DOI: 10.1126/science.6387910. Disulfide Bond Engineered into T4 Lysozyme: Stabilization of the Protein Toward Thermal Inactivation

Disulfide Bond Engineered into T4 Lysozyme: Stabilization of the Protein Toward Thermal Inactivation

10.1126/science.6387910

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

By recombinant DNA techniques, a disulfide bond was introduced at a specific site in T4 lysozyme, a disulfide-free enzyme. This derivative retained full enzymatic activity and was more stable toward thermal inactivation than the wild-type protein. The derivative, T4 lysozyme (Ile 3 → Cys), was prepared by substituting a Cys codon for an Ile codon at position 3 in the cloned lysozyme gene by means of oligonucleotide-dependent, site-directed mutagenesis. The new gene was expressed in Escherichia coli under control of the ( trp-lac ) hybrid tac promoter, and the protein was purified. Mild oxidation generated a disulfide bond between the new Cys 3 and Cys 97 , one of the two unpaired cysteines of the native molecule. Oxidized T4 lysozyme (Ile 3 → Cys) exhibited specific activity identical to that of the wild-type enzyme when measured at 20°C in a cell-clearing assay. The cross-linked protein was more stable than the wild type during incubation at elevated temperatures as determined by recovered enzymatic activity at 20°C.

Bibliography

Perry, L. J., & Wetzel, R. (1984). Disulfide Bond Engineered into T4 Lysozyme: Stabilization of the Protein Toward Thermal Inactivation. Science, 226(4674), 555â557.

Authors 2

L. Jeanne Perry (first)
Ronald Wetzel (additional)

References 43 Referenced 261

ANFINSEN, C.B., ADVANCES IN PROTEIN CHEMISTRY 29: 205 (1975). (10.1016/S0065-3233(08)60413-1) / ADVANCES IN PROTEIN CHEMISTRY (1975)
DEBOER, H, GENE PROTEIN TRANSLA 19: 309 (1982). / GENE PROTEIN TRANSLA (1982)
de Boer, H. A., Proceedings of the National Academy of Sciences of the United States of America 80: 21 (1983). / Proceedings of the National Academy of Sciences of the United States of America (1983)
DESMADRIL, M, EXISTENCE OF INTERMEDIATES IN THE REFOLDING OF T4 LYSOZYME AT PH 7.4, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 101: 563 (1981). (10.1016/0006-291X(81)91296-1) / BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (1981)
DESMADRIL, M, EVIDENCE FOR INTERMEDIATES DURING UNFOLDING AND REFOLDING OF A 2-DOMAIN PROTEIN, PHAGE-T4 LYSOZYME - EQUILIBRIUM AND KINETIC-STUDIES, BIOCHEMISTRY 23: 11 (1984). (10.1021/bi00296a003) / BIOCHEMISTRY (1984)
DESMADRIL, M, IMMUNOCHEMICAL STUDY OF PHAGE T-4 LYSOZYME, FEBS LETTERS 133: 183 (1981). (10.1016/0014-5793(81)80501-7) / FEBS LETTERS (1981)
ELWELL, M, PHAGE-T4 LYSOZYME - PHYSICAL-PROPERTIES AND REVERSIBLE UNFOLDING, BIOCHIMICA ET BIOPHYSICA ACTA 386: 309 (1975). (10.1016/0005-2795(75)90273-1) / BIOCHIMICA ET BIOPHYSICA ACTA (1975)
ELWELL, M.L., STABILITY OF PHAGE-T4 LYSOZYMES .1. NATIVE PROPERTIES AND THERMAL-STABILITY OF WILD-TYPE AND 2 MUTANT LYSOZYMES, BIOCHIMICA ET BIOPHYSICA ACTA 494: 367 (1977). (10.1016/0005-2795(77)90166-0) / BIOCHIMICA ET BIOPHYSICA ACTA (1977)
ELWELL, M.L., STABILITY OF PHAGE-T4 LYSOZOMES .2. UNFOLDING WITH GUANIDINIUM CHLORIDE, BIOCHIMICA ET BIOPHYSICA ACTA 580: 327 (1979). (10.1016/0005-2795(79)90145-4) / BIOCHIMICA ET BIOPHYSICA ACTA (1979)
FREEDMAN, R.B., ENZYMOLOGY POST TRAN 1: 157 (1980). / ENZYMOLOGY POST TRAN (1980)
GRUETTER, M.G., BACTERIOPHAGE T4: 356 (1983). / BACTERIOPHAGE T4 (1983)
Grutter, M. G., Nature 277:667 (1979). (10.1038/277667a0) / Nature (1979)
JOHNSON, R.E., THERMODYNAMICS OF PROTEIN CROSS-LINKS, BIOCHEMISTRY 17: 1479 (1978). (10.1021/bi00601a019) / BIOCHEMISTRY (1978)
KONISHI, Y, REGENERATION OF RIBONUCLEASE-A FROM THE REDUCED PROTEIN .1. CONFORMATIONAL-ANALYSIS OF THE INTERMEDIATES BY MEASUREMENTS OF ENZYMATIC-ACTIVITY, OPTICAL-DENSITY, AND OPTICAL-ROTATION, BIOCHEMISTRY 19: 1308 (1980). (10.1021/bi00548a008) / BIOCHEMISTRY (1980)
KONISHI, Y, REGENERATION OF RIBONUCLEASE-A FROM THE REDUCED PROTEIN - ISOLATION AND IDENTIFICATION OF INTERMEDIATES, AND EQUILIBRIUM TREATMENT .3., BIOCHEMISTRY 20: 3945 (1981). (10.1021/bi00517a001) / BIOCHEMISTRY (1981)
KONISHI, Y, REGENERATION OF RIBONUCLEASE-A FROM THE REDUCED PROTEIN - RATE-LIMITING STEPS .4., BIOCHEMISTRY 21: 4734 (1982). (10.1021/bi00262a033) / BIOCHEMISTRY (1982)
10.1126/science.7455704
MATTHEWS, B.W., RELATION BETWEEN HEN EGG-WHITE LYSOZYME AND BACTERIOPHAGE-T4 LYSOZYME - EVOLUTIONARY IMPLICATIONS, JOURNAL OF MOLECULAR BIOLOGY 147: 545 (1981). (10.1016/0022-2836(81)90399-5) / JOURNAL OF MOLECULAR BIOLOGY (1981)
MATTHEWS, B.W., 3 DIMENSIONAL STRUCTURE OF LYSOZYME FROM BACTERIOPHAGE-T4, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 71: 4178 (1974). / PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA (1974)
MESSING, J, NEW M13 VECTORS FOR CLONING, METHODS IN ENZYMOLOGY 101: 20 (1983). (10.1016/0076-6879(83)01005-8) / METHODS IN ENZYMOLOGY (1983)
MOREHEAD, H, ROLES OF THE 29-138 DISULFIDE BOND OF SUBTYPE-A OF HUMAN ALPHA-INTERFERON IN ITS ANTIVIRAL ACTIVITY AND CONFORMATIONAL STABILITY, BIOCHEMISTRY 23: 2500 (1984). (10.1021/bi00306a028) / BIOCHEMISTRY (1984)
PERRY L.J. unpublished data.
10.1128/jb.153.1.27-32.1983
RASTETTER, W.H., TRENDS IN BIOTECHNOLOGY 1: 1 (1983). (10.1016/0167-7799(83)90016-1) / TRENDS IN BIOTECHNOLOGY (1983)
REMINGTON, S.J., STRUCTURE OF LYSOZYME FROM BACTERIOPHAGE-T4 - ELECTRON-DENSITY MAP AT 2.4 A RESOLUTION, JOURNAL OF MOLECULAR BIOLOGY 118: 81 (1978). (10.1016/0022-2836(78)90245-0) / JOURNAL OF MOLECULAR BIOLOGY (1978)
RICHARDSON, J.S., ADVANCES IN PROTEIN CHEMISTRY 34: 167 (1981). (10.1016/S0065-3233(08)60520-3) / ADVANCES IN PROTEIN CHEMISTRY (1981)
ROSSMANN, M.G., EXPLORING STRUCTURAL HOMOLOGY OF PROTEINS, JOURNAL OF MOLECULAR BIOLOGY 105: 75 (1976). (10.1016/0022-2836(76)90195-9) / JOURNAL OF MOLECULAR BIOLOGY (1976)
SCHEELE, G, CONFORMATIONAL-CHANGES ASSOCIATED WITH PROTEOLYTIC PROCESSING OF PRE-SECRETORY PROTEINS ALLOW GLUTATHIONE-CATALYZED FORMATION OF NATIVE DISULFIDE BONDS, JOURNAL OF BIOLOGICAL CHEMISTRY 257: 12277 (1982). (10.1016/S0021-9258(18)33711-6) / JOURNAL OF BIOLOGICAL CHEMISTRY (1982)
SCHELLMAN, J.A., MUTATIONS AND PROTEIN STABILITY, BIOPOLYMERS 20: 1989 (1981). (10.1002/bip.1981.360200921) / BIOPOLYMERS (1981)
SCHELLMAN, J.A., PROTEIN FOLDING: 331 (1980). / PROTEIN FOLDING (1980)
THORNTON, J.M., DISULFIDE BRIDGES IN GLOBULAR-PROTEINS, JOURNAL OF MOLECULAR BIOLOGY 151: 261 (1981). (10.1016/0022-2836(81)90515-5) / JOURNAL OF MOLECULAR BIOLOGY (1981)
TSUGITA, A, ENZYMES 5: 344 (1971). / ENZYMES (1971)
TSUGITA, A, PURIFICATION OF BACTERIOPHAGE T4 LYSOZYME, JOURNAL OF BIOLOGICAL CHEMISTRY 243: 391 (1968). (10.1016/S0021-9258(18)99306-3) / JOURNAL OF BIOLOGICAL CHEMISTRY (1968)
10.1126/science.6572017
10.1126/science.6356360
WETZEL, R, BIOL INTERFERON SYST: 101 (1983). / BIOL INTERFERON SYST (1983)
WETZEL, R, EXPRESSION IN ESCHERICHIA-COLI OF A CHEMICALLY SYNTHESIZED GENE FOR A MINI-C ANALOG OF HUMAN PROINSULIN, GENE 16: 63 (1981). (10.1016/0378-1119(81)90061-5) / GENE (1981)
WETZEL, R, ASSIGNMENT OF THE DISULFIDE BONDS OF LEUKOCYTE INTERFERON, NATURE 289: 606 (1981). (10.1038/289606a0) / NATURE (1981)
WETZEL, R, PEPTIDES ANAL SYNTHE 5: 1 (1983). / PEPTIDES ANAL SYNTHE (1983)
WILKINSON, A.J., SITE-DIRECTED MUTAGENESIS AS A PROBE OF ENZYME STRUCTURE AND CATALYSIS - TYROSYL-TRANSFER RNA-SYNTHETASE CYSTEINE-35 TO GLYCINE-35 MUTATION, BIOCHEMISTRY 22: 3581 (1983). (10.1021/bi00284a007) / BIOCHEMISTRY (1983)
WILKINSON, A.J., A LARGE INCREASE IN ENZYME SUBSTRATE AFFINITY BY PROTEIN ENGINEERING, NATURE 307: 187 (1984). (10.1038/307187a0) / NATURE (1984)
WINTER, G, REDESIGNING ENZYME STRUCTURE BY SITE-DIRECTED MUTAGENESIS - TYROSYL TRANSFER-RNA SYNTHETASE AND ATP BINDING, NATURE 299: 756 (1982). (10.1038/299756a0) / NATURE (1982)
ZOLLER, M.J., OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS OF DNA FRAGMENTS CLONED INTO M13 VECTORS, METHODS IN ENZYMOLOGY 100: 468 (1983). (10.1016/0076-6879(83)00074-9) / METHODS IN ENZYMOLOGY (1983)

Dates

Type	When
Created	18 years, 10 months ago (Oct. 5, 2006, 3:41 p.m.)
Deposited	1 year, 7 months ago (Jan. 14, 2024, 6:20 p.m.)
Indexed	3 weeks, 6 days ago (Aug. 2, 2025, 12:38 a.m.)
Issued	40 years, 9 months ago (Nov. 2, 1984)
Published	40 years, 9 months ago (Nov. 2, 1984)
Published Print	40 years, 9 months ago (Nov. 2, 1984)

Funders 0

None

BibTeX

@article{Perry_1984, title={Disulfide Bond Engineered into T4 Lysozyme: Stabilization of the Protein Toward Thermal Inactivation}, volume={226}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.6387910}, DOI={10.1126/science.6387910}, number={4674}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Perry, L. Jeanne and Wetzel, Ronald}, year={1984}, month=nov, pages={555–557} }

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