Protein stabilization via hydrophilization

Mozhaev, Vadim V.; Šikšnis, Virginius; Melik‐Nubarov, N. S.; Galkantaite, Nida Z.; Denis, Gervydas J.; Butkus, Eugenijus; Zaslavsky, Boris Y.; Mestechkina, N. M.; Martínek, Karel

doi:10.1111/j.1432-1033.1988.tb13978.x

Cited by 98 publications

(34 citation statements)

References 48 publications

(33 reference statements)

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“…36). It has been observed that extremozymes are often less active at lower temperatures than their mesophilic counterparts (37,39,40). The present results, however, lead to the unexpected but important conclusion that it is possible to engineer extremozymes that retain their full activity at lower temperatures.…”

Section: Fig 4 Hydrolysis Of Protease-resistant ␣-Amylase Frommentioning

confidence: 53%

Engineering an enzyme to resist boiling

Burg

Vriend

Veltman

et al. 1998

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

264

143

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In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100°C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state (''rigidifying'' mutations such as Gly 3 Ala, Ala 3 Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37°C.Denaturation of proteins at elevated temperatures is usually the result of unfolding, which is followed by an irreversible process, most often aggregation (1). The notion that the unfolding processes involved in irreversible denaturation often have a partial (as opposed to global) character has been confirmed experimentally in several cases (2-4). We have studied the thermal stability and denaturation of a broadspecificity metalloprotease produced by Bacillus stearothermophilus CU21 (ref. 5; called TLP-ste; EC 3.4.24.4) that shares 85% sequence identity with its more stable and better known counterpart thermolysin (ref. 6; Table 1). Thermal denaturation of thermolysin-like proteases (TLPs) also depends on partial unfolding processes that, however, are not followed by aggregation but by autolytic degradation starting at unknown sites in the partially unfolded molecule (7-9). An extensive mutation study in which residues in TLP-ste were replaced by the corresponding amino acid in thermolysin showed that only a few of the 43 substitutions between the two enzymes are important for stability (10). All important substitutions are clustered in the N-terminal domain of the protein, in particular in the 55-69 surface loop (refs. 10-12; Fig. 1). Remarkably, combination of only a few of the stabilizing substitutions identified in the 55-59 region resulted in a TLP-ste variant that was more stable than thermolysin itself (11).Based on the observation that the difference in stability between thermolysin and TLP-ste is mainly determined by mutations in the 55-69 area, we set out to search for several additional stabilizing mutations in this same area. Indeed, several stabilizing mutations involving residues in the 55-69 region have been identified. These include Xaa 3 Pro mutations (13,14), the introduction of a salt bridge (15), and the introduction of a disulfide bridge ...

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Section: Fig 4 Hydrolysis Of Protease-resistant ␣-Amylase Frommentioning

confidence: 53%

Engineering an enzyme to resist boiling

Burg

Vriend

Veltman

et al. 1998

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

264

143

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“…These roles are consistent with localization of tyrosine sulfate residues to very hydrophilic sites in proteins (10) and occurrence of this modification on secretory proteins that are exposed to the oxidizing extracellular environment. Chemical modification of surface-exposed tyrosine residues in proteins has demonstrated that significant stabilization of protein structure occurs when prosthetic groups are added to render tyrosine residues more hydrophilic (47).…”

Section: Discussionmentioning

confidence: 99%

Sulfation of tyrosine residues increases activity of the fourth component of complement.

Hortin

Farries

Graham

et al. 1989

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

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Sulfation of tyrosine residues recently has been recognized as a biosynthetic modification of many plasma proteins and other secretory proteins. Effects of this sitespecific modification on protein function are not known, but the activity of several peptides such as cholecystokinin is greatly augmented by sulfation. Here, we examine the role of sulfation in the processing and activity of C4 (the fourth component of complement), one of the few proteins in which sites and stoichiometry of tyrosine sulfation have been characterized.Our results, with C4 as a paradigm, suggest that sulfation of tyrosine residues can have major effects on the activity of proteins participating in protein-protein interactions. Sulfation of C4 synthesized by Hep G2 cells was blocked by incubating the cells with NaCIO3 and guaiacol. These sulfation inhibitors did not alter secretion or other steps in the processing of C4. However, hemolytic activity of C4 was decreased more than 50%. The inhibitors' effect on C4 activity was prevented by adding Na2SO4 to restore sulfation of C4. Activity of C3, a complement component homologous to C4 but lacking tyrosine sulfate residues, was minimally reduced (19%) by the inhibitors. Decreased hemolytic activity of nonsulfated C4 apparently resulted from impaired interaction with complement subcomponent Cli (EC 3.4.21.42), the protease that physiologically activates C4. Purified Cli was able to cleave nonsulfated C4, but 410-fold higher concentrations of Cli were required for that cleavage than to yield equivalent cleavage of sulfated C4. Our results suggest that activation of C4, a central component in the classical pathway of complement activation, is influenced by the level of sulfation of the protein. Thus, sulfation of C4 provides a potential locus for physiological or pharmacological modulation of complement-mediated opsonization and inflammation.

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“…Modification with cyclic anhydrides followed Mozhaev et al (1988). To 900 L of 0.15 mM horseradish peroxidase was added, with stirring, 100 L of 15 mM anhydride (phthalic, trimellitic, or pyromellitic) in DMSO (i.e., 100-fold molar excess of modifier over protein).…”

Section: Chemical Modificationsmentioning

confidence: 99%

“…Mozhaev et al (1988) reported dramatic increases in ␣-chymotrypsin's thermal stability following modification of the -amino groups of lysine residues with aromatic carboxylic acid anhydrides. Phthalic, trimellitic, and pyromellitic anhydrides introduced one, two, or three negatively charged carboxylic groups, respectively, to each Lys residue modified; stability increases were 2-to 3-fold with phthalic anhydride and >300-fold with the other two.…”

Section: Introductionmentioning

confidence: 97%

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

O'Brien

Smith

Ó’Fágáin

2002

Biotech & Bioengineering

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Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected.

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Protein stabilization via hydrophilization

Cited by 98 publications

References 48 publications

Engineering an enzyme to resist boiling

Engineering an enzyme to resist boiling

Sulfation of tyrosine residues increases activity of the fourth component of complement.

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

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