title: The Effect of Hydration on Enzyme Activity and Dynamics

authors: Lopez, Murielle

subjects: protein hydration

abstract: Water has long been assumed to be essential for biological function. To understand the molecular basis of the role of water in protein function, several studies have established a correlation between enzyme activity and hydration level. While a threshold of hydration of 0.2 h (grams of water per gram of dried protein) is usually accepted for the onset of enzyme activity, recent works show that enzyme activity is possible at water contents as low as 0.03 h (Lind et al., 2004). Diffusion limitation in these experiments was avoided by monitoring enzyme-catalyzed hydrolysis of gas-phase esters. However, since water is also a substrate for the enzyme used in these experiments, they cannot be used to probe the possibility of activity at zero hydration. However, the pig liver esterase and C. rugosa lipase B are able to catalyse alcoholysis reactions in which an acyl group is transferred between an ester and an alcohol. Therefore, by following this reaction and using a gas phase catalytic system, we have been able to show that activity can occur at 0 g/g. These results led to the question of the accuracy of determinations of very low water concentrations; i.e., how dry is 0 g/g? Although gravimetric measurements of the hydration level do not allow us to define the anhydrous state of the protein with sufficient sensitivity, using 18O-labeled water, we have been able to quantify the small number of water molecules bound to the protein after drying, using a modification of the method of Dolman et al. (1997). Testing different drying methods, we have been able to determine a level of hydration as low as 2 moles of water per mole of protein (equivalent to 0.0006 h in the case of pig liver esterase) and have shown that in the case of the pig liver esterase, activity can occur at this hydration level. 
At the molecular level, if the hydration level affects activity, we can expect an effect on the protein dynamics. Neutron scattering spectra of hydrated powders, for instance, show that diffusive motions of the protein increase with the hydration (Kurkal et al., 2005) To address the question of the protein motions involved in the onset of enzyme activity at low hydration, we performed neutron scattering experiments on a pico-second time scale on dried powders. Preliminary results show a dynamical transition at hydration levels as low as 3 h. Molecular dynamic simulations have also been used in this study to access the dynamics of the active site.
Overall, the results here show that pig liver esterase can function at zero hydration, or as close to zero hydration as current methods allow us to determine. Since the experimental methodology restricts this work to a small number of enzymes, it is unlikely that it will ever be possible to determine if all enzymes can function in the anhydrous state: however, the results here indicate that water is not an obligatory requirement for enzyme function.

text: The Effect of Hydration on Enzyme Activity and Dynamics Water has long been assumed to be essential for biological function. To understand the molecular basis of the role of water in protein function, several studies have established a correlation between enzyme activity and hydration level. While a threshold of hydration of 0.2 h (grams of water per gram of dried protein) is usually accepted for the onset of enzyme activity, recent works show that enzyme activity is possible at water contents as low as 0.03 h (Lind et al., 2004). Diffusion limitation in these experiments was avoided by monitoring enzyme-catalyzed hydrolysis of gas-phase esters. However, since water is also a substrate for the enzyme used in these experiments, they cannot be used to probe the possibility of activity at zero hydration. However, the pig liver esterase and C. rugosa lipase B are able to catalyse alcoholysis reactions in which an acyl group is transferred between an ester and an alcohol. Therefore, by following this reaction and using a gas phase catalytic system, we have been able to show that activity can occur at 0 g/g. These results led to the question of the accuracy of determinations of very low water concentrations; i.e., how dry is 0 g/g? Although gravimetric measurements of the hydration level do not allow us to define the anhydrous state of the protein with sufficient sensitivity, using 18O-labeled water, we have been able to quantify the small number of water molecules bound to the protein after drying, using a modification of the method of Dolman et al. (1997). Testing different drying methods, we have been able to determine a level of hydration as low as 2 moles of water per mole of protein (equivalent to 0.0006 h in the case of pig liver esterase) and have shown that in the case of the pig liver esterase, activity can occur at this hydration level. 
At the molecular level, if the hydration level affects activity, we can expect an effect on the protein dynamics. Neutron scattering spectra of hydrated powders, for instance, show that diffusive motions of the protein increase with the hydration (Kurkal et al., 2005) To address the question of the protein motions involved in the onset of enzyme activity at low hydration, we performed neutron scattering experiments on a pico-second time scale on dried powders. Preliminary results show a dynamical transition at hydration levels as low as 3 h. Molecular dynamic simulations have also been used in this study to access the dynamics of the active site.
Overall, the results here show that pig liver esterase can function at zero hydration, or as close to zero hydration as current methods allow us to determine. Since the experimental methodology restricts this work to a small number of enzymes, it is unlikely that it will ever be possible to determine if all enzymes can function in the anhydrous state: however, the results here indicate that water is not an obligatory requirement for enzyme function.

year: 2008

title: Fresh moves dance festival

authors: Cheesman, Sue

abstract: Fresh Moves Dance Festival is an independent local initiative held in the Bay of Plenty region of New Zealand. In 2021 Fresh Moves reached a 20-year milestone. Part of a larger research inquiry which chronicles the festival’s journey through the years and seeks to understand how such an initiative has been sustained over time, this article presents a snapshot of the initiative. This snapshot describes some of the history, successes, challenges and plans for future development of this initiative. As a dance educator, observer and member of the expert panel of Fresh Moves Dance Festival, I find the festival provides much food for thought about our own practice and community connections through dance education.

text: Fresh moves dance festival Fresh Moves Dance Festival is an independent local initiative held in the Bay of Plenty region of New Zealand. In 2021 Fresh Moves reached a 20-year milestone. Part of a larger research inquiry which chronicles the festival’s journey through the years and seeks to understand how such an initiative has been sustained over time, this article presents a snapshot of the initiative. This snapshot describes some of the history, successes, challenges and plans for future development of this initiative. As a dance educator, observer and member of the expert panel of Fresh Moves Dance Festival, I find the festival provides much food for thought about our own practice and community connections through dance education.

year: 2022-08-03

title: Te tātari i te kaupapa

authors: Ka'ai, Tānia M.

abstract: The primary objective of this thesis focuses on the linking of the National Qualifications Framework and standards-based assessment as an emancipatory mechanism for Māori in Kaupapa Māori schooling and the wider Māori community in New Zealand.

Argued from an eclectic theoretical approach incorporating Kaupapa Māori theory, it espouses a position which rejects structural inequalities that perpetuate the subordinate positioning of Māori people through the development of culturally responsive assessment theories and practices in written language for teachers and children in Kura Kaupapa Māori which purport a culturally responsive pedagogy.

The thesis also links curriculum, assessment and pedagogy using Kaupapa Māori theory which provides insights into Māori views of emancipation in education.

text: Te tātari i te kaupapa The primary objective of this thesis focuses on the linking of the National Qualifications Framework and standards-based assessment as an emancipatory mechanism for Māori in Kaupapa Māori schooling and the wider Māori community in New Zealand.

Argued from an eclectic theoretical approach incorporating Kaupapa Māori theory, it espouses a position which rejects structural inequalities that perpetuate the subordinate positioning of Māori people through the development of culturally responsive assessment theories and practices in written language for teachers and children in Kura Kaupapa Māori which purport a culturally responsive pedagogy.

The thesis also links curriculum, assessment and pedagogy using Kaupapa Māori theory which provides insights into Māori views of emancipation in education.

year: 1995