Quaternary Structure and Stability
The quaternary structure of macromolecules is stabilized by the same non-covalent interactions and disulfide bonds as the tertiary structure, and can also be affected by formulation conditions. A number of chromatographic, hydrodynamic, electrophoretic and electron microscopic methods for the characterization of the quaternary structure of a macromolecule are available at our center, and several of them have been adapted to high-throughput formats. Chief among these are size exclusion chromatography coupled with MALLS detector, multiple dynamic light scattering instruments (DLS), agarose and polyacrylamide gel electrophoresis, electron microscopy (SEM, TEM and cryo) as well as analytical ultracentrifuge (AUC) in both its velocity and equilibrium modes. A number of methods span the nanometer to micron size range and can be used in the context of monitoring aggregation as well.
- Wyatt Dynapro DLS system
- Brookhaven DLS system
- Analytical ultracentrifuge (Beckman ProteomeLab XL-10)
- SEM and TEM in KU Electron Microscopy facility
Size Exclusion Chromatography coupled with MALS Detector
Because of its ease of use and high sample throughput, size exclusion high performance liquid chromatography (SEC) is the most commonly used technique for sizing and quantifying soluble protein aggregates in the molecular weight range of 5-1000 kDa. We have two Shimadzu® HPLC systems that are used exclusively for SE-HPLC. One of the HPLC systems is connected to a Wyatt Dawn Heleos II MALS detector. The Wyatt system is an 18-angle light scattering detector for the measurement of absolute molecular weight, size, and conformation of macromolecules in solution.
We have access to the KU Microscopy Center, which has both scanning and transmission electron microscopes with energy-dispersive X-ray analysis (EDX) capabilities, as well as cryo-EM.
Agarose (AGE) and Polyacrylamide Gel Electrophoresis (PAGE)
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids that differ in size, charge or conformation. We have in our center both AGE and PAGE capabilities for protein and nucleic acid detection, separation, and quantitation.
Dynamic Light Scattering (DLS)
The fluctuations of light intensity scattered from particles undergoing Brownian motion is the basis for DLS. To calculate the hydrodynamic diameter, an autocorrelation function yields a diffusion constant based on the Brownian motion of the particles. Our center is equipped with a Wyatt Dynapro DLS system that performs DLS in sample volumes as small as 30 µl in 96- or 384-well plates, as well as two Brookhaven DLS systems that can also measure the Zeta potential. A variety of different data analysis methods are available, ranging from cumulant analysis to various deconvolution methods for multicomponent resolution.
Analytical Ultracentrifugation (AUC)
While DLS determines the effective diameter of any particle that scatters light, analytical ultracentrifugation offers the benefit of limiting the measurement to particles that absorb light at a specific wavelength (e.g. 260 nm for DNA, 280 nm for proteins). Instead of calculating the effective hydrodynamic diameter, AUC measures the particle sedimentation coefficient or the molecular weight (including that of associating systems) using equilibrium sedimentation. Our center has a Beckman ProteomeLab XL-1 analytical ultracentrifuge equipped with integrated Rayleigh Interference and Scanning UV/VISIBLE detection systems.
He, F., Joshi, S.B., Moore, D.S., Shinogle, H.E. and Middaugh, C.R. (2009) Using spectroscopic and microscopic methods to probe the structural stability of human adenovirus type 4. Human Vaccines 6(2), 202-211
PMID 19946219 http://www.ncbi.nlm.nih.gov/pubmed?term=19946219
Zheng, Y., Ramsey, J.D., King, R., Leviten, M., McGuire, R., Volkin, D.B., Joshi, S.B. and Middaugh, C.R. (2010) Identifying stabilizers of plasmid DNA for pharmaceutical use. J. Pharm. Sci. 100(3), 904-914 PMID 20740679 http://www.ncbi.nlm.nih.gov/pubmed/20740679