using 112.05 as the mean residue weight for canine lysozyme.
Stopped-flow fluorescence measurements were performed on a SX.18 MV stopped-flow spectrometer from Applied Photophysics. After excitation at 280 nm, we measured the fluorescence intensity either by recording the total fluorescence emission above 320 nm by using a cut-off filter, or by monitoring the fluorescence intensity at distinct wavelengths between 300 and 420 nm. Kinetics were measured 10 to 20 times each, averaged and fitted by exponential functions using the manufacturer’s software. Further experimental details on the stopped-flow experiments and the use of the MeU-triNAG probe can be found in our previous publications (Haezebrouck et al. 1999; Noyelle et al. 2001).
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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), 228 nm (
), 287 nm (
) and with Trp-fluorescence (
). The latter feature is depicted by the wavelength at which the maximum intensity is observed (
