Genetic Studies of the lac Repressor. XIV. Analysis of 4000 Altered Escherichia coli lac Repressors Reveals Essential and Non-essential Residues, as well as "Spacers" which do not Require a Specific Sequence

Markiewicz, Peter; Kleina, Lynn G.; Cruz, Chris; Ehret, S.; Miller, Jeffery H.

doi:10.1006/jmbi.1994.1458

Cited by 261 publications

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“…To compare impact scores with experimental data, we identified four mutagenesis studies (on Escherichia coli LacI (Markiewicz et al 1994;Suckow et al 1996), T4 Lysozyme (Rennell et al 1991), HIV Protease (Loeb et al 1989), and HIV Reverse Transcriptase (RT) (Wrobel et al 1995) in which a large number of single-substitution protein variants were assayed for their degree of functional impairment. For each of these studies, we obtained a multiple sequence alignment of homologs and calculated the impact score for each variant that was assayed experimentally.…”

Section: Mapp Methodologymentioning

confidence: 99%

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Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity

Stone¹,

Sidow²

2005

Genome Res.

358

319

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We find that the degree of impairment of protein function by missense variants is predictable by comparative sequence analysis alone. The applicable range of impairment is not confined to binary predictions that distinguish normal from deleterious variants, but extends continuously from mild to severe effects. The accuracy of predictions is strongly dependent on sequence variation and is highest when diverse orthologs are available. High predictive accuracy is achieved by quantification of the physicochemical characteristics in each position of the protein, based on observed evolutionary variation. The strong relationship between physicochemical characteristics of a missense variant and impairment of protein function extends to human disease. By using four diverse proteins for which sufficient comparative sequence data are available, we show that grades of disease, or likelihood of developing cancer, correlate strongly with physicochemical constraint violation by causative amino acid variants.[Supplemental material is available online at www.genome.org. A Java executable of MAPP and documentation are freely available for download at http://mendel.stanford.edu/supplementarydata/stone_MAPP_2005.]Missense mutations that impair protein function may result in disease. For diseases caused by such deleterious mutations, a simple but plausible model presents itself: The type of disease is dependent on when and where the protein's function is required in the organism. Given the type of disease, its severity is likely determined by at least three parameters: (1) the degree to which the function of the protein is impaired by the missense mutation; (2) variants of other genes that modulate the effect of the major locus, also referred to as genetic background; and (3) the environment. We present here a predictive statistical framework for the first of these parameters, impairment of protein function by missense mutations.Our study was motivated by several observations relevant to protein structure, function, and evolution, and by previous studies that addressed the relationship between the nature of missense variants, impairment of protein function, and resulting disease ( In aggregate, these studies suggest that mutations in evolutionarily conserved sites tend to impair protein function and lead to disease. There is also weak evidence that protein impairment and disease severity are somewhat correlated with the physicochemical difference between the original amino acid and the missense variant. However, no transparent, quantitative relationship between evolutionary constraint, functional impairment, and disease severity has been described. We believe that there has been a methodological barrier to uncovering such a relationship.Our analysis rests on two complementary ideas: (1) that differences in standard physicochemical properties between the "wild-type" amino acid and the missense variant are the root cause of functional impairment; and (2) that evolutionary variation among orthologs in the affected position is a sample ...

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Section: Mapp Methodologymentioning

confidence: 99%

“…We explored this hypothesis by restricting the analysis to a subset of positions involved in inducer binding (Markiewicz et al 1994;Lewis et al 1996). MAPP's analysis of the deep, paralogous alignment was substantially less accurate for this restricted set of positions than for the rest of the protein (Fig.…”

Section: Paralogous Vs Orthologous Prediction Accuracymentioning

confidence: 99%

Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity

Stone¹,

Sidow²

2005

Genome Res.

358

319

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“…More broadly, in larger and independent data sets, correlations between the Evolutionary Action and the fraction of dysfunctional mutants in vivo or the average loss of activity in vitro range from 0.73 to 0.96 ( Fig. 2B-E) in 4041 lac repressor mutations in E. coli assayed for their impact on b-galactosidase repression (Markiewicz et al 1994); 2015 lysozyme mutations in bacteriophage T4 assayed for plaque formation due to degradation of the host cell walls by lysozyme (Rennell et al 1991); 336 HIV-1 protease mutations assayed by the cleavage products (Loeb et al 1989); and 2314 TP53 mutants assayed for transactivation (see Methods) (Kato et al 2003). The Spearman's rank correlation coefficient is at least 0.98.…”

Section: The Evolutionary Action Correlates With Experimental Loss Ofmentioning

confidence: 97%

“…Mutational action correlates with experimental impact. Each panel shows along the x-axis the action predicted from Equation (2) and along the y-axis the fractional activity or fitness measured experimentally as (A) the average loss of recombination activity in 31 point mutants of E. coli RecA protein; (B) the nonfunctional fraction of 4041 point mutants in E. coli lac repressor in a b-galactosidase repression assay (Markiewicz et al 1994); (C ) the nonfunctional fraction of 2015 point mutants in bacteriophage T4 lysozyme in a plaque formation assay (Rennell et al 1991); (D) the nonfunctional fraction of 336 HIV-1 protease point mutants in substrate cleavage (Loeb et al 1989); and (E) the average transactivation activity of 2314 human TP53 point mutants assayed in yeast over eight response-elements (Petitjean et al 2007). The data are binned into action deciles, the R 2 values indicate Pearson product-moment correlation coefficients following linear fitting, and the standard error of the mean is shown with error bars.…”

Section: The Evolutionary Action Correlates With Severity In Inheritementioning

confidence: 99%

“…Mutants with no (À) and difficult to discern (À/+) plaque formation were considered as deleterious, while mutants with normal (+) and small plaque formation (+/À) were considered as neutral. The set of 4041 E. coli lac repressor mutations were assayed in Markiewicz et al (1994) by the protein's repression activity. Mutations with phenotypes less than 20-fold (À and À/+) repression activity were considered as deleterious, while mutants with more than 20-fold (+ and +/À) repression activity were considered as neutral.…”

Section: Experimental Data Setsmentioning

confidence: 99%

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A formal perturbation equation between genotype and phenotype determines the Evolutionary Action of protein-coding variations on fitness

Katsonis¹,

2014

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The relationship between genotype mutations and phenotype variations determines health in the short term and evolution over the long term, and it hinges on the action of mutations on fitness. A fundamental difficulty in determining this action, however, is that it depends on the unique context of each mutation, which is complex and often cryptic. As a result, the effect of most genome variations on molecular function and overall fitness remains unknown and stands apart from population genetics theories linking fitness effect to polymorphism frequency. Here, we hypothesize that evolution is a continuous and differentiable physical process coupling genotype to phenotype. This leads to a formal equation for the action of coding mutations on fitness that can be interpreted as a product of the evolutionary importance of the mutated site with the difference in amino acid similarity. Approximations for these terms are readily computable from phylogenetic sequence analysis, and we show mutational, clinical, and population genetic evidence that this action equation predicts the effect of point mutations in vivo and in vitro in diverse proteins, correlates disease-causing gene mutations with morbidity, and determines the frequency of human coding polymorphisms, respectively. Thus, elementary calculus and phylogenetics can be integrated into a perturbation analysis of the evolutionary relationship between genotype and phenotype that quantitatively links point mutations to function and fitness and that opens a new analytic framework for equations of biology. In practice, this work explicitly bridges molecular evolution with population genetics with applications from protein redesign to the clinical assessment of human genetic variations.

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Modelling repressor proteins docking to DNA

et al. 1998

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The docking of repressor proteins to DNA starting from the unbound protein and model-built DNA coordinates is modeled computationally. The approach was evaluated on eight repressor/DNA complexes that employed different modes for protein/ DNA recognition. The global search is based on a protein-protein docking algorithm that evaluates shape and electrostatic complementarity, which was modified to consider the importance of electrostatic features in DNA-protein recognition. Complexes were then ranked by an empirical score for the observed amino acid /nucleotide pairings (i.e., protein-DNA pair potentials) derived from a database of 20 protein/ DNA complexes. A good prediction had at least 65% of the correct contacts modeled. This approach was able to identify a good solution at rank four or better for three out of the eight complexes. Predicted complexes were filtered by a distance constraint based on experimental data defining the DNA footprint. This improved coverage to four out of eight complexes having a good model at rank four or better. The additional use of amino acid mutagenesis and phylogenetic data defining residues on the repressor resulted in between 2 and 27 models that would have to be examined to find a good solution for seven of the eight test systems. This study shows that starting with unbound coordinates one can predict three-dimensional models for protein/DNA complexes that do not involve gross conformational changes on association.

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Genetic Studies of the lac Repressor. XIV. Analysis of 4000 Altered Escherichia coli lac Repressors Reveals Essential and Non-essential Residues, as well as "Spacers" which do not Require a Specific Sequence

Cited by 261 publications

References 0 publications

Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity

Physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity

A formal perturbation equation between genotype and phenotype determines the Evolutionary Action of protein-coding variations on fitness

Modelling repressor proteins docking to DNA

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