title: Directed evolution and structural analysis of an OB-fold domain towards a specifc binding reagent

authors: Steemson, John Durand

subjects: OB-fold

abstract: Interactions between proteins are a central concept in biology, and understanding and manipulation of these interactions is key to advancing biological science. Research into antibodies as customised binding molecules provided the foundation for development of the field of protein “scaffolds” for molecular recognition, where functional residues are mounted on to a stable protein platform. Consequently, the immunoglobulin domain has been describes as “nature’s paradigm” for a scaffold, and has been widely researched to make engineered antibodies better tools for specific applications. However, limitations in their use have lead to a number of non-immunoglobulin domains to be investigated as customisable scaffolds, to replace or complement antibodies. To be considered a scaffold, a protein domain must show an evolutionarily conserved hydrophobic core in diverse functional contexts. The study presented here investigated the oligosaccharide/oligonucleotide-binding (OB) fold as scaffold, which is a 5-standed β-barrel seen in diverse organisms with no sequence conservation. The term “Obody” was coined to describe engineered OB-folds. This thesis examined a previously engineered Obody with affinity for lysozyme (KD = 40 μM) in complex with its ligand by x-ray crystallography (resolution 2.75 Å) which revealed the atomic details of binding. Affinity maturation for lysozyme was undertaken by phage display directed evolution. Gene libraries were constructed by combinatorial PCR incorporating site-specific randomised codons identified by examination of the structure in complex with lysozyme, or by random generation of point mutations by error-prone PCR. Overall a 100-fold improvement in affinity was achieved (KD = 600 nM). To investigate the structural basis of the affinity maturation, two further Obody-lysozyme complexes were solved by x-ray crystallography, one at a KD of 5 μM (resolution 1.96 Å), one at 600 nM (resolution 1.86 Å). Analysis of the structures revealed changes in individual residue arrangements, as well as rigid-body changes in the relative orientation of the Obody and lysozyme molecules in complex. Directed evolution of Obodies as protein binding reagents remains a challenge, but this study demonstrates their potential. The structures presented here will contribute invaluable insights for the future design of improved Obodies.

text: Directed evolution and structural analysis of an OB-fold domain towards a specifc binding reagent Interactions between proteins are a central concept in biology, and understanding and manipulation of these interactions is key to advancing biological science. Research into antibodies as customised binding molecules provided the foundation for development of the field of protein “scaffolds” for molecular recognition, where functional residues are mounted on to a stable protein platform. Consequently, the immunoglobulin domain has been describes as “nature’s paradigm” for a scaffold, and has been widely researched to make engineered antibodies better tools for specific applications. However, limitations in their use have lead to a number of non-immunoglobulin domains to be investigated as customisable scaffolds, to replace or complement antibodies. To be considered a scaffold, a protein domain must show an evolutionarily conserved hydrophobic core in diverse functional contexts. The study presented here investigated the oligosaccharide/oligonucleotide-binding (OB) fold as scaffold, which is a 5-standed β-barrel seen in diverse organisms with no sequence conservation. The term “Obody” was coined to describe engineered OB-folds. This thesis examined a previously engineered Obody with affinity for lysozyme (KD = 40 μM) in complex with its ligand by x-ray crystallography (resolution 2.75 Å) which revealed the atomic details of binding. Affinity maturation for lysozyme was undertaken by phage display directed evolution. Gene libraries were constructed by combinatorial PCR incorporating site-specific randomised codons identified by examination of the structure in complex with lysozyme, or by random generation of point mutations by error-prone PCR. Overall a 100-fold improvement in affinity was achieved (KD = 600 nM). To investigate the structural basis of the affinity maturation, two further Obody-lysozyme complexes were solved by x-ray crystallography, one at a KD of 5 μM (resolution 1.96 Å), one at 600 nM (resolution 1.86 Å). Analysis of the structures revealed changes in individual residue arrangements, as well as rigid-body changes in the relative orientation of the Obody and lysozyme molecules in complex. Directed evolution of Obodies as protein binding reagents remains a challenge, but this study demonstrates their potential. The structures presented here will contribute invaluable insights for the future design of improved Obodies.

year: 2011

title: Keeping our children safe around dogs: Guidelines for safe interaction between children and pet dogs - English

authors: Carter, Jennifer

subjects: dog safety tips

abstract: This information booklet for parents and caregivers will provide the best level of understanding of how to avoid pet dog attacks which can injure, maim and even kill children.    This research-based educational resource provides practical, valid and reliable guidelines for safe interaction between children and pet dogs in their homes or known to them. The content of this booklet has been drawn from ‘Stories with happy endings: Preventing pet dog attacks on children’, the 2007-2008 Summer Scholarship report prepared for the Child Accident Prevention Foundation of New Zealand (CAPFNZ). Obtain a copy of the report from http://hdl.handle.net/10289/768

text: Keeping our children safe around dogs: Guidelines for safe interaction between children and pet dogs - English This information booklet for parents and caregivers will provide the best level of understanding of how to avoid pet dog attacks which can injure, maim and even kill children.    This research-based educational resource provides practical, valid and reliable guidelines for safe interaction between children and pet dogs in their homes or known to them. The content of this booklet has been drawn from ‘Stories with happy endings: Preventing pet dog attacks on children’, the 2007-2008 Summer Scholarship report prepared for the Child Accident Prevention Foundation of New Zealand (CAPFNZ). Obtain a copy of the report from http://hdl.handle.net/10289/768

year: 2008

title: Māori data governance model

authors: Kukutai, Tahu

abstract: This report describes the Māori Data Governance Model that has been designed by Māori data experts for use across the Aotearoa New Zealand public service. Māori data is a taonga that requires culturally grounded models of protection and care. The Model provides guidance for the system-wide governance of Māori data, consistent with the Government’s responsibilities under te Tiriti o Waitangi. The Model is intended to assist all agencies to undertake Māori data governance in a way that is values-led, centred on Māori needs and priorities, and informed by research. This is important because existing government data processes and practices are failing to meet Māori informational needs.

The Vision, Tuia te korowai o Hine-Raraunga – Data for self-determination, enables iwi, hapū and Māori organisations, businesses and communities to pursue their own goals for cultural, social, economic and environmental wellbeing. Eight Data Pou or pillars define critical areas of data governance and specify the actions that should be undertaken to realise six desired outcomes. The Model does not cover every element of data governance; instead, it focuses on key priorities and actions, against which agencies can assess their level of data maturity. The Model explicitly recognises the need for changes to system leadership, policies and legal settings so that Māori can exercise authority over Māori data to reduce unethical data use and strengthen outcomes for individuals, whānau and communities. The report also identifies the need for strategic investment in a Mana Motuhake data system that sits outside of the public sector to ensure iwi and hapū sovereignty over iwi and hapū data.

text: Māori data governance model This report describes the Māori Data Governance Model that has been designed by Māori data experts for use across the Aotearoa New Zealand public service. Māori data is a taonga that requires culturally grounded models of protection and care. The Model provides guidance for the system-wide governance of Māori data, consistent with the Government’s responsibilities under te Tiriti o Waitangi. The Model is intended to assist all agencies to undertake Māori data governance in a way that is values-led, centred on Māori needs and priorities, and informed by research. This is important because existing government data processes and practices are failing to meet Māori informational needs.

The Vision, Tuia te korowai o Hine-Raraunga – Data for self-determination, enables iwi, hapū and Māori organisations, businesses and communities to pursue their own goals for cultural, social, economic and environmental wellbeing. Eight Data Pou or pillars define critical areas of data governance and specify the actions that should be undertaken to realise six desired outcomes. The Model does not cover every element of data governance; instead, it focuses on key priorities and actions, against which agencies can assess their level of data maturity. The Model explicitly recognises the need for changes to system leadership, policies and legal settings so that Māori can exercise authority over Māori data to reduce unethical data use and strengthen outcomes for individuals, whānau and communities. The report also identifies the need for strategic investment in a Mana Motuhake data system that sits outside of the public sector to ensure iwi and hapū sovereignty over iwi and hapū data.

year: 2023