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Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond

Journal Article · · Biochemistry; (United States)
DOI:https://doi.org/10.1021/bi00382a002· OSTI ID:5558604
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Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in an 1.3 A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1/sup 0/C higher than that of the wild-type protein and 5.8/sup 0/C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiment performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers.
Research Organization:
Genex Corp., Gaithersburg, MD
OSTI ID:
5558604
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:8; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201 -- Biochemistry-- Tracer Techniques
550602* -- Medicine-- External Radiation in Diagnostics-- (1980-)
59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
BACILLUS
BACILLUS SUBTILIS
BACTERIA
BIOCHEMICAL REACTION KINETICS
CALORIMETRY
CARBOXYLIC ACIDS
CHEMICAL BONDS
COHERENT SCATTERING
CYSTEINE
DIFFRACTION
ELECTROPHORESIS
ENZYMES
GENES
HYDROLASES
INACTIVATION
KINETICS
MICROORGANISMS
MUTAGENESIS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDE HYDROLASES
REACTION KINETICS
SCATTERING
SERINE PROTEINASES
TEMPERATURE DEPENDENCE
THIOLS
X-RAY DIFFRACTION