How Protein Stability and New Functions Trade Off

Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (ΔΔG), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (ΔΔG>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied ΔΔG computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to ΔΔG changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average ΔΔG = +0.9 kcal/mol), and are almost as destabilizing as the “average” mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non-adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently “silent” mutations in regions of the protein that are irrelevant to its function

Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor

Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs—reduced sensitivity to all hormones and increased selectivity for glucocorticoids—are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR–MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and reinforce the importance of permissive mutations in protein evolution

Engineered mononuclear variants in Bacillus cereus metallo-beta-lactamase BcII are inactive.

Metallo-beta-lactamases (MbetaLs) are zinc enzymes able to hydrolyze almost all beta-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of MbetaLs has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-beta-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms. Despite extensive studies, there is still controversy about the nature of the active BcII species. Here we have designed two mutant enzymes in which each one of the metal binding sites was selectively removed. Both mutants were almost inactive, despite preserving most of the structural features of each metal site. These results reveal that neither site isolated in the MbetaL scaffold is sufficient to render a fully active enzyme. This suggests that only the dinuclear species is active or that the mononuclear variants can be active only if aided by other residues that would be metal ligands in the dinuclear species

Evaluación del daño en el ADN y vigilancia biológica de la exposición laboral a solventes orgánicos, 2006

Introducción. La exposición a solventes es uno de los mayores riesgos potenciales para millones de trabajadores en el mundo; los solventes generan contaminación ambiental y desencadenan problemas de salud pública. Objetivo. Determinar los niveles de los metabolitos benceno, tolueno y xileno, los polimorfismos de las enzimas CYP2E1, GSTM1, GSTT1 y el daño del ADN mediante el ensayo del cometa. Materiales y métodos. Se llevó a cabo un estudio descriptivo de corte transversal para la determinación de polimorfismos genéticos y prueba del cometa en 90 trabajadores pertenecientes a cinco empresas. Se aplicó una encuesta, se tomaron muestras de sangre y de orina, se midieron las concentraciones de fenol, ácido hipúrico orto y meta-metilhipúrico. Se hizo el análisis estadístico y se exploraron posibles asociaciones. Resultados. El 34,4% eran trabajadores con exposición directa a solventes. En este grupo se evidenciaron concentraciones superiores a los límites permisibles en 3,3% para fenol, en 6,6% para ácido hipúrico, en 3,3% para ácido orto-metilhipúrico y en 36,7% para ácido meta-metilhipúrico, mayor longitud promedio de la cola del cometa (19,5 µm) y un incremento del porcentaje de células con daño medio (19,0%) (p=0,0007). El porcentaje de individuos expuestos con genotipos ausentes para las enzimas GSTT1 y GSTM1 fue de 46,7% y de 56,8%, respectivamente. Conclusión. El uso de biomarcadores de exposición, efecto y susceptibilidad, se ha convertido en una herramienta fundamental para la evaluación del riesgo asociado con la exposición a agentes tóxicos