Correct protein function is critically dependent upon conformation - if a protein is not correctly folded, it will not execute its function or worse, it may be toxic. Circular dichroism spectroscopy (CD) is an exquisitely sensitive probe of protein conformation but is often perceived as a technique limited by its lack of productivity perhaps because, until recently, CD spectrometers have remained resolutely manual in operation.
This study describes a new automated CD technique for measuring protein conformation and how it changes with pH and, by extension, with other variables. The study confirms the robustness of the technique and, with unattended running of up to 200 samples per day, demonstrates very considerable improvements in productivity over manual CD measurements.
Method
Stock solutions bovine serum albumin (BSA), sodium phosphate and sodium citrate were used to prepare buffered samples from pH2.2 to pH8. The concentration of BSA was 1mg/ml; the samples were prepared directly in a 96-well plate and each protein solution was paired with its buffer.
Buffers and samples were transferred in turn by a fixed-probe, x, y, z linear-motion robot to a Chirascan-plus automated CD spectrometer. CD and absorption spectra were measured simultaneously and in duplicate for each.
The robot transferred 40µl of solution from the well to a 0.1mm pathlength flow-cell via an injection port. The CD and absorption spectra were measured before the sample was then removed from the cell and the cell washed and dried, ready for the next solution (Fig. 1). Buffers and samples were treated identically. Each fully automated measurement/wash cycle took 7.5 minutes.
Results
The short-wavelength limit is governed by increasingly high absorption at shorter wavelengths. The highest total absorption occurred at pH5, reaching 2AU at 188nm, defining the effective lower wavelength limit for the experiment.
There are two isosbestic points, one occurring at 206nm in the range pH2.2 to pH3.8 and the second occurring at 201nm in the range pH4.0 to pH8.0 (Fig. 2). This agrees with findings reported in the literature1 which suggest that BSA adopts three different conformations in the range of pH covered in this study: the N-form which occurs at physiological pH, the F-form which may also be physiologically active and the extended or E-form, which dominates at lower pH values.
The CD spectra of the buffers show no significant drift during the experiment, nor is there any evidence of cross-contamination, indicating that the inter-sample cleaning regime is very effective (Fig. 3).
Analysis
Simply plotting a contour map of the raw data would give an immediate, qualitative feel for the stability of the protein; a histogram of the ratio of positive to negative peak-heights would give a semi-quantitative picture and highlight conformational changes in a concentration-independent way. For quantitative results, however, some modelling of the data is required.
The presence of two isosbestic points indicates that a three-state model would be appropriate to describe the data. Global fitting of the model to the data yielded end-point spectra and the concentration profiles of the three species. The pKa values were calculated to be 2.34 for the E- and F-form and 4.44 for the F- and N-form equilibria respectively. The concentration profiles can be used to determine the onset of conformational change.
The conformational stability (contour) map for the three-state model as a function of pH is shown in Fig. 4. The region of conformational stability (pH>5.8) of the calculated spectra is well-defined, unambiguous and needs no expert interpretation.
Conclusion
A new automated method to measure and analyse the conformational stability of proteins has been described. The model protein BSA was used in this study but automated CD spectroscopy could be equally well applied to study the relative conformational stabilities of bio-therapeutic candidates in response to changing pH, ionic strength, ligand binding and other variables. The improvement in reproducibility, productivity and sample economy brought to CD by automation give it the qualities needed to help identify promising bio-therapeutic candidates at the pre-formulation phase of product development.
Enter √ at www.scientistlive.com/elab
David Gregson is Managing Director and Lindsay Cole is Senior Applications Scientist, Applied Photophysics Ltd, Leatherhead Surrey, UK. www.photophysics.com
References:
1. Carter D C and Ho J X, Structure of Serum Albumin pp153-203, Advances in Protein Chemistry, Vol.45, Lipoproteins, Apolipoproteins and Lipases, Academic Press 1994, Ed. Verne Schumaker. ISBN: 0-12-034245-6.
