Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Monitoring Ca 2+ -Calmodulin Concentration 373
Fmax – F [CaM] tot
————— = —————— (1)
Fmax – Fmin [CaM] tot – Kd If Michaelis-Menten conditions do not apply, then the data must be fit to Eq. 2.
Fmax – F [I] tot – [CaM] tot – Kd – √([I] tot + [CaM] tot + Kd) 2 – 4[I] tot[CaM] tot
————— = —————————————————————————— (2)
Fmax – Fmin 1[I] tot
For example, FIP-CB SM–35 has a K d of 1 nM for Ca 2+ –CaM, but in our hands, 2 nM
is the minimum indicator concentration producing acceptable fluorescence data.
Thus, over most of the range of added CaM concentrations the bound and free
concentrations of the protein are quite different, so a quadratic is required to fit
the data (see Eq. 2). A drawback to using Eq. 2 is that it requires a precise knowledge
of the indicator concentration, so it is important to perform titrations at two
or three different indicator concentrations to ensure that consistent results are
obtained. In addition, we should emphasize that little useful information about
binding can be extracted from an essentially linear isotherm, such as would be
obtained if we were to titrate the response of FIP-CB SM–35 at a concentration of
100 nM. Binding data are fit directly to Eq. 1 or 2 using a standard nonlinear
least-squares analysis. F max and F min are fluorescence emission measurements
made at the acceptor (EYFP) emission maximum (approx 530 nm) when the indicator
is CaM-free and CaM-saturated, respectively. [I] tot and [CaM] tot are the
total concentrations of indicator and CaM (see Table 1).
3.2. Stable Expression of Indicators in Mammalian Cells
The procedures described here have been developed using HEK-293 cells, a
line derived from human embryonic kidney epithelium (ATCC #1573). To construct
mammalian expression vectors indicators, we simply excise the DNA
encoding it from the bacterial expression vector using BamHI and XhoI and
ligate the fragment into a pcDNA3 vector (Invitrogen, Inc., Carlsbad, CA)
cleaved with these enzymes. Expression of the cloned indicator in these vectors
is under control of a cytomegalovirus (CMV) promoter, and a nonfusion
protein is produced. The supplier provides variants of this vector that carry
selectable markers for zoecin, G418 or blasticidin. A vector map for the construct
used to express FIP-CB SM–35 is shown in Fig. 2.
3.2.1. Transfection and Selection to Produce HEK–293 Cells
Stably-Expressing CaM Indicators
LipofectAMINE ® (Life Technologies, Inc., Gaithersburg, MD) is used to introduce
vector DNA into HEK-293 cells essentially as described by the manufacturer.
1. Cells are plated at a density of 5 × 10 5 per 60-mm dish 2 d before transfection.
Cells should be 50–80% confluent at the time of transfection.