Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10-50 degrees C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity

GATTI LANFRANCONI, P., Natalello, A., Rehm, S., Doglia, S., Pleiss, J., Lotti, M. (2010). Evolution of Stability in a Cold-Active Enzyme Elicits Specificity Relaxation and Highlights Substrate-Related Effects on Temperature Adaptation. JOURNAL OF MOLECULAR BIOLOGY, 395(1), 155-166 [10.1016/j.jmb.2009.10.026].

Evolution of Stability in a Cold-Active Enzyme Elicits Specificity Relaxation and Highlights Substrate-Related Effects on Temperature Adaptation

GATTI LANFRANCONI, PIETRO FRANCESCO;NATALELLO, ANTONINO;DOGLIA, SILVIA MARIA;LOTTI, MARINA
2010

Abstract

Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10-50 degrees C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity
Articolo in rivista - Articolo scientifico
circular dichroism; differential scanning fluorimetry; directed evolution; molecular dynamics; temperature adaptation;
English
2010
395
1
155
166
none
GATTI LANFRANCONI, P., Natalello, A., Rehm, S., Doglia, S., Pleiss, J., Lotti, M. (2010). Evolution of Stability in a Cold-Active Enzyme Elicits Specificity Relaxation and Highlights Substrate-Related Effects on Temperature Adaptation. JOURNAL OF MOLECULAR BIOLOGY, 395(1), 155-166 [10.1016/j.jmb.2009.10.026].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/9210
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