1Department of Chemistry and Biotechnology, Agricultural University of Norway, PO Box 5040, 1432 Ås, Norway and 2Center for Molecular and Biomolecular Informatics, University of Nijmegen, PO Box 9010, 6500 GL Nijmegen, The Netherlands
3 To whom correspondence should be addressed. e-mail: vincent.eijsink{at}ikb.nlh.no
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Abstract |
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Keywords: chitinase/proline/thermal stability/unfolding
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Introduction |
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Studies of the thermodynamic stability of small proteins and their mutated variants have provided some fundamental insights into factors that determine protein stability (Fersht and Serrano, 1993; Matthews, 1995
; Vieille and Zeikus, 2001
). From these studies, several important strategies for protein stabilization have arisen. Successful stabilization strategies include the introduction of disulphide bridges (Matsumura et al., 1989
; Mansfeld et al., 1997
) and the optimization of helices and helix caps (Serrano et al., 1992
; Blaber et al., 1993
). Another strategy, termed entropic stabilization (Matthews et al., 1987
), involves the introduction of mutations that reduce the conformational freedom of the unfolded state. This may be achieved by the introduction of disulphide bridges, but also by simpler types of mutations that reduce the conformational freedom of the residue in question. These simpler mutations concern removal of glycine residues (thus restricting the
and
dihedral angles) and introduction of proline residues (thus strongly restricting the
angle) (Matthews et al., 1987
; Hardy et al., 1993
; Van den Burg et al., 1998
; Bryan, 2000
; Watanabe et al., 2000
).
ChiB unfolds irreversibly at higher temperatures, meaning that only the kinetic (and not thermodynamic) stability of this enzyme may be addressed. It has been suggested that kinetic stability is determined by the rate of a partial (as opposed to global) unfolding event that triggers an irreversible process, e.g. aggregation or, in the case of proteases, autolysis (Vriend and Eijsink, 1993; Vriend et al., 1998
; Zhao and Arnold, 1999
; Bryan, 2000
; Machius et al., 2003
). Stabilizing mutations are most likely to be effective if they affect these stability-determining parts (weak spots) of the protein (Mansfeld et al., 1997
; Vriend et al., 1998
; Machius et al., 2003
). Thus, it is useful to start a mutagenesis strategy aimed at improvement of kinetic stability by a search for crucial unfolding regions in a protein (Eijsink et al., 1995
; Machius et al., 2003
). It has been suggested that limited proteolysis may be used for the identification of such regions, since this technique may reveal areas that are particularly flexible and/or prone to local unfolding (Fontana, 1988
; Arnold et al., 1996
, 1999; Fontana et al., 1997
).
Here we describe attempts to stabilize ChiB by introducing prolines and removing glycines. Since limited proteolysis experiments did not reveal any potential weak spot (see below), mutations were introduced all over the protein. Mutations were designed using semi-automated design methods implemented in the molecular modelling package of WHAT IF (Vriend, 1990). The results show that this strategy worked well in the sense that more stable variants of ChiB were obtained. On the other hand, many of the mutations had no or hardly any effect on ChiB stability. Possible explanations for these observations are discussed.
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Materials and methods |
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Mutations were designed with the WHAT IF automatic mutation prediction options. These options predict how well each of the 20 possible amino acids fit at specific positions in the protein and calculate a quality parameter for each fit. Special options exist for scanning proteins to find good positions for prolines, cysteines or alanines. These options are all based on the position-specific rotamer technology as originally described by Jones and Thirup (Jones and Thirup, 1986) and Filippis et al. (Filippis et al., 1994
). For each putative rotamer, the fitness in the local environment is calculated using the same scoring functions as used in the WHAT IF homology modelling procedure (Chinea et al., 1995
).
Note that the WHAT IF options used in this study do not explicitly predict thermostabilizing mutations, but merely mutations that are likely to be compatible with the structure. Other knowledge is needed to select those mutations that are likely to stabilize the protein among all predicted mutations. In this case, for reasons explained above, we focused on mutations of the GlyAla and Xxx
Pro type.
All automatically detected mutations were visually inspected and a further selection of mutations was made on the basis of the following criteria:
1. The residue is surface located. This criterion was based on the notion that irreversible thermal inactivation is likely to be governed by unfolding processes that primarily involve surface-located regions of the protein. Also, surface-located residues make fewer contacts, reducing the chance for mutation design errors and side effects (see next criterion).
2. The mutation does not lead to removal of favourable interactions or to introduction of unfavourable (steric hindrance) interactions.
3. The mutation does not affect the catalytic centre of the enzyme.
This manual process yielded 15 mutations, which were all made. Some details of the structural context of the mutated residues are provided in Table I. The pictures presented in this report were made with PyMOL (DeLano Scientific, San Carlos, CA, USA).
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Thermolysin (Roche Molecular Biochemicals, Basel, Switzerland) was dissolved to 10 mg/ml in 50 mM TrisHCl, pH 8.0, 10 mM CaCl2 and stored in aliquots at 20°C. For proteolysis, 20 µg of purified ChiB (see below) were incubated with various amounts of thermolysin in 50 mM TrisHCl, pH 8 at various temperatures (reaction volume 100 µl). Reactions were stopped by adding 50 mM EDTA to a final concentration of 5.5 mM, followed by addition of one volume of an SDSPAGE sample buffer containing 62.5 mM TrisHCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) ß-mercaptoethanol and 0.025% (w/v) bromophenol blue. After incubation of the samples at 95°C for 4 min, SDSPAGE was conducted essentially according to Laemmli (Laemmli, 1970), using a 12% seperating gel, in a Bio-Rad Mini Protean II system.
Mutagenesis
ChiB mutants were made with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) essentially as described by the manufacturer. DNA sequences of mutated gene fragments were sequenced with an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit and an ABI PRISM 377 DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). Mutated gene fragments were used to construct variants of the wild-type production plasmid pMAY2-10 (Brurberg et al., 1996). Wild-type and mutant chib genes were expressed in Escherichia coli DH5
(Life Technologies, Rockville, MD, USA) and ChiB variants were purified as described earlier (Brurberg et al., 1996
).
Catalytic activities of ChiB variants were determined using the substrate 4-methylumbelliferyl-ß-D-N,N'-diacetylchitobioside [4MU-NAG2 (Sigma-Aldrich, St. Louis, MO), an analogue of the natural substrate (NAG)3], as described previously (Brurberg et al., 1996). Protein concentrations were determined with the Protein Assay solution from Bio-Rad, based on staining with Coomassie blue.
Stability (t1/2)
Purified ChiB variants (20 nM) were incubated in 50 mM citrate-phosphate buffer, pH 6.2 (Stoll and Blanchard, 1990) and 0.1 mg/ml BSA at 57°C (BSA was added to prevent unspecific binding of proteins to the test tube). At regular intervals (depending on the enzyme tested), 25 µl of the enzyme solution was transferred to ice. After the last sample had been removed from the water bath, residual catalytic activity was tested. Five microlitres of the 20 nM enzyme reaction was transferred to a test tube containing 95 µl of a solution of 68 µM substrate (4MU-NAG2) and 0.1 mg/ml BSA in 50 mM citrate-phosphate buffer, pH 6.2. After 10 min, the reaction was stopped with 1.9 ml of 0.2 M Na2CO3 and the fluorescence measured with a Hoefer DyNA Quant 200 fluorimeter. Thermal inactivation followed first-order kinetics and plots of relative remaining activity versus t were fitted to a first-order exponential curve. The fit normally gave curves with a correlation higher than 0.99. Half-lives were calculated using the equation t1/2 = 0.6931/kapp, where kapp is the apparent rate constant of the reaction. The t1/2 values presented are average values derived from three independent measurements; the error margins reported correspond to one standard deviation.
Circular dichroism (CD) measurements
CD measurements were performed using a Jasco J-810 spectropolarimeter (Jasco) calibrated with ammonium d-camphor-10-sulfonate (Icatayama Chemicals, Tokyo, Japan). The experimental conditions were: protein concentration, 0.1 mg/ml; buffer, 20 mM sodium phosphate, pH 8.0; pathlength, 1 mm; wavelength, 222 nm; scan rate, 1°C/min. The apparent fraction of unfolded protein (Fapp) was calculated using the equation Fapp = (Y YN)/(YU YN), where Y is the observed signal and YU and YN are the formulae of the linear baselines of unfolded and native protein, respectively [see Pace (Pace, 1990) for details on the calculations]. Apparent Tm values were calculated from a plot of the first derivative of the two-state unfolding curve.
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Results |
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In an attempt to identify unstable regions (weak spots) of ChiB, we conducted a series of limited proteolysis experiments with thermolysin. The rate of proteolysis increased upon increasing the incubation temperature from 50 to 60°C (Figure 2). At 50°C, no proteolysis was detected, indicating that ChiB is stable. At 60°C, however, most of the enzyme was degraded during the 20 min incubation period. Thus, ChiB seems to unfold, and become more prone to proteolysis, in the 5060°C interval (see also the stability measurements described below).
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Because we had no clue as to which parts of ChiB could be involved in the partial unfolding processes that lead to thermal inactivation, mutations were deliberately scattered over the complete ChiB molecule. Thus, we optimized the chance of hitting a weak spot with the mutations.
Production and characterization of mutants
All mutants made in this study could be produced in normal amounts under standard conditions and purified using the standard procedure for ChiB. The half-lives and specific activities of the purified ChiB variants are presented in Table II.
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Highly solvent-exposed hydrophobic residues such as Trp252 are often observed in glycoside hydrolases because they contribute to the binding of longer sugar chains (van Aalten et al., 2000; Uchiyama et al., 2001
). Such highly exposed hydrophobic residues do not seem favourable for stability and, thus, we wondered whether the stabilizing effect of the W252P mutation could be a result of non-entropic effects (see below for further discussion). Therefore, as a control, we replaced Trp252 with a polar, charged residue, glutamate, which we assumed to be beneficial for stability. Indeed, the W252E mutant showed a considerable increase in stability, surpassing that of the W252P mutant (Table II).
Melting curves of ChiB and its G188A_A234P mutant (Figure 4) showed an irreversible transition that led to almost complete loss of the CD signal at 222 nm and to aggregation. The melting curves permitted the determination of apparent Tm values of 59.9 and 64.1°C, respectively.
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Discussion |
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In the design of mutants, we aimed at selecting mutants with relatively clean effects. That is, we discarded mutations which we expected to have major effects in terms of removal or introduction of interactions. We cannot exclude though that seemingly minor changes in beneficial or unfavourable interactions contribute to some of the observed mutational effects, for example, in the case of the A234P and G188A mutations.
The Cß of Ala234 is highly solvent exposed and has few contacts. Its closest contact is with the C of the solvent-accessible hydrophobic residue Leu438 (distance 3.84 Å). A model of the A234P mutant shows that the introduction of proline leaves the hydrophobic contact between residues 234 and 438 intact, and that a new contact (Pro234C
Lys362C
; 3.5 Å) is created. Since such hydrophobic contacts at the surface may be beneficial for stability (Van den Burg et al., 1994
; Machius et al., 2003
) they may contribute to the stabilizing effect of the A234P mutation. Likewise, the destabilizing effect of the A234G mutation may in part be a result of the loss of the contact between Ala234Cß and Leu438C
.
The Cß of Ala188 is expected to have a hydrophobic contact with the C2 of Leu216 (3.69 Å), which may contribute to the stability of the G188A mutant. Interestingly, and unexpectedly, the stabilizing G188A mutation also yielded an
2-fold increase in activity. Leu216 is next to Asp215, which is known to be important in catalysis (van Aalten et al., 2001
). One might thus speculate that small structural adjustments of and around Asp215 account for the beneficial effect of the G188A mutation on activity.
In general, one could expect that at least some of the rigidifying mutations would reduce catalytic efficiency, based on the general notion that enzyme flexibility is important for catalysis. In some cases, small reductions in activity were indeed observed (Table I), but the general picture is that the mutations had only minor effects on catalytic activity. This was expected since mutations located close to the catalytic centre were discarded during the mutant design process.
The two mutations present in the most stable ChiB variant (G188A, A234P) as well as all other mutations with larger effects on stability (A234G, W252P, W252E, E253P) are clustered on one side of the ChiB molecule. Apart from Gly188, all these mutated residues are located in an exposed surface meander running from residue 230 to 265 (Figure 5). Gly188 is located just in front of an -helix on which the surface meander packs (there are many contacts between residues 189190 and residues 239243; Ala189Cß also has a contact with Phe263C
). Thus, it is conceivable that mutation of residue 188 somehow affects the 230265 surface meander. The presence of a region in which mutations have relatively strong effects on stability can be explained by assuming that the unfolding process that leads to thermal inactivation involves only part of the protein. Only mutations involved in these stability-determining parts will have noticeable stability effects, as observed in protein engineering work on proteases and amylases (Vriend and Eijsink, 1993
; Vriend et al., 1998
; Zhao and Arnold, 1999
; Machius et al., 2003
). The present results suggest that the partial unfolding process that precedes irreversible thermal inactivation (aggregation) of ChiB involves the 230265 meander, which thus may be a weak spot. In this respect it is interesting to note the large stabilizing effect of the W252E mutation. One might speculate that the reduction of surface hydrophobicity in an area that supposedly unfolds relatively easily will lead to a decreased tendency to aggregate.
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In general, it cannot be excluded that the designed mutations have small negative side effects that outweigh the beneficial effects of the mutation and that have been overlooked in the design process. It seems unlikely though that this explains the fact that all mutations outside the 188252 region had such small effects on the stability of ChiB. We thus conclude that ChiB has a weak spot (Vriend and Eijsink, 1993) or nucleation site for unfolding [a term derived from helixcoil theory as explained by Machius et al. (Machius et al., 2003
)]. As shown previously for proteases (Eijsink et al., 1995
) and amylases (Machius et al., 2003
) and as shown in this study, the identification and mutation of such a nucleation site for unfolding is a prerequisite for successful attempts to increase the kinetic stability of a protein.
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Acknowledgements |
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Received June 18, 2003; revised September 3, 2003; accepted September 12, 2003.