title: Protein Dynamics and its Correlation to Protein Activity and Stability

authors: Clement, David

subjects: Incoherent neutron scattering

abstract: The aim of this thesis is to investigate the role of fast protein dynamics (picosecond
timescale) in enzyme activity and stability, and specifically to test the
hypothesis that enzyme activity and stability are inversely related by their internal
dynamics.
Activity   Dynamics (flexibility)   1/Stability
In order to test this hypothesis, the well known anti-cancer drug: methotrexate
was used as an informative ligand in the network established between these
properties. A multidisciplinary approach combining neutron scattering, circular
dichroism, UV absorption, isothermal titration calorimetry and X-ray crystallography
was undertaken to examine the current paradigm using the enzyme: dihydrofolate
reductase as a model.
As inferred by neutron spectroscopy, the binding of MTX influences the
dynamical behavior of DHFR. Macromolecular dynamics such as the resilience: lt;kgt;
(i.e. structural rigidity) was found to be increased and, inversely, the flexibility
decreased upon MTX binding. In addition, as revealed by circular dichroism, this
dynamical dependency upon MTX binding was correlated with an enhanced thermal
stability. Compared to the free enzyme, the melting temperature was found to be
increased by 13.8  C in the presence of MTX. The inhibitory power of MTX was also
examined by steady state kinetics and isothermal titration calorimetry. The Ki for
MTX was found to be in the nanomolar range Ki= 10.9 nM. Using isothermal
titration calorimetry, the binding thermodynamic signature between MTX and DHFR
was characterized. The binding event was found to be largely favourable (DGb=-12.1
Kcal mol-1), enthalpy driven (DHb= -16.8 Kcal mol-1) with an unfavourable entropy
DSb=-15.6 cal K-1mol-1.
In conclusion, the modulation of the macromolecular dynamics may reflect
how specific conformations are favoured for subsequent protein function in response
of the binding of specific ligand and how conformational substates approach to
protein function. In this context the unprecedented power of transition state analogs
such as MTX on protein function might therefore be dependent on fast protein
dynamics.

text: Protein Dynamics and its Correlation to Protein Activity and Stability The aim of this thesis is to investigate the role of fast protein dynamics (picosecond
timescale) in enzyme activity and stability, and specifically to test the
hypothesis that enzyme activity and stability are inversely related by their internal
dynamics.
Activity   Dynamics (flexibility)   1/Stability
In order to test this hypothesis, the well known anti-cancer drug: methotrexate
was used as an informative ligand in the network established between these
properties. A multidisciplinary approach combining neutron scattering, circular
dichroism, UV absorption, isothermal titration calorimetry and X-ray crystallography
was undertaken to examine the current paradigm using the enzyme: dihydrofolate
reductase as a model.
As inferred by neutron spectroscopy, the binding of MTX influences the
dynamical behavior of DHFR. Macromolecular dynamics such as the resilience: lt;kgt;
(i.e. structural rigidity) was found to be increased and, inversely, the flexibility
decreased upon MTX binding. In addition, as revealed by circular dichroism, this
dynamical dependency upon MTX binding was correlated with an enhanced thermal
stability. Compared to the free enzyme, the melting temperature was found to be
increased by 13.8  C in the presence of MTX. The inhibitory power of MTX was also
examined by steady state kinetics and isothermal titration calorimetry. The Ki for
MTX was found to be in the nanomolar range Ki= 10.9 nM. Using isothermal
titration calorimetry, the binding thermodynamic signature between MTX and DHFR
was characterized. The binding event was found to be largely favourable (DGb=-12.1
Kcal mol-1), enthalpy driven (DHb= -16.8 Kcal mol-1) with an unfavourable entropy
DSb=-15.6 cal K-1mol-1.
In conclusion, the modulation of the macromolecular dynamics may reflect
how specific conformations are favoured for subsequent protein function in response
of the binding of specific ligand and how conformational substates approach to
protein function. In this context the unprecedented power of transition state analogs
such as MTX on protein function might therefore be dependent on fast protein
dynamics.

year: 2009

title: Low-dose alum application trialled as a management tool for internal nutrient loads in Lake Okaro, New Zealand

authors: Paul, Wendy J.

subjects: flocculation

abstract: Aluminium sulfate (alum) was applied to Lake Okaro, a eutrophic New Zealand lake with recurrent cyanobacterial blooms, to evaluate its suitability for reducing trophic status and bloom frequency. The dose yielded 0.6 g aluminium m–3 in the epilimnion. Before dosing, pH exceeded 8 in epilimnetic waters but was optimal for flocculation (6–8) below 4 m depth. After dosing, there was no significant change in water clarity, hypolimnetic pH decreased to 5.5, and soluble aluminium exceeded recommended guidelines for protection of freshwater organisms. Epilimnetic phosphate concentrations decreased from 40 to 5 mg m–3 and total nitrogen (TN):total phosphorus (TP) mass ratios increased from 7:1 to 37:1. The dominant phytoplankton species changed from Anabaena spp. before dosing, to Ceratium hirudinella , then Staurastrum sp. after dosing. Detection of effectiveness of dosing may have been limited by sampling duration and design, as well as the low alum dose. The decrease in hypolimnetic pH and epilimnetic TP, and increase in Al3+ and chlorophyll a, are attributed to the low alkalinity lake water and coincidence of alum dosing with a cyanobacterial bloom and high pH.

text: Low-dose alum application trialled as a management tool for internal nutrient loads in Lake Okaro, New Zealand Aluminium sulfate (alum) was applied to Lake Okaro, a eutrophic New Zealand lake with recurrent cyanobacterial blooms, to evaluate its suitability for reducing trophic status and bloom frequency. The dose yielded 0.6 g aluminium m–3 in the epilimnion. Before dosing, pH exceeded 8 in epilimnetic waters but was optimal for flocculation (6–8) below 4 m depth. After dosing, there was no significant change in water clarity, hypolimnetic pH decreased to 5.5, and soluble aluminium exceeded recommended guidelines for protection of freshwater organisms. Epilimnetic phosphate concentrations decreased from 40 to 5 mg m–3 and total nitrogen (TN):total phosphorus (TP) mass ratios increased from 7:1 to 37:1. The dominant phytoplankton species changed from Anabaena spp. before dosing, to Ceratium hirudinella , then Staurastrum sp. after dosing. Detection of effectiveness of dosing may have been limited by sampling duration and design, as well as the low alum dose. The decrease in hypolimnetic pH and epilimnetic TP, and increase in Al3+ and chlorophyll a, are attributed to the low alkalinity lake water and coincidence of alum dosing with a cyanobacterial bloom and high pH.

year: 2008

title: The Long Bay Restaurant site, R10/1374: Volume 1, the archaeology (HNZPTA authorities 2015/19 and 2016/81): Report to Heritage New Zealand Pouhere Taonga and Auckland Council

authors: Campbell, M

abstract: Auckland Council had proposed refurbishing the Long Bay Restaurant at Long Bay Regional Park (Lot 2 DP 54616), amounting to a near full rebuild of the structure apart from the in situ foundations. Council began the project in mid-2013 with the partial demolition of the existing structure and sawing of the concrete floor slab to install new utilities. Following the discovery of human remains beneath the floor slab on 2 July 2013, work was halted. The remains were inspected by the New Zealand Police who confirmed that they were pre-European kōiwi tangata. An assessment of the site was then undertaken by Beatrice Hudson of ArchOs Archaeology (Hudson 2013) and the find was recorded as site R10/1374 in the New Zealand Archaeological Association (NZAA) Site Recording Scheme (SRS) (this site number applies to any other archaeological features associated with the wider area). Auckland Council applied to the New Zealand Historic Places Trust (now Heritage New Zealand Pouhere Taonga, HNZPT) for an archaeological authority under section 18 of the Historic Places Act 1993 to undertake archaeological test excavations to investigate the nature, extent and condition of the site, and how it would be affected by the proposed development project. Authority 2014/506 was granted on 28 November 2013 and the investigation was carried out on the 20th and 21st of January 2014 (Campbell et al. 2014) (the 2014 excavation is summarised in Chapter 3).

Auckland Council then applied to HNZPT for an authority under section 44 of the Heritage New Zealand Pouhere Taonga Act 2014 to modify or destroy any further material found during the refurbishment project, including associated car parks and footpaths. Authority 2015/19 was granted on 7 August 2014 and works resumed. On 6 November 2014 further kōiwi were found within the project footprint and work once again ceased. 

Following extensive consultation with mana whenua and HNZPT, Auckland Council determined that the refurbishment would continue but it was agreed that the entire footprint of the development would be cleared archaeologically in order to provide assurance that no further kōiwi would remain within the project area. As the scope of the archaeological investigation had changed, a new archaeological authority, superseding authority 2015/19, was required and a full archaeological assessment of efects was also required in support of the authority application (Campbell and Hudson 2015). Authority 2016/81 was granted by HNZPT on 25 August 2015. This report fulfils the conditions of both authority 2015/19 and 2016/81.

Excavation began on 5 October 2015 with site clearance and removal of the concrete pad but not the ground beam foundations, while full excavation commenced on 12 October under the direction of Matthew Campbell (Excavation Director, CFG Heritage) and Beatrice Hudson (Osteologist, ArchOs Archaeology). Excavation continued until 3 November, by which stage kōiwi of a further 10 individuals had been found, bringing the total to 12. Following an onsite hui on 4 November between representatives of mana whenua, HNZPT, Auckland Council and the excavation team, the excavation was halted but mana whenua requested that the 3 possible burials that had been located but not excavated be lifted, and on 9 November the site was shut down. At a second on site hui on 9 December Auckland Council announced that they had resolved to cancel the restaurant project, but mana whenua expressed a desire that the 13 x 12 m excavation area opened up at the time be excavated to a sterile layer to lift all kōiwi. Excavation resumed on 18 January 2016 and continued until 4 March, when all kōiwi and archaeological deposits and features had been excavated.

text: The Long Bay Restaurant site, R10/1374: Volume 1, the archaeology (HNZPTA authorities 2015/19 and 2016/81): Report to Heritage New Zealand Pouhere Taonga and Auckland Council Auckland Council had proposed refurbishing the Long Bay Restaurant at Long Bay Regional Park (Lot 2 DP 54616), amounting to a near full rebuild of the structure apart from the in situ foundations. Council began the project in mid-2013 with the partial demolition of the existing structure and sawing of the concrete floor slab to install new utilities. Following the discovery of human remains beneath the floor slab on 2 July 2013, work was halted. The remains were inspected by the New Zealand Police who confirmed that they were pre-European kōiwi tangata. An assessment of the site was then undertaken by Beatrice Hudson of ArchOs Archaeology (Hudson 2013) and the find was recorded as site R10/1374 in the New Zealand Archaeological Association (NZAA) Site Recording Scheme (SRS) (this site number applies to any other archaeological features associated with the wider area). Auckland Council applied to the New Zealand Historic Places Trust (now Heritage New Zealand Pouhere Taonga, HNZPT) for an archaeological authority under section 18 of the Historic Places Act 1993 to undertake archaeological test excavations to investigate the nature, extent and condition of the site, and how it would be affected by the proposed development project. Authority 2014/506 was granted on 28 November 2013 and the investigation was carried out on the 20th and 21st of January 2014 (Campbell et al. 2014) (the 2014 excavation is summarised in Chapter 3).

Auckland Council then applied to HNZPT for an authority under section 44 of the Heritage New Zealand Pouhere Taonga Act 2014 to modify or destroy any further material found during the refurbishment project, including associated car parks and footpaths. Authority 2015/19 was granted on 7 August 2014 and works resumed. On 6 November 2014 further kōiwi were found within the project footprint and work once again ceased. 

Following extensive consultation with mana whenua and HNZPT, Auckland Council determined that the refurbishment would continue but it was agreed that the entire footprint of the development would be cleared archaeologically in order to provide assurance that no further kōiwi would remain within the project area. As the scope of the archaeological investigation had changed, a new archaeological authority, superseding authority 2015/19, was required and a full archaeological assessment of efects was also required in support of the authority application (Campbell and Hudson 2015). Authority 2016/81 was granted by HNZPT on 25 August 2015. This report fulfils the conditions of both authority 2015/19 and 2016/81.

Excavation began on 5 October 2015 with site clearance and removal of the concrete pad but not the ground beam foundations, while full excavation commenced on 12 October under the direction of Matthew Campbell (Excavation Director, CFG Heritage) and Beatrice Hudson (Osteologist, ArchOs Archaeology). Excavation continued until 3 November, by which stage kōiwi of a further 10 individuals had been found, bringing the total to 12. Following an onsite hui on 4 November between representatives of mana whenua, HNZPT, Auckland Council and the excavation team, the excavation was halted but mana whenua requested that the 3 possible burials that had been located but not excavated be lifted, and on 9 November the site was shut down. At a second on site hui on 9 December Auckland Council announced that they had resolved to cancel the restaurant project, but mana whenua expressed a desire that the 13 x 12 m excavation area opened up at the time be excavated to a sterile layer to lift all kōiwi. Excavation resumed on 18 January 2016 and continued until 4 March, when all kōiwi and archaeological deposits and features had been excavated.

year: 2019