A Multiwavelength Analytical Centrifuge platform for Solute Characterisation
Lead Research Organisation:
University of East Anglia
Department Name: Biological Sciences
Abstract
Sedimentation of samples by an analytical ultracentrifuge (AUC) is rightly described as the gold standard for the characterisation of a wide range of particles in solution, including proteins, oligonucleotides, polysaccharides, micelles and nanoparticles. By monitoring the migration of particles in an applied centrifugal field, information about the size, shape and distribution of samples can be directly obtained. This information can be used to identify the distribution of oligomeric states, characterise protein-ligand binding, and provide structural information about different complexes.
The Beckman XL-I/A AUC at the University of East Anglia (UEA) is now over 25 years old and many iterations behind current state of the art equipment. While it is still being used, data aquisition is slow and can only be reliably collected at a single wavelength. The latest Beckman Coulter Optima AUC allows for multiple wavelengths to be measured during the experiment, with faster measurements allowing for higher resolution data to be collected with greater accuracy. Access to this state of the art instrument would greatly improve the quality and scope of experiments that investigate the properties of proteins in solution.
Within the Norwich Research Park (NRP) and beyond, there are many research projects that are linked to BBSRC strategic goals and require information about the properties of samples in solution. These range from initial studies confirming the multimeric state of newly discovered proteins and protein complexes, to more detailed studies that investigate interactions between proteins and a different of ligands, including metal containing cofactors such as: hemes, iron-sulfur clusters and cobalalamin derivatives; organic cofactors such as flavin groups; ATP derivatives and caroteniods or oligonucleotides of DNA and RNA. By recording the sedimentation over time of a sample at different wavelengths a better understanding of protein-ligand interactions can be obtained. This information can include whether the ligan remains bound to the protein, the distribution of the ligand within different multimeric protein forms, or how the shape of the protein is affected by the presence of the ligand.
Data obtained by AUC can greatly accelerate the understanding of the impact of different ligands on different conformational states of proteins, and how proteins behave in different solutions. This information is essential for studies that investigate the molecular mechanism of proteins and how conformational state is linked to function. For advanced techniques such as CryoEM, this information can help identify relevant protein forms for detailed analysis, support the screening of drug ligands in stabilising discrete conformations of transporters, and provide a tool for the optimisation of biotechnologically important systems, where knowledge of the conformation and oligomeric state of a system is essential for fully defining it's biophysical properties.
Analytical Ultracentrifugation at the UEA has a proven track record of use by groups across the NRP and the wider scientific community to drive exceptional research, and has proved invaluable for the characterisation of protein systems. The proposed new AUC will open new avenues of research, while providing high quality experimental data and training experience for new investigators.
The Beckman XL-I/A AUC at the University of East Anglia (UEA) is now over 25 years old and many iterations behind current state of the art equipment. While it is still being used, data aquisition is slow and can only be reliably collected at a single wavelength. The latest Beckman Coulter Optima AUC allows for multiple wavelengths to be measured during the experiment, with faster measurements allowing for higher resolution data to be collected with greater accuracy. Access to this state of the art instrument would greatly improve the quality and scope of experiments that investigate the properties of proteins in solution.
Within the Norwich Research Park (NRP) and beyond, there are many research projects that are linked to BBSRC strategic goals and require information about the properties of samples in solution. These range from initial studies confirming the multimeric state of newly discovered proteins and protein complexes, to more detailed studies that investigate interactions between proteins and a different of ligands, including metal containing cofactors such as: hemes, iron-sulfur clusters and cobalalamin derivatives; organic cofactors such as flavin groups; ATP derivatives and caroteniods or oligonucleotides of DNA and RNA. By recording the sedimentation over time of a sample at different wavelengths a better understanding of protein-ligand interactions can be obtained. This information can include whether the ligan remains bound to the protein, the distribution of the ligand within different multimeric protein forms, or how the shape of the protein is affected by the presence of the ligand.
Data obtained by AUC can greatly accelerate the understanding of the impact of different ligands on different conformational states of proteins, and how proteins behave in different solutions. This information is essential for studies that investigate the molecular mechanism of proteins and how conformational state is linked to function. For advanced techniques such as CryoEM, this information can help identify relevant protein forms for detailed analysis, support the screening of drug ligands in stabilising discrete conformations of transporters, and provide a tool for the optimisation of biotechnologically important systems, where knowledge of the conformation and oligomeric state of a system is essential for fully defining it's biophysical properties.
Analytical Ultracentrifugation at the UEA has a proven track record of use by groups across the NRP and the wider scientific community to drive exceptional research, and has proved invaluable for the characterisation of protein systems. The proposed new AUC will open new avenues of research, while providing high quality experimental data and training experience for new investigators.
