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incorporated by this method is currently being evaluated. In addition, more expedient methods for preparing the suppressor tRNA and for expressing greater quantities of protein are being investigated.

Acknowledgments This workwas supportedby the U.S. Officeof NavalResearchGrantN00014-86-K-0522) and by the Materials and Chemicals Science Division of the U.S. Departmentof Energy, Officeof EnergyResearch, undercontractNo. DE-AC03-76SF-0098. J.E. acknowledgesthe National ScienceFoundationfor postdoctoralfellowshipfunding(CHE-8907488) and D.M. thanks the AmericanCancer Societyfor postdoctoralfellowshipfunding(PF-4014).

[16] S t a b i l i z a t i o n o f F u n c t i o n a l P r o t e i n s b y I n t r o d u c t i o n o f Multiple Disulfide Bonds By MASAZUMI MATSUMURA and BRIAN W. MATTHEWS

Introduction The instability of proteins often limits their utility in medical and commercial applications. Therefore, the development of rational methods to increase protein stability is of considerable interest. Is it possible to stabilize proteins by modifying just a few amino acid residues in the primary sequence? The answer to this question lies in the fact that the native conformations of proteins are only marginally stable. The net stability of a protein is determined by the free energy difference between the native and denatured states. The factors that contribute to the stability of the folded protein are very large but are offset by the almost equally large factors that favor the unfolded form. As a consequence, the net free energy of stabilization of native proteins is unexpectedly small (5-20 kcal/mol). 1-3 On the other hand, the contribution of individual interactions such as T. E. Creighton, "Proteins: Structure and Molecular Properties." F r e e m a n , N e w York, 1983. 2p. L. Privalov, Ado. Protein Chem. 33, 168 (1979). 3 j. A. Schellman, Annu. Reo. Biophys. Biophys. Chem. 16, 115 (1987).

METHODS IN ENZYMOLOGY, VOL. 202

Copyright © 1991by AcademicPress, Inc. All fights of reproduction in any form reserved.

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hydrogen bonds and salt bridges to protein stabilization is estimated to be 1-3 kcal/mol) Therefore the addition of a few extra interactions can significantly enhance the stability of proteins. It has been shown that stability can be improved by genetic screens 4-9 and rational modifications. 10-14 Disulfide bonds can make substantial contributions to the stability of proteins) 5,16 The effect is presumed to be due mainly to a decrease in the conformational chain entropy of the unfolded polypeptide. 17-z° This large stabilizing potential has made the engineering of disulfide bonds into proteins an attractive strategy for the improvement of protein stability. To date, disulfide bonds have been introduced in dihydrofolate reductase, 21,22 phage T4 lysozyme, 23-25 subtilisin, 26-28 and h repressor. 29 In all cases the 4 D. Shortle and B. Lin, Genetics 110, 539 (1985). 5 T. Alber and J. Wozniak, Proc. Natl. Acad. Sci. U.S.A. 82, 747 (1985). 6 M. Matsumura and S. Aiba, J. Biol. Chem. 2,60, 15298 (1985). 7 H. Liao, T. McKenzie, and R. Hageman, Proc. Natl. Acad. Sci. U.S.A. 83, 576 (1986). 8 p. N. Bryan, M. L. Rollence, M. W. Pantoliano, J. Wood, B. C. Finzel, G. L. Gilliland, A. J. Howard, and T. L. Poulos, Proteins: Struet. Funet. Genet. 1, 326 (1986). 9 B. C. Cunningham and J. A. Wells, Protein Eng. 1, 319 (1987). l0 C. Mitchinson and R. L. Baldwin, Proteins: Struct. Funct. Genet. 1, 23 (1986). 11 M. H. Hecht, J. M. Sturtevant, and R. T. Sauer, Proteins: Struct. Funct. Genet. 1, 43 (1986). ~z B, W. Matthews, H. Nicholson, and W. J. Becktel, Proc. Natl. Aead. Sci. U.S.A,. 84, 6663 (1987). 13 L. Serrano and A. R. Fersht, Nature (London) 342, 296 (1989). 14 H. Nicholson, W. J. Becktel, and B. W. Matthews, Nature (London) 336, 651 (1988). 15 T. E. Creighton, BioEssays 8, 57 (1988). 16 C. N. Pace, G. R. Grimsley, J. A. Thomson, and B. J. Barnett, J. Biol. Chem. 263, 11820 (1988). 17 j. A. Schellman, C. R. Trao. Lab. Carlsberg Ser. Chim. 29, 230 (1955), 18 p. j. Flory, J. Am. Chem. Soc. 78, 5222 (1956). 19 D. C. Poland and H. A. Scheraga, Biopolymers 3, 379 (1965). 2o H. S. Chan and K. A. Dill, J. Chem. Phys. 90, 492 (1988). 2~ J. E. Villafranca, E. E. Howell, D. H. Voet, M. S. Strobel, R. C. Ogden, J. N. Abelson, and J. Kraut, Science 222, 782 (1983). 22 j. E. Villafranca, E. E. Howell, S. J. Oatley, N-H. Xuong, and J. Kraut, Biochemistry 26, 2182 (1987). 23 L. J. Perry and R. Wetzel, Science 226, 555 (1984). 24 t . J. Perry and R. Wetzel, Biochemistry 25, 733 (1986). 25 R. Wetzel, L. J. Perry, W. A. Baase, and W. J. Becktel, Proc. Natl. Acad. Sci. U.S.A. 85, 401 (1988). 26 j. A. Wells and D. B. Powers, J. Biol. Chem. 261, 6564 (1986). 27 C. Mitchinson and J. A. Wells, Biochemistry 28, 4807 (1989). 28 M. W. Pantoliano, R. C. Ladner, P. N. Bryan, M. L. Rollence, J. F. Wood, and T. L. Poulos, Biochemistry 26, 2077 (1987). 29 R. T. Sauer, K. Hehir, R. S. Stearman, M. A, Weiss, A. J. Nilson, E. G. Suchanek, and C. O. Pabo, Biochemistry 25, 5992 (1986).

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newly introduced cysteines were found to form disulfide bonds. However, the addition of these new disulfides did not always confer an increase in stability. One of the reasons for this modest success appears to be the difficulty in finding optimal sites that are compatible with the strict stereochemical requirements of disulfide bridges) °-32 The introduction of an atypical disulfide can introduce strain into the protein structure and can offset the stabilizing effect of the cross-link. 22,33,34 We have recently introduced a number of single and multiple disulfide bonds in phage T4 lysozyme, and have shown that some of the variants are significantly more stable than wild type. 34-37 In this chapter, we describe the design, construction, and characteristics of these mutant proteins.

Design of Disulfide Bonds There are at least three aspects to be considered in the overall design and introduction of disulfide bonds for the stabilization of proteins: the geometric requirements for the formation of disulfide bonds, the size of the loop formed by the cross-link, and the loss of existing interactions associated with replacement of wild-type residues by cysteines. The stereochemistry of disulfide bridges is quite restricted, which causes such bridges to be relatively inflexible. Therefore, disulfide geometry is important because inappropriate positions and orientations of the two cysteine residues will tend to introduce strain. The loop size is also relevant because polymer theory ~7-2° suggests that the entropic effect on the unfolded state increases with increased loop size. Finally, it is desirable that replacement of existing residues with cysteines should avoid disruption or loss of interactions that stabilize the native structure. For T4 lysozyme, possible sites for the introduction of disulfide bonds were evaluated in three steps as follows35: (1) examples of disulfide bridges taken from the Brookhaven Protein Data Bank were used to find pairs of residues in the protein that could form geometrically reasonable disulfide 3o j. S. Richardson, Adv. Protein Chem. 34, 167 (1981). 31 j. M. Thornton, J. Mol. Biol. 151, 261 (1981). 32 C. O. Pabo and E. G. Suchanek, Biochemistry 25, 5987 (1986). 33 B. A. Katz and A. Kossiakoff, J. Biol. Chem. 261, 15480 (1986). P. E. Pjura, M. Matsumura, J. A. Wozniak, and B. W. Matthews, Biochemistry 29, 2592

(o90). 35 M. Matsumura, W. J. Becktel, M. Levitt, and B. W. Matthews, Proc. Natl. Acad. Sci. U.S.A. 86, 6562 0989). 36 M. Matsumura and B. W. Matthews, Science 243, 792 (1989). 37 M. Matsumura, G. Signor, and B. W. Matthews, Nature (London) 342, 291 (1989).

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bridges when they were replaced with cysteines; (2) the strain energy associated with forming the disulfide bond between each of these pairs of cysteine residues was estimated; and (3) the deleterious effect of each mutation on residue interactions in the wild-type structure was also evaluated. The first step involved comparison of the N, C ~, C ~ , Sv , C, and O coordinates of pairs of cysteine residues in disulfide bridges in all known protein X-ray structures 38 with all possible sites in the structure of wildtype lysozyme)9 The lowest root mean square deviation after superposition was used to rank the selected pairs (see geometry, Table I). The second step was to estimate the strain associated with formation of putative disulfide bonds. Energy minimization, with defined parameters, 4° was applied to a small "molten zone" that included one residue on each side of the pair of residues being replaced, allowing only the total of six residues to move. The strain energy was calculated as the difference between the energy of the protein with and without disulfide bond formation (see strain energy, Table I). The calculated energy of the cross-linked form was found to be always at least 4 kcal/mol higher than that of the noncross-linked form, suggesting that the introduction of any disulfide bond would introduce strain into the T4 lysozyme structure. It should also be noted that the energy calculation was only intended as a rough guide, and in practice was supplemented by visualization of the proposed sites using graphics 4~ or a wire model of the structure. The third step involved an analysis of existing interactions that would be lost on introduction of new cysteine residues. Table I summarizes the data for the five selected sites. It had previously been shown that a single mutation at position 3 (lie3 to Cys) in T4 lysozyme forms a disulfide bond with wild-type Cys-97 and stabilizes the enzyme toward thermal denaturation. 23-25'42 Our procedure correctly predicted the 3-97 pair as one of the best candidates for engineering a disulfide bond. Four of the disulfide bonds were predicted to introduce strain energies of 4-8 kcal/mol. For the 21-142 disulfide, however, the calculated strain energy was much higher (20.9 kcal/mol). In spite of this high strain energy, we nevertheless selected the 21-142 site as a candidate because it was anticipated that the presumed interdomain "hinge-bending" motion 39 at the active-site cleft would facilitate the formation of this cross-link. 38 F. C. Bernstein, T. F. Koetzle, G. J. B. Williams, E. F. Meyer, M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi, and M. Tasumi, J. Mol. Biol. 112, 535 (1977). 39 L. H. Weaver and B. W. Matthews, J. Mol. Biol. 193, 189 (1987). 40 M. Levitt, J. Mol. Biol. 168, 595 (1983). 4l T. A. Jones, in "Crystallographic Computing" (D. Sayre, ed.), p. 303. Oxford Univ. Press, Oxford, 1982. 42 M. Matsumura, W. J. Becktel, and B. W. Matthews, Nature (London) 334, 406 (1988).

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TABLE I PARAMETERS FOR DISULFIDE BONDS IN T4 LYSOZYMEa

Parameter Geometry (fit) Distance C~-C ~ (fit) C/Ov-C/O ~ (fit) Strain energy (kcal/mol) Loop size (residues) Loss of contacts

Ile-9/ Leu-164

Thr-21/ Thr-142

Ser-90/ Gin-122

Asp-127/ Arg-154

Ile-3/ Cys-97

1.35

1.33

0.94

1.10

0.93

7.4 4.4 4.1 156 7 Glu-5 Arg-148 Ala-160 Tyr-161

8.1 4.2 20.9 122 3 Gin- 105 Trp-138

6.2 3.8 6.7 33 5 Pro-86 Val-87 Ser-90 Gln-122

6.0 4.4 8.4 28 5 Trp- 126 Asp-127 Ala-130 Trp-151 Arg-154

71, 69 17, 76

19, 82 29, 14

72, 34 14, 22

0, 56 19, 21

5.6 4.8 7.4 95 7 Met-6 Leu-7 Ala-96 Cys-97 Ile-100 Trp-158 84, 78 15, 11

Side-chain burial (%) Main-chain B value (fit2)

Geometry is the root-mean-square discrepancy with the best corresponding bond bridge found in the Brookhaven Data Bank (see text). The strain energy was estimated using a small "molten zone" consisting of the tripeptides centered on the two residues involved in the disulfide bridge (see text). Side-chain burial is the fraction of the substituted side chain in the wild-type structure inaccessible to solvent. Main-chain B value is the average crystallographic thermal factor of the N, C a, C, and O atoms of the substituted residue.

Figure 1 shows the locations of the engineered disulfide bonds. The 9-164 disulfide, which connects the N-terminal helix with the extreme C terminus, makes an almost circularized protein. The 3-97 disulfide also connects the N-terminal helix with the C-terminal domain. The 21-142 disulfide provides a large loop by connecting the active-site cleft of T4 lysozyme (note that the active-site residues are Glu-11 and Asp-20). Thus, the 3-97, 9-164, and 21-142 disulfide bonds link the N- and C-terminal domains with large loops. In contrast, the 90-122 and the 127-154 disulfide bonds link two helices within the C-terminal domain and give relatively small loops. Experimental

Procedures

Site-Directed Mutagenesis Mutagenic oligonucleotides (21 to 23-mer) are synthesized using a Model 380B DNA synthesizer (Applied Biosystems, Foster City, CA) and

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STABILIZATION OF PROTEINS BY DISULFIDE BONDS

341

140

%

,sel Gh,I )11

127 90

154

122

97 "3

1

142 i

i

164

9

F

eo

'

~o

'

t~o

'

I,;o

,c

FIG. 1. Schematic view of the a-carbon backbone of bacteriophage T4 lysozyme, showing locations of the introduced disulfide bonds. Four pairs of amino acid residues, namely, Ile-9/Leu-164, Thr-21/Thr-142, Ser-90/Gln-122, and Asp-127/Arg-154, were replaced with cysteine residues, respectively. The disulfide bond obtained by mutations Ile-3 --* Cys (and Cys-54 ~ Thr) was also constructed. Gin-11 and Asp-20 are active-site residues. (Inset) Sizes of the loops formed by the respective disulfide bonds. (From Matsumura et al?5).

purified by a Cl8 Sep-Pak cartridge (Millipore, Bedford, MA) according to the manufacturer's protocol. Site-directed mutagenesis is performed essentially according to Kunkel et al. 43 Briefly, uracil-containing singlestranded DNA of a bacteriophage Ml3mpl8 derivative, which carries the T4 lysozyme gene on a 630 base-pair B a m H I - H i n d l I I fragment, is pre43 T. A. Kunkel, J. D. Roberts, and R. A. Zakour, this series, Vol. 154 [19].

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pared from Escherichia coli strain CJ236 [dutl, ungl, thil, relA1/pCJ l05 ( C m r ) ] . The 5'-phosphorylated mutagenic primer is annealed to the uracilcontaining template DNA, and the sequence contained with oligonucleotide is converted to a covalently closed circular DNA molecule by the Klenow fragment of E. coli DNA polymerase I and T4 DNA ligase. The double-stranded DNA synthesized in vitro is used to transform competent E. coli JMI01. The phage progenies are then segregated on a lawn of strain JMI01 to obtain plaques derived from single phage particles. Following the preparation of single-stranded DNAs, the mutants are screened by dideoxy sequencing44 or plaque hybridization45 with the 32p-labeled mutagenic primer. Technical details of DNA manipulations and various routine procedures follow the m a n u a l . 46 Expression and Purification o f Proteins The mutated T4 lysozyme gene on M13 is digested with BamHI and HindlII, then cloned into the expression plasmid p r i S e 5 4 7 that contains tandem lacUV5 and tac promoters, the trp terminator, as well as the lacq gene. Escherichia coli RR1 is then transformed by the recombinant plasmid. The production of protein is carried out by using a 5-liter fermentor (Braun Biostat V) containing 3 liters of LB broth with 100/zg/ml of ampicillin. The culture is agitated with good aeration at 37° until the optical density at 600 nm reaches about 1. Lysozyme synthesis is then induced by addition of isopropyl-/3-thiogalactoside to a final concentration of 1 mM. After an additional cultivation for 2 hr at 32°, the broth is centrifuged. The protein is purified as previously d e s c r i b e d . 47,48 In brief, the ethylenediaminetetraacetic acid (EDTA)-lysed cells and the supernatant from the centrifuged growth medium, which contains most of the lysozyme released from lysed cells, are dialyzed against distilled water to reduce the conductivity. The solution is then applied to a CM-Sephadex column, and the protein is eluted by a 50-300 mM NaC1 gradient in 50 mM phosphate buffer, pH 6.5. The peak fractions are collected, dialyzed, and adsorbed on an SP-Sephadex column. The concentrated protein is eluted with 0.1 M NaC1 in the buffer. Typical yields are 40-70 mg of lysozyme per liter of culture. Protein concentrations are determined spec44 F. Sanger, S. Nickelson, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977). 45 M. J. Zoller and M. Smith, this series, Vol. 100 [32]. 46 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 47 D. C. Muchmore, L. P. Mclntosh, C. B. Russell, D. E. Anderson, and F. W,. Dahlquist, this series, Vol. 177 [3]. 48 T. Alber and B. W. Matthews, this series, Vol. 154 [27].

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trophotometrically (molar absorption coefficient of wild-type T4 lysozyme, ~280 ~0.1% nm, of 1.28; molecular weight of T4 lysozyme, 18,700. 49

Assay of Disulfide Bond Formation The formation of disulfide bonds introduced into T4 lysozyme is monitored by one or a combination of the following four methods. Ellman Titration. The thiol content of the protein is determined by Ellman's reagent. 5°'51 A 0.5-ml aliquot of the protein (0.4 mg, corresponding to - 2 0 nmol) is mixed with 2.5 ml of a solution containing 2% sodium dodecyl sulfate (SDS), 80 mM sodium phosphate (pH 8.0), and 0.5 mg/ml EDTA. To 3 ml of the mixture is immediately added 0.1 ml of 5,5-dithiobis(2-nitrobenzoic acid) (DNTB) (40 mg DTNB in 10 ml of 0.1 M sodium phosphate, pH 8.0). After 15 min, the absorbance at 410 nm is measured. The samples are always calibrated relative to three references: wild-type lysozyme, which contains two cysteine residues at positions 54 and 97; the single-cysteine mutant (Cys-54 ~ Thr); and the cysteine-free mutant (Cys-54 ~ Thr and Cys-97 ~ Ala). Nonreducing SDS-Polyacrylamide Gel Electrophoresis. About 5-10 /zg of protein (1 mg/ml) is treated with 50 mM iodoacetamide in 40 mM Tris-HC1, pH 8.5 (total volume 50/xl), for 1 hr at 23 ° in the dark in order to block free cysteines. Samples are then adjusted to 10% trichloroacetic acid and incubated for 30 min at 23 °. Protein precipitate is collected by centrifugation, washed 3 times with 2 volumes of acetone, and dried. The precipitate is dissolved in 1 × SDS sample buffer without 2-mercaptoethanol. Otherwise the conditions are essentially those of Laemmli. 52 Samples are boiled for 2-5 min and applied on a 12.5% SDS-polyacrylamide gel. Reversed-Phase High-Performance Liquid Chromatography. Protein samples (20-40 tzg) in a volume of 50-100 tzl are injected on a C18 column (Vydac, 4.6 × 150 mm, Rainin, Woburn, MA) and eluted at 0.5 ml/ min with a linear gradient of 305 to 54% acetonitrile in 0.1% aqueous trifluoroacetic acid at 0.33% acetonitrile per minute. The elution of protein is detected at 220 nm. Ion-Exchange High-Performance Liquid Chromatography. The proteins (40 ~g) are incubated in the dark at 23 ° with 0.1 M iodoacetic acid in 40 mM Tris-HCl, pH 8.5 (total volume 100 /xl), for I hr to block all free thiols. The alkylated enzymes are then loaded on a cation-exchange 49 A. Tsugita, in "The Enzymes" (P, D. Boyer, ed.), Vol. 5, p. 343. Academic Press, New York, 1971. 5o G. L. Ellmann, Arch. Biochem. Biophys. 82, 70 (1959). 51 A. F. S. A. Habeeb, this series, Vol. 25 [37]. 52 U. K. Laemmli, Nature (London) 227, 680 (1970).

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column (Aquapore Cation, 4.6 x 100 mm, Rainin) and eluted at 1 ml/min with a linear gradient of 50-300 mM sodium phosphate buffer, pH 6.6, at l0 mM sodium phosphate per minute. The elution of protein is detected at 220 nm.

Preparation of Oxidized and Reduced Forms of Proteins Oxidized (cross-linked) forms of the mutant lysozymes are prepared on exposure to air in vitro by incubating in 0.1 M Tris-HCl, pH 8.0, and 0.15 M NaCI for 1-3 weeks at 4 °, depending on the nature of the disulfide bonds. The reduced (noncross-linked) forms of the mutant lysozymes are prepared by treating the proteins (typically 1 mg/ml) with 6 M guanidine hydrochloride, 20 mM dithiothreitol (DTT), and 1 mM EDTA in 50 mM Tris-HCl, pH 8.3. After incubation for 4 hr at 23 °, the mixture is extensively dialyzed under a nitrogen gas purge at 4 ° against 0.2 M NaCI and 1 mM EDTA solution, pH 2.0. Under the nitrogen atmosphere and at acidic pH, the reduced proteins are stable for at least 1 week at 4 °.

Assay of Enzyme Activity Lysozyme activities are measured at 23 ° using the turbidity assay. 49 A suspension (0.95 ml) of lyophilized E. coli cells in 50 mM Tris-HC1, pH 7.4, is quickly mixed with 0.05 ml of the enzyme (0.05-0.2/xg) in a cuvette in a spectrophotometer. The initial concentration of E. coli cells in the reaction mixture is adjusted to an absorbance at 450 nm of 0.7-0.8. The enzyme activity is calculated from the rate of decrease in absorbance at 450 nm monitored at 23 °. Measurements for the reduced forms of mutant lysozymes are conducted in the buffer containing 5 mM DTT, which produces no effect on the activities of wild-type lysozyme.

Thermal Stability of Proteins The thermal stability of the mutant lysozymes is assessed at pH 2.0 by measuring the circular dichroism (CD) at 223 nm as a function of temperature. 53'54At the temperature of the midpoint of the thermal denaturation transition (Tm), half the protein is unfolded, and the apparent free energy of unfolding (AG) is 0. The CD spectra are monitored with a Jasco J-500C instrument equipped with a Hewlett-Packard 89100A thermoionic controller. The temperature of the sample is changed at a constant rate, typically 1° per minute, under control of a Hewlett-Packard 87 XM computer. The sample solutions contain about 0.02 mg/ml of protein, 0.15 M 5~ M. Elwell and J. Schellman, Biochim. Biophys. Acta 386, 309 (1975). 54 R. Hawkes, M. G. Gr~tter, and J. Schellman, J. Mol. Biol. 175, 195 (1984).

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KC1, and 1 mM EDTA, adjusted to pH 2.0 with HC1. To avoid air oxidation, the experiments are carefully performed in a nitrogen atmosphere, and the KC1-EDTA solutions are extensively bubbled with nitrogen gas before use. 55 Lysozymes with Single Disulfide Bonds Construction of Mutants Wild-type T4 lysozyme contains two cysteines at positions 54 and 97. To avoid possible intermolecular thiol/disulfide interchange reaction with the newly engineered disulfide, 24 Cys-54 and Cys-97 are replaced with Thr and Ala, respectively. The cysteine-free mutant, designated either T54-A97 or pseudo-wild type (WT*), has activity as well as stability essentially identical with those of the wild-type enzyme (see below). In addition, the WT* mutant crystallizes isomorphously with the wild type, and its structure at 1.8-,~ resolution is very similar to that of wild-type lysozyme, except in the vicinity of the mutations. 34 On the template of the WT* gene, the four disulfide mutants are created by site-directed mutagenesis: Ile-9 ~ Cys and Leu-164 ~ Cys (C9-C 164WT*), Thr-21 ~ Cys and Thr-142 ~ Cys (C21-C142-WT*), Ser-90 ~ Cys and Gin-122 ~ Cys (C9°-C122-WT*), and Asp-127 ~ Cys and Arg-154 Cys (C127-C154-WT*).The 3-97 disulfide mutant (C3-T54) is constructed by mutating Ile-3 ~ Cys (and Cys-54 --~ Thr) in wild-type lysozyme. Table II summarizes the amino acid replacements introduced in these mutants. Each of the mutant genes is expressed in E. coli, and the proteins are purified to homogeneity as described under Experimental Procedures. Assessment of Disulfide Bond Formation Immediately after purification, the mutant proteins are found to be a mixture of oxidized (cross-linked) and reduced (noncross-linked) forms, as judged by titration of protein thiols with Ellman's reagent, nonreducing SDS-PAGE, reversed-phage HPLC, and cation-exchange HPLC. Each of these methods (described above) has advantages and disadvantages. The EUman titration is a rapid and accurate procedure to determine the content of free cysteines within the protein. However, the titration requires a relatively large amount of protein (0.4 mg in the case of lysozyme), and the procedure cannot be used in the presence of reducing agents. In contrast, the analysis by SDS-PAGE or HPLC needs only 5-40/xg of 55 W. J. Becktel and W. A Baase,

Biopolymers 26,

619 (1987).

346

PROTEINS

AND PEPTIDES;

PRINCIPLES

AND

METHODS

Z~

¢

Z 0

z

.~

z

~

=

,

Stabilization of functional proteins by introduction of multiple disulfide bonds.

336 PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS [16] incorporated by this method is currently being evaluated. In addition, more expedient method...
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