Step-wise Mutation of Barnase to Binase

Serrano, Luís; Day, Anthony G.; Fersht, Alan R.

doi:10.1006/jmbi.1993.1508

Cited by 166 publications

(69 citation statements)

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“…The mutations L25A and D46N each destabilized FF by 2.5-3 kcal⅐mol Ϫ1 . As ⌬G D-N at 298 K for wild-type FF domain is 2.8-3 kcal⅐mol Ϫ1 , the native form of the double mutant L25A͞D46N should not be populated to more than Ϸ1-2% as energetics are typically additive for noninteracting residues (28,29).…”

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confidence: 99%

Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation

Jemth

Gianni

Day

et al. 2004

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

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102

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confidence: 99%

Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation

Jemth

Gianni

Day

et al. 2004

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

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102

142

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“…This strategy has been developed from earlier studies of the effects of conversion of barnase to its homologue binase, which differs in only 17 positions in the sequence. It was found that six of the sequence differences in binase were stabilizing when substituted into barnase and that the effects of the individual substitution on stability were additive (15). Further, the thermostable mutant was fully active.…”

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confidence: 99%

“…Molecular evolution data had confirmed that Ϸ84% of missense mutation target codons are evolutionary conserved in all p53 proteins and are mapped to the core domain, where the mutational hot spots for human cancer are located (p53 database, http:͞͞ www.iarc.fr). Because conserved residues in the sequence alignment most likely reflect biological selection for function, the risk of introducing changes that would be detrimental to the function by substituting residues in the human p53 protein with the most prevalent evolutionary conserved residue at a specific position should be minimal (15,16,18). Thus, we also may find more stable variants.…”

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confidence: 99%

Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability

Nikolova

Henckel

Lane

et al. 1998

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

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We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from ؉1.27 (less stable Q165K) to ؊1.49 (more stable N239Y) kcal mol ؊1 , respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancerassociated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN-268D), which is stabilized by 2.65 kcal mol ؊1 and T m raised by 5.6°C is of potential interest for trials in vivo.

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“…Serendipity plays a major role (Imanaka et al, 1986;Pantoliano et al, 1989;Mrabet et al, 1992;Hardy et al, 1993), even though the probability of finding a stabilizing mutation by chance is small and methods have to be devised to reduce the search space (Serrano et al, 1993;Heringa et al, 1995). a-Helices may respond favorably to the stabilization of the helix dipole (Nicholson et al, 1988;Goodenough et al, 1991;Eijsink et al, 1992;Walter et al, 1995) or to the provision of N-cap structural elements Reprint requests to: Boris Steipe, Genzentrum, Feodor-Lynen-Str.…”

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β‐Turn propensities as paradigms for the analysis of structural motifs to engineer protein stability

et al. 1997

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The thermodynamic stability of a protein provides an experimental metric for the relationship of protein sequence and native structure. We have investigated an approach based on an analysis of the structural database for stability engineering of an immunoglobulin variable domain. The most frequently occurring residues in specific positions of p-turn motifs were predicted to increase the folding stability of mutants that were constructed by site-directed mutagenesis. Even in positions in which different residues are conserved in immunoglobulin sequences, the predictions were confirmed. Frequently, mutants with increased p-turn propensities display increased folding cooperativities, suggesting pronounced effects on the unfolded state independent of the expected effect on conformational entropy. We conclude that structural motifs with predominantly local interactions can serve as templates with which patterns of sequence preferences can be extracted from the database of protein structures. Such preferences can predict the stability effects of mutations for protein engineering and design.

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Step-wise Mutation of Barnase to Binase

Cited by 166 publications

References 0 publications

Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation

Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation

Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability

β‐Turn propensities as paradigms for the analysis of structural motifs to engineer protein stability

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