Fluorescence Optics for the Analytical Ultracentrifuge
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Biosciences
Abstract
1) Purchase and commission XL-A upgrade to fluorescence optics. 2) Carry out determinations of protein/DNA dissociation constants. 3) Using tracer methodologies, understand better the effect of crowded environments upon macromolecular associations. 4) Use the equipment to derive experimental data which can be described by the new and emerging description of non-ideality described in the proposal.
Technical Summary
The analytical ultracentrifuge can be used to determine both sedimentation coefficients and absolute molecular weights. It is a primary technique, requiring no calibration standards as is the case with gel filtration, and is thermodynamically rigorous; theoretically it is based directly on the second law of thermodynamics. New instrumentation in the early 1990's has lead to a renaissance in the technique with many new theoretical and methodological studies emerging every year. One limitation with the method, however, is that the optical system relies on either absorbance of light or refractive index increment (Rayleigh interference). This limits the minimum concentration to around 10 ug/ml. Practically, this means that dissociation constants can only be determined to around >200 nM. With fluorescence optics we will be able to probe concentrations down to 20-30 pM, and dissociation constants in the range >100 pM. In addition, fluorescently labelled macromolecules can be used to probe the effects of very high co-solute concentrations and therefore be a probe for how a particular interaction is affected by a crowded environment. Such non-ideal interactions can be analysed by an emerging methodology developed in the NCMH with our collaborators.
Publications
Wills PR
(2015)
The osmotic second virial coefficient for protein self-interaction: Use and misuse to describe thermodynamic nonideality.
in Analytical biochemistry
Scott DJ
(2015)
Evaluation of diffusion coefficients by means of an approximate steady-state condition in sedimentation velocity distributions.
in Analytical biochemistry
Wills PR
(2012)
Allowance for effects of thermodynamic nonideality in sedimentation equilibrium distributions reflecting protein dimerization.
in Analytical biochemistry
Scott DJ
(2008)
The shock of the old: hydrodynamics for the masses.
in Biochemical Society transactions
Scott DJ
(2010)
Allowance for the effect of protein charge in the characterization of nonideal solute self-association by sedimentation equilibrium.
in Biophysical chemistry
Scott DJ
(2009)
Comparison of methods for characterizing nonideal solute self-association by sedimentation equilibrium.
in Biophysical journal
Patel TR
(2016)
Analytical ultracentrifugation: A versatile tool for the characterisation of macromolecular complexes in solution.
in Methods (San Diego, Calif.)
Scott DJ
(2015)
Characterization of Intrinsically Disordered Proteins by Analytical Ultracentrifugation.
in Methods in enzymology
Scott DJ
(2012)
Sedimentation velocity of intrinsically disordered proteins: what information can we actually obtain?
in Molecular bioSystems
Machón C
(2010)
RepD-mediated recruitment of PcrA helicase at the Staphylococcus aureus pC221 plasmid replication origin, oriD.
in Nucleic acids research