Dr David Scott of the National Centre for Macromolecular Hydrodynamics, based at the University of Nottingham, has been visiting Birkbeck for a few days to give a course. He also gave a seminar in which he explained one of the biophysical techniques used in the Centre, analytical centrifugation, and how it is used to help determine something of the "structure" inherent in unstructured, or partially structured, proteins.
The analytical ultracentrifuge was invented by Theodor Svedberg in 1923; three years later, he won the Nobel Prize in Chemistry for research using it. It is simply a centrifuge that spins very fast (from about 1,000 - 60,000 rpm); the sample being spun is monitored optically over a period of time. The normal settling of particles in solution under gravity (sedimentation) is speeded up by spinning in a centrifuge, which essentially replaces the gravitational force by a centrifugal force. The speed of sedimentation depends on the masses and shapes of the particles involved. The maths is far too complex to be described here.
One of the first uses of ultracentrifugation was in the determination of molecular mass. Sedimentation times are measured in Svedberg units (S); 1S is exactly equivalent to 10-13 seconds. These times are related rather inexactly to molecular mass and often used to characterise large proteins and protein complexes; you have come across these in the PPS course in our discussion of ribosomal subunits. The small subunit of the Thermus thermophilus ribosome, illustrated there, is described as "30S".
Ultracentrifugation is now used routinely to determine whether a sample is homogeneous; if it is, all particles will have the same mass and shape, and therefore the same sedimentation time. A plot of sedimentation velocity for a sample can show whether the solution is homogenous or heterogenous, and whether protein is aggregated (in which case, aggregates will consist of different numbers of molecules and have different masses). Sedimentation equilibration experiments, which investigate the final steady state where sedimentation is balanced by diffusion, can be used to determine ligand binding and chemical reactions.
Scott has used ultracentrifugation and other biophysical techniques to investigate the structures of unstructured regions of a bacterial DNA-binding protein, KorB. This protein has a DNA-binding domain and a dimerisation domain each with a known structure, and it is known to interact with RNA polymerase as well as with DNA. Other parts of the protein, however, are only known to be "intrinsically unstructured". A combination of ultracentrifugation with other techniques useful for studying unstructured proteins, including circular dichroism and small angle X-ray scattering, were used to investigate the range of structures adopted by these unstructured regions. Results so far indicate that KorB forms a range of relatively compact structures when isolated in solution but that the unstructured regions extend when it binds to a related protein, KorA.
Analytical ultracentrifugation, CD spectroscopy and other biophysical techniques will be described in much more detail in the second year MSc module, Techniques in Structural Molecular Biology (TSMB).