Department of Biochemistry University of Oxford Department of Biochemistry
University of Oxford
South Parks Road
Oxford OX1 3QU

Tel: +44 (0)1865 613200
Fax: +44 (0)1865 613201
Anaphase bridges in fission yeast cells
Whitby lab
Lactose permease represented using bending cylinders in Bendix software
Caroline Dahl, Sansom lab
Epithelial cells in C. elegans showing a seam cell that failed to undergo cytokinesis
Serena Ding, Woollard lab
Collage of Drosophila third instar larva optic lobe
Lu Yang, Davis lab
First year Biochemistry students at a practical class
Image showing the global movement of lipids in a model planar membrane
Matthieu Chavent, Sansom lab
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Biophysical Instrument Facility available in New Biochemistry

The Biophysical Instrument Facility was set up in the New Biochemistry building at the beginning of 2009 as a central resource for the biophysical analysis of proteins and other macromolecules.

By bringing together a range of techniques we have established a central facility that provides full technical support for both new and expert users working in the departments of Oxford University and for those at other institutions. We are especially happy to discuss how we can help researchers to quantitate interactions and suggest techniques best suited to their needs.

New users are taken through the application of the instrument and help is on hand for the analysis of results. Introductory courses are run annually for post-graduates, and other researchers are welcomed to attend.

Among the most popular techniques available are electrospray ionisation (ESI) mass spectrometry, analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These techniques allow the characterisation of a macromolecule in terms of intact mass, solution behaviour and interactions with other moleclues.

ESI mass spectrometry is routinely used to determine molecular weights of intact recombinant proteins to within a Dalton accuracy, allowing sequence confirmation and the identification of any post-translational modifications such as disulphide bonds and phosphorylation (Fig. 1).

Figure 1. ESI mass spectrometry of recombinant protein from size exclusion chromatography (A) and the mass distribution from this spectrum (B). The measured mass is compared to the one predicted from the expected sequence and allows confirmation of the protein and the identification of any post-ttranslational modifications. For this protein the close agreement of the two values indicates no modifications.

Figure 1. ESI mass spectrometry of recombinant protein from size exclusion chromatography (A) and the mass distribution from this spectrum (B). The measured mass is compared to the one predicted from the expected sequence and allows confirmation of the protein and the identification of any post-ttranslational modifications. For this protein the close agreement of the two values indicates no modifications.

AUC characterises the solution state of a macromolecule and can determine its molecular weight and oligomeric state (Fig. 2).

Figure 2. Sedimentation velocity analytical ultracentrifugation. Tile stoichiometry of a protein complex formed by weak interactions can be difficult to determine as it may be too unstable to be characterised under normal laboratory conditions. By varying the molar ratio and by measuring the sedimentation coefficients of the constituents the stoichiometry can be determined by plotting the molar ratio against weight-average sedimentation coefficients. Size distribution profiles for each diUb titration are shown in (A) for the Ub binding protein (UBP) and K48-linked diUb and (B) for the UBP and K63-linked diUb. The total protein concentration was kept constant at 300 M and the diUb:UBP ratio was varied from 0.2 (black), 0.5 (blue), 1 (red), 1.5 (purple), 2.5 (magenta)to 4.0 (cyan). (C). Plots of the experimentally determined weight-average sedimentation coefficients against the molar ratio indicate in each case formation of a 1:1 complex at the given concentrations, Values for the K48-and K63-linked diUb titrations are plotted as blue triangles and black circles respectively.

Figure 2. Sedimentation velocity analytical ultracentrifugation. Tile stoichiometry of a protein complex formed by weak interactions can be difficult to determine as it may be too unstable to be characterised under normal laboratory conditions. By varying the molar ratio and by measuring the sedimentation coefficients of the constituents the stoichiometry can be determined by plotting the molar ratio against weight-average sedimentation coefficients. Size distribution profiles for each diUb titration are shown in (A) for the Ub binding protein (UBP) and K48-linked diUb and (B) for the UBP and K63-linked diUb. The total protein concentration was kept constant at 300 µM and the diUb:UBP ratio was varied from 0.2 (black), 0.5 (blue), 1 (red), 1.5 (purple), 2.5 (magenta)to 4.0 (cyan). (C). Plots of the experimentally determined weight-average sedimentation coefficients against the molar ratio indicate in each case formation of a 1:1 complex at the given concentrations, Values for the K48-and K63-linked diUb titrations are plotted as blue triangles and black circles respectively.

SPR and ITC are two powerful techniques that complement each other and can provide detailed information about both the kinetics and thermodynamics of macromolecular interactions (Figs. 3 and 4).

Figure 3. SPR sensograms for the interaction of a monoclonal antibody and its antigen were analysed on the Biacore T100 using both multi and single cycle kinetics. The antibody was immobilised on the sensor chip and various concentrations of antigen injected over the surface in multiple (A) or a single cycle (B). The sensograms from these injections are shown as coloured lines. Models of binding were fitted to this data (black lines) and the association rate (k<sub>a</sub>), dissociation rate (k<sub>d</sub>) and equilibrium dissociation constant (k<sub>D</sub>) were determined for these interactions.

Figure 3. SPR sensograms for the interaction of a monoclonal antibody and its antigen were analysed on the Biacore T100 using both multi and single cycle kinetics. The antibody was immobilised on the sensor chip and various concentrations of antigen injected over the surface in multiple (A) or a single cycle (B). The sensograms from these injections are shown as coloured lines. Models of binding were fitted to this data (black lines) and the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (kD) were determined for these interactions.

Figure 4. ITC Experiment following ligand binding to a protein. Top sections shows raw data from the iTC200 instrument and in the lower section the ITC isotherm of this data integrated and fitted to a one independent site model. The three variables stoichiometry (N), equilibrium binding constant (K) and enthalpy of interaction (ΔH) that best fit this data are determined, and the entropy of the interaction (ΔS) is derived from the free energy (ΔG=-RTlnk) and enthalpy (ΔG=ΔH-TΔS).

Figure 4. ITC Experiment following ligand binding to a protein. Top sections shows raw data from the iTC200 instrument and in the lower section the ITC isotherm of this data integrated and fitted to a one independent site model. The three variables stoichiometry (N), equilibrium binding constant (K) and enthalpy of interaction (ΔH) that best fit this data are determined, and the entropy of the interaction (ΔS) is derived from the free energy (ΔG=-RTlnk) and enthalpy (ΔG=ΔH-TΔS).

These are only a few of the techniques available. For a comprehensive list of instruments go to the Facility website:

www.bioch.ox.ac.uk/services/equipmentbooking

The Biophysics Facility is currently organised using a web-based booking system that is accessible from any computer in the University. New users can contact David Staunton (david.staunton@bioch.ox.ac.uk) to set up an account.

We also organise demonstrations of new applications and international workshops on biophysical techniques. Information about these are advertised in our newsletter that is published quarterly. The most recent edition (January 2010) can be downloaded here:

http://users.ox.ac.uk/~bioc0126/biophysical_instrument_facility_newsletter_1.pdf

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