The evolution of multiple active site configurations in a designed enzyme

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis

Artificial neural network analysis of thermally stimulated depolarisation currents in Sb2O3-WO3-Li2O-Na2O glasses

AbstractThe mixed alkaline effect (MAE) is a well-known anomaly in glasses. It results in a nonlinear response of various physical properties on mixing of lkali ions in the glass. In this work, the thermal depolarization currents (TSDC) were studied in antimony oxides based glasses 60Sb2O3-20WO3-(20-x)Li2O-xNa2O (in mol%) for x= 0, 5,10, 15 a 20. TSDC methods are standardly used for characterization of different types of polarization in solids

Single fluorescent protein-based Ca2+ sensors with increased dynamic range

<p>Abstract</p> <p>Background</p> <p>Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca²⁺sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).</p> <p>Results</p> <p>Here we report significant progress on the development of the latter type of Ca²⁺sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca²⁺concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca²⁺-free and Ca²⁺-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca²⁺response to a prolonged glutamate treatment in cortical neurons.</p> <p>Conclusion</p> <p>We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.</p