Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

52 Martin and Bayley
discussed by Manning (19). One of the most important points to remember (17)
is that, with the exception of nonconstrained least squares analysis, all the methods
of analysis require a precise knowledge of protein concentration (see Subheading
3.2.).
6. When working with mutant proteins, it is essential to examine the effect of the
mutation on the overall conformation and stability of the protein. CD provides a
convenient means of doing this with limited amounts of material. Differences
observed in the far-UV spectra are generally an indication that the mutation has
produced a significant change in the secondary structure (see Note 13). However,
differences observed in the near-UV region may derive from subtle changes in
the environment of particular aromatic residues that are not necessarily associated
with any major structural change.
4. Notes
1. d10-CSA has a second CD band at 192.5 nm (∆εM,192.5 =–4.72 M –1cm –1 ). The
far-UV performance of a CD instrument can, therefore, be checked by recording
the spectrum of d10-CSA (approx 5 mM) using a 1-mm path length cuvet. If the
intensity ratio of the two peaks (–Signal[192.5]/Signal[290.5]) is significantly
less than 1.95, then the machine is not performing correctly. This spectrum also
provides a useful check on the wavelength calibration of the instrument.
2. This is particularly important if the solution contains any unusual components.
For example, dithiothreitol (DTT) (oxidized), phenyl methyl sulfonyl fluoride
(PMSF), and high concentrations of common chelators will distort the absorption
spectrum if not accounted for.
3. A more elaborate method, easily implemented in a spreadsheet program, is to
plot Ln(Aλ) against Ln(λ) and perform a least-squares fit to the straight line. This
has the advantage that significant deviations from linearity may indicate the presence
of contaminants rather than light scattering, and the actual value of the wavelength
exponent (n) can be calculated.
4. Pace et al. (8) have shown that ε280nm can be predicted using
ε280nm (M –1cm –1 ) = (#Trp)(5500) + (#Tyr)(1490) + (#cystine)(125)
This equation works best for proteins that contain tryptophan. For proteins that
lack both Trp and Tyr one may use (with appropriate caution)
ε257.5nm (M –1cm –1 ) = (#Phe)(195) + (#cystine)(295)
5. The majority of simple buffer components will permit far-UV CD measurements
to below 200 nm. However, high concentrations of chloride and (especially)
nitrate (use perchlorate if possible), certain solvents (dioxane, dimethyl sulfoxide
[DMSO]), high concentrations (>25 mM) of some biological buffers (HEPES,
PIPES, Mes), and high concentrations (>1 mM) of chelators (ethylene glycol-bis
N,N,N',N'-tetraacetic acid [EGTA]/ethylenediaminetetracetic acid [EDTA])
should be avoided. It is also worth noting that distilled water stored in a polyeth-