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

Pantoliano, Michael W.; Ladner, Robert C.; Bryan, Philip N.; Rollence, Michele L.; Wood, Jay F.; Poulos, T.L.

doi:10.1021/bi00382a002

Cited by 223 publications

(118 citation statements)

References 30 publications

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“…The weak stabilization of this crosslink may derive from a poor model of the N-state (the Escherichia coli enzyme crystal structure was used) or from a nonadherence to a two-state equilibrium. Novel disulfide bonds have been introduced into subtilisin BPN' (Wells & Powers, 1986;Pantoliano et al, 1987;Mitchinson & Wells, 1989), subtilisin E (Takagi et al, 1990), and troponin C (Gusev et al, 1991), but irreversible denaturation is observed for either the crosslinked or non-crosslinked forms or both, obviating thermodynamic treatment based on Equation 1.…”

Section: Novel Disulfidesmentioning

confidence: 99%

Disulfide bonds and the stability of globular proteins

1993

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An understanding of the forces that contribute to stability is pivotal in solving the protein-folding problem. Classical theory suggests that disulfide bonds stabilize proteins by reducing the entropy of the denatured state. More recent theories have attempted to expand this idea, suggesting that in addition to configurational entropic effects, enthalpic and native-state effects occur and cannot be neglected. Experimental thermodynamic evidence is examined from two sources: (1) the disruption of naturally occurring disulfides, and (2) the insertion of novel disulfides. The data confirm that enthalpic and native-state effects are often significant. The experimental changes in free energy are compared to those predicted by different theories. The differences between theory and experiment are large near 300 K and do not lend support to any of the current theories regarding the stabilization of proteins by disulfide bonds. This observation is a result of not only deficiencies in the theoretical models but also from difficulties in determining the effects of disulfide bonds on protein stability against the backdrop of numerous subtle stabilizing factors (in both the native and denatured states), which they may also affect. Keywords: disulfide bonds; protein stability; thermodynamicsThe determination of the forces that govern protein stability is of fundamental importance for our ability to understand and control the interactions of complex biological molecules. It has been known since the 1960s that the primary structure of a protein dictates its threedimensional fold (see Anfinsen, 1973), yet a comprehensive understanding of the factors that impart thermodynamic stability to proteins is elusive. This is because the tertiary folds of native proteins are defined by a large Reprint requests to: Stephen F. Betz, The DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0328.Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; C,, the concentration of GdmCl at which half the protein is denatured; D-state, the denatured state of a protein; GdmCI, guanidinium chloride; hew, hen egg white; mden, the slope of the line of -AGd versus denaturant concentration; N-state, the native state of a protein; RNase, ribonuclease; Tm, temperature at which half the protein is denatured; t l I z , time required for enzymatic activity to decrease 50%; AC,, the difference in heat capacity between the denatured and native states; AGd, AG for protein denaturation; AGd,HZO, AGd determined from chemical denaturation; AGd, T, AGd determined from thermal denaturation; A H , , , the change in conformational enthalpy between the native and denatured states; N H , , AH for protein denaturation; AH&,,, the change in solvational enthalpy between the native and denatured states; AH,,,, AH for protein denaturation at T,,,; AS,,,, the change in conformational entropy between the native and denatured states; AS,, AS for protein denaturation; Ashyd, the change in solvational entropy between the native and denatured states.

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Section: Novel Disulfidesmentioning

confidence: 99%

Disulfide bonds and the stability of globular proteins

1993

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“…This structure was subsequently refined at 1.8 A resolution with phenyl methyl sulfonyl fluoride (PMSF) inhibitor covalently bound (Bott, Ultsch, Kossiakoff, Graycar, Katz & Power, 1988), in complex with Streptomyces inhibitor at 2.6 A (Hirono, Akagawa, Mitsui & Iitaka, 1984) and at 2.1 A as a complex with the chymotrypsin inhibitor 2 from barley seed (McPhalen, Svendsen, Jonassen & James, 1985;McPhalen & James, 1988). High-resolution structures of site-directed mutants of SBPN (Pantoliano et al, 1987(Pantoliano et al, , 1988 have also been reported. The three-dimensional structures of other Bacilli subtilisins have been published: the SBCARL-eglin-C complex at 1.8/k (McPhalen, Schnebli & Jamesl 1985;McPhalen & James, 1988) and independently at a nominal resolution of 1.2 A (Bode, Papamokos, Musil, Seemfiller & Fritz, 1986;Bode, Papamokos & Musil, 1987), and native SBCARL at 2.5A (Neidhart & Petsko, 1988).…”

Section: Bacteriummentioning

confidence: 99%

“…Genetically engineered mutants of subtilisin BPN' have been extensively investigated (e.g. Russell, Thomas & Fersht, 1987;Pantoliano, Ladner, Bryan, Rollence, Wood & Poulos, 1987;Pantoliano, Whitlow, Wood, Rollence, Finzel, Gilliland, Poulos & Bryan, 1988). In addition the subtilisins have extensive practical uses, mainly as additives to laundry detergents to improve their cleaning power by hydrolysis of protein stains.…”

Section: Introductionmentioning

confidence: 99%

Complex between the subtilisin from a mesophilic bacterium and the Leech inhibitor eglin-C

Dauter

Betzel²,

Genov³

et al. 1991

Acta Crystallogr Sect B

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“…This knowledge has, therefore, stimulated several attempts to introduce new disulfide bonds to improve protein stability. To date, disulfide bonds have been engineered into dihydrofolate reductase (2), T4 lysozyme (3,4), subtilisin (5,6), and A repressor (7). The addition of new disulfides has not, however, always increased stability.…”

mentioning

confidence: 99%

“…Nonreducing SDS/PAGE (5,6) and reversed-phase HPLC (3,4) have been used to assay the formation of disulfide bonds. However, neither SDS/PAGE nor reversed-phase HPLC was always effective in distinguishing between the oxidized (crosslinked) and reduced (noncrosslinked) form of the mutant lysozymes.…”

mentioning

confidence: 99%

Stabilization of phage T4 lysozyme by engineered disulfide bonds.

Matsumura

Becktel

Levitt

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

242

181

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Four different disulfide bridges (inking positions 9-164, 21-142, 90-122, and 127-154) were introduced into a cysteine-free phage T4 lysozyme at sites suggested by theoretical calculations and computer modeling. The new cysteines spontaneously formed disulfide bonds on exposure to air in vitro. In all cases the oxidized (crosslinked) lysozyme was more stable than the corresponding reduced (noncrosslinked) enzyme toward thermal denaturation. Relative to wild-ype lysozyme, the melting temperatures of the 9-164 and 21-142 disulfide mutants were increased by 6.4CC and 11.0°C, whereas the other two mutants were either less stable or equally stable. Measurement of the equilibrium constants for the reduction of the engineered disulfide bonds by dithiothreitol indicates that the less thermostable mutants tend to have a less favorable crosslink in the native structure. The two disulfide bridges that are most effective in increasing the stability of T4 lysozyme have, in common, a large loop size and a location that includes a flexible part of the molecule. The results suggest that stabilization due to the effect of the crosslink on the entropy of the unfolded polypeptide is offset by the strain energy associated with formation of the disulfide bond in the folded protein. The design of disulffide bridges is discussed in terms of protein flexibility.The design of proteins with enhanced stability is one of the major goals of protein engineering. Among the physical forces that maintain the tertiary structure of proteins, disulfide bonds can make a substantial contribution (1). This knowledge has, therefore, stimulated several attempts to introduce new disulfide bonds to improve protein stability. To date, disulfide bonds have been engineered into dihydrofolate reductase (2), T4 lysozyme (3, 4), subtilisin (5, 6), and A repressor (7). The addition of new disulfides has not, however, always increased stability. One aspect of this problem is that knowledge of the mechanism by which crosslinks, such as disulfide bonds, stabilize or destabilize proteins is limited.In this study, we have designed and constructed four different disulfide mutants of phage T4 lysozyme. The results illustrate the principles by which the engineered disulfide bonds stabilize the enzyme and suggest a strategy for the selection of potential disulfides that are most likely to increase the stability of a protein. MATERIALS AND METHODSDesign of Disulfide Bridges. Possible sites for the introduction of disulfide bridges were sought by the following three steps. (i) The coordinates of pairs of cysteine residues in disulfide bridges (total of 295 pairs) in known protein x-ray structures were collected (8). Next, all potential pairs of residues to be engineered in T4 lysozyme (9) were scanned to find pairs that satisfied the following three criteria: The residues must be separated by at least 20 residues along the polypeptide chain, both residues must have y atoms (This criterion excluded alanine and glycine, which may, in retrospect, have been unnecessa...

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

Cited by 223 publications

References 30 publications

Disulfide bonds and the stability of globular proteins

Disulfide bonds and the stability of globular proteins

Complex between the subtilisin from a mesophilic bacterium and the Leech inhibitor eglin-C

Stabilization of phage T4 lysozyme by engineered disulfide bonds.

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