28 chromatography • spectroscopy
Mapping protein stability using automated circular dichroism spectroscopy
David Gregson and Lindsay Cole describe a new automated circular dichroism technique for measuring protein conformation.
C
orrect 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.
Tis study describes a new automated CD technique for measuring protein conformation and how it changes with pH and, by extension, with other variables. Te 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.
Fig. 1. Buffer-subtracted CD and absorption spectra of BSA showing change with pH.
Method Stock solutions bovine serum albumin (BSA), sodium phosphate and sodium citrate were used to prepare buffered samples from pH2.2 to pH8. Te 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.
Te robot transferred 40µl of solution from the well to a 0.1mm pathlength flow-cell via an injection port. Te 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 Te short-wavelength limit is governed by increasingly high absorption at shorter wavelengths. Te highest total absorption occurred at pH5, reaching 2AU at 188nm, defining the effective lower
wavelength limit for the experiment. Tere 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). Tis 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.
Te 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
Fig. 2. CD spectra showing isosbestic points at 206nm (top) and 201nm (bottom).
www.scientistlive.com
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36