Conformation and stability of barley chymotrypsin inhibitor-2 (CI-2) mutants containing multiple lysine substitutions

Keith R. Roesler and A. Gururaj Rao1

Pioneer Hi-Bred International, Inc., 7300 NW 62nd Avenue, PO Box 1004, Johnston, IA 50131-1004, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A major goal of agricultural biotechnology is to increase the nutritional value of maize seed through the expression of heterologous proteins enriched in lysine. One promising candidate is barley chymotrypsin inhibitor-2 (CI-2), a plant protein that has been extensively characterized with respect to structure and function. Based on the tertiary structure of wild-type (WT) CI-2, five mutants with lysine contents ranging from 20 to 25 mol percent were designed, expressed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Inasmuch as previous transgenic experiments suggested that proper folding and stability may be essential for in vivo accumulation of the engineered proteins in plant cells, we first undertook an in vitro study of the conformation and thermodynamic stability of the CI-2 mutants in order to select an ideal candidate for plant expression. Mutant and WT CI-2 proteins had similar circular dichroism spectra, suggesting similar secondary structures. However, differences in the accessibility of the sole tryptophan residue, Trp24, indicated that the local conformation differed among the mutants. The thermodynamic stability of the mutants ranged from <2 to 4.9 kcal/mol compared with ~7 kcal/mol for the wild-type protein. In conjunction with proteolytic stability studies, we have identified one mutant that has the potential to be expressed in a stable manner in plant cells.

Keywords: barley chymotrypsin inhibitor-2/nutritional enhancement/protein conformation/protein engineering/protein stability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Maize and other cereal crops are deficient in the essential amino acid lysine. This deficiency has serious nutritional consequences, because these crops comprise a significant part of human and animal diets worldwide. In the US, where maize is a major component in livestock feed, lysine is routinely added as a feed supplement for swine and poultry. Over the years, conventional plant breeding approaches have had only limited success in producing varieties of maize enriched in lysine and with acceptable agronomic properties.

As an alternative to conventional breeding and with the availability of improved plant transformation protocols, several biotechnological approaches for increasing lysine content of plant seeds have been reported. These include manipulation of the lysine biosynthetic pathway to increase the levels of free lysine (Falco et al., 1995Go), de novo design of synthetic high lysine proteins (Keeler et al., 1998Go) and rational engineering of naturally occurring plant proteins to enrich for lysine (Rao et al., 1994Go).

As an extension of this latter concept, in the present study we have explored the potential for engineering barley chymotrypsin inhibitor-2 (CI-2) to increase the lysine content. Native CI-2 is an 83-amino acid monomer that contains no disulfide bonds and shares homology with a large number of sequences within the potato inhibitor 1 family of proteins (Svendsen et al., 1982Go). The three-dimensional structure of the truncated form of the protein containing 64 residues has been determined by X-ray crystallography (McPhalen and James, 1987Go) and NMR (Clore et al., 1987Go; Ludvigsen et al., 1991Go). CI-2 contains a four-stranded ß-sheet that is flanked by an {alpha}-helix on one side and a rigid reactive-site loop stabilized by hydrogen bonding and a salt bridge network, on the opposite side. The additional N-terminal 19 residues of the full-length, mature protein are unstructured and apparently do not contribute to either stability or inhibitory activity (Kjaer et al., 1987Go). CI-2 has been the subject of many studies concerning protein folding and protein structure–function relationships (Jandu et al., 1990Go; Jackson et al., 1993Go; Jackson and Fersht, 1994Go; Otzen and Fersht, 1995Go; Otzen et al., 1995Go; Ladurner and Fersht, 1997Go). This extensive structural characterization made CI-2 seem especially attractive as a candidate for protein engineering for nutritional enhancement. To this end, we have introduced several lysine residues into the CI-2 molecule and have examined the effect of these multiple substitutions on the function, conformation and stability of the mutants in comparison with the wild type. These mutants have been designated as barley high lysine (BHL) proteins.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Vector construction

Gene constructs for all sequences shown in Figure 1Go were made using the expression vector pET28 from Novagen (Madison, WI). The BHL1 gene was prepared by annealing and ligating two pairs of complementary oligonucleotides that together encoded the BHL1 sequence. Unique 5' RcaI and 3' HindIII restriction sites were included to allow ligation into the NcoI and HindIII sites of the expression vector. The gene encoding BHL2 sequence was prepared by overlap PCR, using the BHL1 gene as template. Primers were chosen to replace three amino acids in the BHL1 active site loop region, and to create unique AgeI and HindIII sites flanking the active site loop, to facilitate loop replacement. Unique 5' RcaI and 3' XhoI sites were created in the BHL2 gene to allow ligation into the NcoI and XhoI sites of the expression vector. The BHL3 and BHL4 constructs were prepared by replacing the AgeI to HindIII loop region with the appropriate annealed complementary oligonucleotide pairs. The BHL3N gene was prepared from BHL3 by PCR with a forward primer that included the N-terminal additional 18 amino acids. The unique RcaI and XhoI sites were retained for compatibility with the NcoI and XhoI sites of pET28. The WT CI-2 construct was prepared by PCR, using as template a CI-2 clone kindly provided by Dr Peter Shewry (ICAR, Long Ashton, UK). The WT CI-2 sequence was reported previously (Williamson et al., 1987Go). Insert regions of all constructs were verified by DNA sequencing.



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Fig. 1. Amino acid sequences of WT CI-2 and mutants. Underlined residues were confirmed by N-terminal protein sequencing.

 
Protein expression and purification

The Novagen vector pET28 was used to express the WT and engineered proteins in E.coli. Typically, the strain BL21(DE3)pLysS was grown at 37°C with the appropriate antibiotics in 2x YT medium to an OD600 of 0.8–1.0. Cultures were then induced with 1 mM IPTG, and allowed to grow for an additional 3 h before harvest. Mutants BHL2, BHL3, BHL3N and BHL4 were purified by SP-Sepharose (Amersham-Pharmacia, Piscataway, NJ) cation exchange chromatography in 50 mM sodium phosphate, pH 7.0. Following step elution with NaCl in the same buffer, the proteins were concentrated by ultrafiltration and subjected to Superdex-75 (Amersham-Pharmacia, Piscataway, NJ) gel permeation chromatography twice in 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. The purified proteins were quick frozen in liquid nitrogen, without glycerol, for long-term storage. BHL1 was purified by cation exchange chromatography with an SP-Sepharose FF 16/10 FPLC column, eluting with a NaCl gradient in 50 mM sodium phosphate, pH 7.0. The WT protein was precipitated in 70% ammonium sulfate and the resuspended pellet was dialyzed against 50 mM Tris–HCl, pH 8.6. The protein was then loaded on to a Hi-Trap Q column (Amersham-Pharmacia, Piscataway, NJ) and the unbound protein was collected and precipitated in 70% ammonium sulfate. The resuspended pellet was further purified by Superdex-75 chromatography.

Protein concentration

Molar extinction coefficients of 7040 and 7368 M–1cm–1 at 280 nm were determined for WT CI-2 and BHL4, respectively, by relating absorbance to protein concentration as determined by amino acid analysis of the purified proteins. The WT value was used for quantitation of all of the proteins except BHL4.

SDS–PAGE

SDS–PAGE was performed using Bio-Rad (Hercules, CA) precast Tris-Tricine 16.5% gels as per manufacturer's instructions. Proteins were visualized by staining with Coomassie brilliant blue.

N-Terminal sequencing and MALDI mass spectrometry

N-Terminal sequencing and MALDI mass spectrometry analyses were performed by the Iowa State University Protein Facility (Ames, IA).

Protease inhibition assays

Apparent Ki values were determined for the WT and engineered proteins using the equation Vo/Vi = 1 + [I]/Ki(app), where Vo is the reaction rate in the absence of inhibitor, and Vi is the reaction rate in the presence of inhibitor (Nicklin and Barrett, 1984Go). Reactions without inhibitor were started by addition of substrate, and the linear increase in absorbance at 405 nm was monitored over time and the reaction rate calculated from the slope. A known quantity of inhibitor was then added to the same reaction, and the new reaction rate was determined. The following proteases were used: bovine pancreatic chymotrypsin, bovine pancreatic trypsin, porcine pancreatic elastase and subtilisin Carlsberg from Bacillus licheniformis (all from Sigma). Assays were done at 37°C for chymotrypsin, and at 25°C for the other proteases. Reaction volumes were typically 200 µl. The following substrates were used at a concentration of 1 mM: N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma) for chymotrypsin and subtilisin, N-benzoyl-2-Ile-Glu-Gly-Arg-p-nitroanilide (Chromogenix S-2222) for trypsin and N-succinyl-Ala-Ala-Ala-p-nitroanilide (Sigma) for elastase. Chymotrypsin, elastase and subtilisin assays were done in 200 mM Tris–HCl, pH 8.0, with 1 µM bovine serum albumin included. Trypsin assays were done in 50 mM Tris–HCl, 2 mM NaCl, 2 mM CaCl2, 0.005% TritonX-100, pH 7.5.

Protease digests

Trypsin and chymotrypsin digests were done for 30 min at 37°C. Three micrograms of WT or engineered CI-2 were incubated with 0.3 µg protease in 100 mM Tris–HCl, 50 mM NaCl, 1 mM CaCl2, pH 8.0, in a volume of 15 µl. Control samples with protease only were incubated in the same buffer. Reactions were stopped by adding an equal volume of Bio-Rad 2x Tris-Tricine SDS sample buffer containing 6 mM PMSF, followed by boiling for 5 min and SDS–PAGE performed as described earlier.

Circular dichroism

CD analyses were performed by the University of Michigan, Protein and Carbohydrate Structure Facility (Ann Arbor, MI). The CD spectra of the proteins were recorded in the 195–250 nm range at 25°C with a JASCO J-710 spectrometer using 10 mm cells. The protein concentration was 22 µM in 10 mM sodium phosphate, pH 7.0, and scans were done at 50 nm/min for each protein. The contribution of the buffer to the spectra was electronically subtracted. Mean residue ellipticity was calculated using as the mean residue weight, the value derived from the amino acid sequence and molecular weight of each protein.

Fluorescence spectra

Emission spectra (300–400 nm) were recorded in a Perkin-Elmer Model LS-50 luminescence spectrometer after excitation at 280 nm in a thermostated 1 cm cuvette at 25°C. Excitation and emission slit-widths were set at 10 and 15 nm, respectively. Protein samples in 10 mM sodium phosphate, pH 7.0, at a concentration of 20 µM were used in these measurements.

Quenching of protein fluorescence

The quenching of intrinsic fluorescence of the proteins was followed by sequential addition of small aliquots of a 1 M acrylamide solution. The excitation wavelength was set at 295 nm to ensure optimal absorption by the tryptophan residue. In the absence of denaturant, an emission wavelength of 337 nm and a protein concentration of 20 µM were used. In the presence of 6 M GuHCl, the emission wavelength was 356 nm and the protein concentration was lowered to 2 µM because of the increase in the quantum yield of fluorescence after denaturation. The fluorescence intensities were corrected for the self-absorption of incident light (McClure and Edelman, 1967Go) by using a molar extinction coefficient of 0.23 for acrylamide (Parker, 1968Go). The quenching data were plotted as a direct Stern–Volmer plot, F0/F versus the molar concentration of acrylamide, where F0 is the fluorescence intensity in the absence of quencher and F is the fluorescence intensity in the presence of quencher. The Stern–Volmer quenching constant Ksv was determined from the slope of this plot.

Conformational stability

The unfolding of CI-2 follows a reversible two-state transition and can be monitored by fluorescence spectroscopy (Jackson and Fersht, 1991Go). Protein at a concentration of 2 µM was incubated for 18 h at 25°C in 10 mM sodium phosphate pH 7.0, with various concentrations of GuHCl (JT Baker, Phillipsburg, NJ). Subsequently, intrinsic fluorescence was measured in a thermostated cuvette at 25°C, using an excitation wavelength of 280 nm and an emission wavelength of 356 nm. Differences in the magnitude of the fluorescence increase accompanying denaturation were observed among these proteins. Approximately 25-fold increases were observed upon denaturation of WT CI-2 and BHL4. In contrast, less than 10-fold increases were observed for BHL3N and BHL2, respectively. Analysis of the data was done as described (Pace, 1986Go). For WT CI-2, BHL1 and BHL4, relative fluorescence intensity was plotted as a function of denaturant concentration and the linear pre- and post-transition regions extrapolated by linear regression to calculate the fluorescence values for the folded (Yf) and unfolded forms (Yu), respectively. BHL2, BHL3 and BHL3N were analyzed in the same manner except that no corrections were made for pre- and post-transition regions. Fraction unfolded (Fu) at each concentration of denaturant was calculated by the relationship Fu = YfY/YfYu, where Y is the measured value of fluorescence at a given denaturant concentration. The equilibrium constant, Ku, for unfolding and the corresponding Gibbs free energy, {Delta}Gu, were then calculated for each denaturant concentration in the transition region. The linear relationship of {Delta}G versus GuHCl concentration in the transition region (Figure 5Go) was used to calculate the conformational stability of the protein in the absence of GuHCl, {Delta}G(H2O), by assuming that this linear dependency continues to zero protein concentration according to the equation {Delta}Gu = {Delta}Gu (H2O) – m (denaturant) (Schellman, 1978Go; Pace, 1986Go).



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Fig. 5. Equilibrium unfolding of WT and engineered CI-2 by GuHCl. WT CI-2 ({circ}), BHL1 ({blacksquare}), BHL2 ({triangleup}), BHL3 ({square}), BHL3N ({blacktriangleup}), BHL4 (•).

 

    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutant design

The sequences of the WT CI-2 and the engineered derivatives used in this study are presented in Figure 1Go. The rationale for the mutations were derived from an examination of the three-dimensional structure of the protein, calculated solvent accessible surface areas of side chains (Table IGo) and homologous sequence data. We reasoned that surface residues and residue positions where a number of different side chains were accepted at a given position would be more likely to tolerate substitutions (Bowie et al., 1990Go). In the first derivative BHL1, a total of eight residues were substituted with lysine. Five of these occurred at Asn19, Gln41, Ile56, Met59 (reactive-site residue) and Leu73 and were made in accordance with the surface accessibility of these residues (percent solvent accessible surface area >70, Table IGo). Glu34, despite having less than 50% surface accessibility, was replaced with Lys owing to the diversity of side chains at this position in homologous sequences.


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Table I. Percentage exposed surface area (SA) of amino acid residues that were mutated in the three-dimensional structure of barley CI-2a
 
In addition to enriching for lysine, our goal was to ensure that the engineered proteins would not inhibit intestinal digestive proteases, an undesirable property in a protein intended for nutritional enhancement. For example, the soybean Kunitz trypsin inhibitor is a well characterized anti-nutritional protein and a potent allergen (Astwood et al., 1996Go). Previous studies on CI-2 and other members of the potato inhibitor 1 family established that hydrogen bonding and electrostatic interactions involving Thr58, Glu60, Arg62, Arg65 and Arg67 impart stability to the reactive-site loop and are essential for the inhibitory activity of the protein (Jackson and Fersht, 1994Go). Therefore, in an attempt to destabilize the loop, two of the key residues involved in non-covalent interactions, Arg62 and Arg67, were also replaced with Lys in BHL1. Derivatives BHL2 and BHL3 were designed to have completely non-rigid active site loops wherein the three additional residues involved in hydrogen bond interactions of the loop were mutated, i.e. Arg65->Lys, Thr58->Ala or Gly, and Glu60->Ala or His. BHL3N was designed to test whether a Lys-enriched N-terminal extension to BHL3 would be tolerated. The sequence of this extension was identical to the first 18 residues of the full-length native CI-2 except that it contained four Lys substitutions and a starting Met residue (Figure 1Go). The BHL4 sequence was identical to that of BHL1, except that the reactive site residue at position 59 was replaced with Gly, an amino acid with no side chain, to assess effects on inhibitory activity. The Lys content of the engineered proteins ranged from 20 mol% for BHL4 to 25 mol% for BHL3N, well above the 9 mol% lysine content of WT CI-2.

Confirmation of sequence of recombinant proteins

All five of the high lysine proteins had lower mobilities than WT CI-2 during SDS–PAGE, despite four of the five having predicted molecular masses similar to that of WT CI-2. To determine whether the mobility differences reflected post-translation modifications, the purified recombinant proteins were subjected to N-terminal sequencing and MALDI mass spectrometry. The determined N-terminal sequences were identical to the predicted sequences (Figure 1Go) and revealed that the start Met was retained in all of these proteins. The molecular masses of the purified proteins were determined to be 7.54, 7.50, 7.39, 7.44, 9.33 and 7.45 kDa for WT CI-2, BHL1, BHL2, BHL3, BHL3N and BHL4, respectively. These values are similar to the predicted values of 7.53, 7.52, 7.40, 7.45, 9.33 and 7.44 kDa, calculated assuming that the start Met was present, with no formyl group retained.

Circular dichroism analysis

The secondary structural features of WT and engineered proteins were analyzed by far UV circular dichroism spectroscopy (Figure 2Go). The CD spectrum of WT showed a minimum at 206 nm and was comparable to that reported earlier for CI-2 (de Prat Gay and Fersht, 1994Go). Indeed, even though the wild-type protein consists of 20% {alpha}-helix and 45% ß-sheet, the spectrum is atypical of any particular secondary structure element. With the exception of BHL4 which had relatively reduced ellipticity values, the high lysine proteins had CD profiles with similar ellipticities and identical minima at ~206 nm suggesting that overall there were no gross conformational changes in the mutants. The BHL4 spectrum, although quantitatively different from the others, was still very similar and indicative of a highly structured protein. The negative band at 233 nm previously observed in WT CI-2 and attributed to Trp24 (de Prat Gay and Fersht, 1994Go) was also evident in these proteins.



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Fig. 2. Far UV CD spectra of WT CI-2 and mutants. WT CI-2 ({circ}), BHL1 ({blacksquare}), BHL2 ({triangleup}), BHL3 ({square}), BHL3N ({blacktriangleup}), BHL4 (•). Analyses were done in 10 mM sodium phosphate, pH 7.0, at a protein concentration of 22 µM.

 
Intrinsic fluorescence

It is well established that Trp fluorescence is a function of the three-dimensional structure of a protein and is sensitive to subtle interactions with neighboring residues (Lakowicz, 1983Go). Therefore, in addition to CD that measures gross conformation, fluorescence spectroscopy was used to ascertain more subtle structural differences among mutant proteins. A single Trp residue is buried in the hydrophobic core of CI-2, between the {alpha}-helix and ß-sheet (McPhalen and James, 1987Go). The intrinsic fluorescence of the WT protein arising from this Trp showed a {lambda}max of emission at 330 nm (Figure 3Go). However, the mutant proteins differed in their emission peaks as well as intensities suggesting clear differences in the microenvironment of the Trp residue. BHL1 showed a {lambda}max of emission that was indistinguishable from the wild type but the quantum yield of fluorescence was approximately twofold higher. In BHL2, the threefold increase in fluorescence intensity was accompanied by a red-shift in the emission maximum to 348 nm. BHL3 on the other hand showed a spectrum with an emission maximum at ~334 nm that was comparable in intensity with the spectrum of BHL1. However, a shoulder around 310 nm was indicative of a contribution from the sole tyrosine residue in the protein. BHL3N showed the highest fluorescence intensity with a peak at 340 nm. Interestingly, BHL4 showed a spectrum that was most similar to the wild-type protein in intensity albeit with a blue-shifted {lambda}max of emission at 322 nm.



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Fig. 3. Fluorescence emission spectra of WT CI-2 and mutants in 10 mM sodium phosphate, pH 7.0 (excitation at 280 nm). (a) WT CI-2; (b) BHL1; (c) BHL2; (d) BHL3; (e) BHL3N and (f) BHL4.

 
Fluorescence quenching

One approach to discriminating Trp environments in proteins is by probing the exposure of the indole ring using fluorescence quenchers such as acrylamide (Eftink and Ghiron, 1975Go, 1976Go). Thus, we measured the relative accessibility of the sole Trp residue in WT and mutant proteins by monitoring the quenching of fluorescence in the presence of acrylamide (Figure 4Go and Table IIGo). The WT protein had a Stern–Volmer quenching constant, Ksv, of ~ 1.7 M–1, reflective of the relative inaccessibility of the sole Trp residue. In BHL1, containing multiple lysine substitutions, a Ksv of 3.5 ± 0.3 M–1 indicated an increased exposure of the Trp residue. However, derivatives BHL2 and BHL3N appeared to have the most `open' Trp residue with a Ksv of 5.5 ± 0.4 M–1. In contrast, BHL3, which is identical to BHL3N but does not have the N-terminal extension of 18 residues, has a Ksv of 2.4 ± 0.2 M–1, a value that is closer to the WT protein. Unexpectedly, in BHL4, which is different from BHL1 only in that Lys59 is replaced with Gly, the Trp residue is even less accessible than in WT or BHL1 and has a Ksv of 0.65 M–1. Upon unfolding in 6 M GuHCl, the tryptophan residue was completely and equally accessible in all of the proteins, as expected (average Ksv of 17 M–1).



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Fig. 4. Acrylamide quenching of tryptophan fluorescence plotted as a direct Stern–Volmer plot. WT CI-2 ({circ}), BHL1 ({blacksquare}), BHL2 ({triangleup}), BHL3 ({square}), BHL3N ({blacktriangleup}), BHL4 (•).

 

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Table II. Stern–Volmer constants determined by acrylamide quenching of tryptophan fluorescence (mean ± standard deviation)
 
Thermodynamic stability

Previous experiments demonstrated that CI-2 is reversibly unfolded by GuHCl, and that this protein follows a simple two-state folding and unfolding model (Jackson and Fersht, 1991Go). We used similar equilibrium denaturation experiments to assess the thermodynamic stabilities of the WT and engineered proteins (Figure 5Go and Table IIIGo). The WT protein had a {Delta}Gu(H2O) of about 7 kcal/mol, with an unfolding midpoint of about 4 M GuHCl and an m value of 1.77 kcal/mol. These values are almost identical to those previously reported for WT CI-2 (Jackson and Fersht, 1991Go; Jackson et al., 1993Go; Otzen et al., 1995Go). In contrast, BHL2, BHL3 and BHL3N were much less stable, with {Delta}Gu(H2O) = ~1.5 kcal/mol and unfolding midpoints of less than 1 M GuHCl for all three mutants. However, BHL1 and BHL4 had stabilities intermediate to WT CI-2 and the other proteins with {Delta}Gu(H2O) = ~4.5 to 4.9 kcal/mol. It is also clear from the summarized data in Table IIIGo, that, within experimental error, the value of m which measures the free energy of unfolding as a function of denaturant concentration, is similar for wild-type and mutant proteins.


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Table III. Equilibrium unfolding parameters (mean ± standard deviation)
 
Proteolytic stability

Tryptic and chymotryptic digests of WT and engineered proteins were performed at an enzyme–substrate ratio of 1:10 for 30 min and analyzed by SDS–PAGE (Figure 6Go). Under these conditions, WT CI-2 and BHL1 were resistant to trypsin, and BHL4 was somewhat resistant (Figure 6Go, top panel, lanes 3–6, 15 and 16). The other proteins were completely digested by trypsin into fragments too small to be detected by SDS–PAGE (Figure 6Go, top panel, lanes 7, 11 and 13). With respect to chymotrypsin, WT CI-2 was completely resistant, as is to be expected for a chymotrypsin inhibitor (Figure 6Go, center panel, lane 3). BHL1 (Figure 6BGo, lane 5) and BHL4 (Figure 6Go, center panel, lane 15) were partially resistant to chymotrypsin whereas derivatives BHL2, BHL3 and BHL3N were completely digested into detectable fragments (Figure 6Go, center panel, lanes 7, 11 and 13).



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Fig. 6. SDS–PAGE analysis of WT and engineered CI-2 treated with trypsin, chymotrypsin. Lanes 1 and 9, molecular mass markers; lanes 2 and 10, protease only (no WT or engineered CI-2); lanes 3 and 4, WT CI-2; lanes 5 and 6, BHL1; lanes 7 and 8, BHL2; lanes 11 and 12, BHL3; lanes 13 and 14, BHL3N; lanes 15 and 16, BHL4.

 
Protease inhibition assays

Inhibition constants for the WT and engineered proteins were determined, using three mammalian digestive proteases and one bacterial protease with broad substrate specificity (Table IVGo). WT CI-2 was an effective inhibitor of chymotrypsin, subtilisin and elastase, but not of trypsin, with Ki values in the low nanomolar range similar to those determined previously (Longstaff et al., 1990Go). BHL1, containing a lysine residue in place of Met59, now inhibited trypsin (Ki = 65 nM) and also retained some inhibitory activity against subtilisin (Ki = 281 nM). The three mutants with a non-rigid reactive site loop, BHL2, BHL3 and BHL3N, did not appear to be effective inhibitors of any of these proteases, although Ki values in the micromolar range could be obtained for BHL2 against trypsin and subtilisin. BHL4 still possessed detectable, but considerably reduced, inhibitory activity against chymotrypsin and subtilisin, with Ki values of 4180 and 707 nM, respectively.


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Table IV. Inhibition of proteases by wild-type and engineered CI-2 Ki[(nM)]
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Proteins engineered for high lysine content must accumulate to high levels in transgenic plants to be effective in nutritional enhancement. For example, to double the lysine content of maize seed, an engineered protein containing 25 weight % lysine must comprise about 10% of the total seed protein (assuming maize seed normally contains 10% protein and 0.25% lysine on a dry weight basis). Previous studies have suggested that proper folding of proteins may be essential to achieve the desired expression levels. For instance, when malfolded or unstructured proteins enriched in essential amino acids were expressed in plants, they did not accumulate to useful levels (Yang et al., 1989Go; Pueyo et al., 1995Go). Furthermore, there appears to be a direct correlation between thermodynamic stability of proteins and their expression levels, not only during intracellular expression in E.coli (Parsell and Sauer, 1989Go; Inoue and Rechsteiner, 1994aGo,bGo) but also when expressed in the secretory pathway in both E.coli and yeast (Kwon et al., 1996Go; Kowalski et al., 1998aGo,bGo). This relationship may hold true for plant cells as well. We suggest that an in vitro measurement of the thermodynamic and conformational stability of an engineered protein is a useful means of selecting a candidate for transgenic expression in plants and perhaps of predicting its potential for success. In order to make this assessment, therefore, we designed a series of high lysine mutants of CI-2 and measured their physico-chemical properties.

The CD analyses (Figure 2Go) demonstrated that all of the engineered proteins had secondary structures very similar to that of WT CI-2. The Lys substitutions for Glu34 and Gln41, both in the {alpha}-helix, apparently did not disrupt helix formation. Likewise, ß-sheet formation did not appear to be perturbed by substitutions for two ß-strand residues, Arg65 and Arg67, nor for active site loop residues involved in hydrogen bonds with them, Thr58 and Glu60.

The fluorescence quenching experiments (Figure 4Go) did reveal structural differences among the proteins that were not apparent from the CD analyses, but these differences may represent relatively small changes in the microenvironment surrounding Trp24. It was surprising that a Gly for Lys substitution at position 59 resulted in a quenching difference in BHL4, relative to BHL1 (Table IIGo and Figure 4Go). The residue at position 59 is in the center of the reactive site loop, distal from the hydrophobic core where the Trp is located and, a priori, would not be expected to influence the accessibility of the Trp residue. It is possible that the greater conformational flexibility of Gly, with many permissible torsion angles, allowed some minor structural rearrangements to occur in the vicinity of Trp24. Similarly, the Gly58 of BHL3 may be responsible for the smaller Stern–Volmer constant of this protein relative to BHL2 (Table IIGo). Future NMR studies may provide definitive explanations for the differences in fluorescence quenching among these proteins.

Significant insights into the structures of globular proteins may also be obtained by using proteinases such as trypsin and chymotrypsin as probes of protein conformation (Fontana et al., 1989Go; Price and Johnson, 1990Go). In general, proteolysis of a polypeptide chain requires the binding of 6–8 amino acid residues into the active site cleft of the acting protease (Berger and Shechter, 1970Go). In compactly folded proteins these determinants are usually not accessible and as such are poor substrates for proteolysis. Thus, WT CI-2 is resistant to trypsin even though the molecule contains six Lys-X and four Arg-X bonds (Figure 6Go). This may not be entirely surprising considering that the majority of these residues are relatively buried within CI-2, with the exception of Lys37, Lys72 and Arg81 which have >50% solvent accessibility. In BHL1, the introduction of six new Lys residues does not alter the stability of the protein towards trypsin but one cannot unequivocally attribute this to compactness of folding since the mutant protein is an inhibitor of the protease. However, BHL4 which differs from BHL1 in having a Gly at position 59 instead of Lys, is no longer an inhibitor of trypsin and yet is relatively resistant to the enzyme. Aggregation of BHL4 was ruled out as an explanation for this resistance to trypsin (and for the inaccessibility of Trp24 in fluorescence quenching) based on the similar elution profile to WT CI-2 during gel permeation chromatography (data not shown). In contrast, the destabilization of the loop structure in BHL's 2, 3 and 3N must clearly affect the accessibility of the susceptible bonds since these derivatives are completely digested within 30 min. Indeed, these three derivatives are also susceptible to chymotrypsin whereas BHL's 1 and 4 are more resistant. Thus, these results indicate that even though the gross conformations of the proteins appear to be the same as measured by CD, there may be greater flexibility associated with BHL's 2, 3 and 3N and consequent increased exposure of proteolytic nick sites.

In general, mutations at surface residues have little or no effect on protein stability whereas mutations of buried residues can often cause drastic destabilization (Reidhaar-Olson and Sauer, 1988Go). Thus, the substitution at Arg67, which is buried in the loop structure and has 15% solvent accessibility, probably accounted for much of the decreased stabilities of BHL1 and BHL4. A single Arg to Ala mutation at position 67 of WT CI-2 resulted in a decrease in stability of about 1.5 kcal/mol (Jandu et al., 1990Go). That is over half of the reduction we observed here for BHL1 and BHL4 (Table IIIGo). The additional mutations of Thr58, Glu61 and Arg65 caused large additional reductions in stabilities of BHL2, BHL3 and BHL3N (Table IIIGo). An important contribution of Arg65 to stability can be inferred, because a previous double mutant with Ala substitutions for Thr58 and Glu60 resulted in only a 0.67 kcal/mol decrease in stability (Jackson and Fersht, 1994Go). The importance of Arg65 was also evident from its complete conservation in the sequences of over 25 CI-2 homologs from diverse organisms (data not shown).

An inadvertent albeit important consequence of the multiple lysine substitutions was the overall increase in positive charge and a concomitant increase in the pI of the mutant proteins, from a pI of 6.85 for WT CI-2 to >10.5 for the mutants. However, our results indicate that the folding of the engineered proteins was not compromised by the increased charge repulsions, consistent with the notion that electrostatic interactions do not have a dominant role in protein folding (Dill, 1990Go). It is worth noting that engineering of {alpha}-hordothionin for high lysine content also resulted in proper folding (Rao et al., 1994Go). However, the WT hordothionin is intrinsically very basic and, unlike CI-2, has four disulfides that contribute significantly to the stability of the folded protein. In contrast, lysozyme was engineered from a very basic protein to a more acidic one, with minimal effects on structure and stability (Dao-pin et al., 1991Go).

In conclusion, we have demonstrated that through an iterative cycle of mutagenesis and characterization of mutants, it is possible to design a high lysine protein that meets the specifications introduced earlier in this section. Thus, the mutant BHL4 has 20 mol% Lys, appears to have a native-like conformation and has thermodynamic stability comparable with many naturally occurring globular proteins (Pace, 1990Go) albeit less than that of WT CI-2. Furthermore, the protein retains little protease inhibitory activity, yet is relatively resistant to proteases and therefore has the potential to accumulate to high levels in plant cells.


    Acknowledgments
 
We thank Dr Peter Shewry (ICAR, Long Ashton, UK) for kindly providing the WT CI-2 clone, Laura Tagliani for constructing the vectors for WT CI-2 and BHL1 and Dr Bipin Dalmia for expression of the two proteins in E.coli. We also thank Dr Heidi Sleister for help with the overlap PCR strategy and Drs Rudolf Jung and Larry Beach of this department for critiquing the manuscript.


    Notes
 
1 To whom correspondence should be addressed; email: raog{at}phibred.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received January 5, 1999; revised April 28, 1999; accepted August 10, 1999.





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