Six sites likely to report on the calcium-induced conformational change were selected based on the known apo and holo structures of calmodulin (). Two sites were selected in the N-terminal lobe, and four in the structurally homologous C-terminal lobe. The nitroxide spin probe R1 was introduced at each site by means of site-directed spin labeling (). The effect of calcium binding on the EPR lineshape at each site was determined by acquiring room-temperature spectra at X-band in the presence and absence of calcium (). Two mutants showed substantive lineshape changes upon calcium binding: the lineshape of I100R1 changed from highly mobile in the absence of calcium to immobile in its presence, whereas the reverse was observed for the lineshape of M109R1. The lineshape of I27R1, the homologous mutant to I100R1 in the N-terminal lobe, showed a similar transition from mobile to immobile as its counterpart, albeit with less dynamic range. The remaining three mutants (V35R1, L105R1, M145R1) showed only discrete lineshape changes upon calcium binding and were not further studied.
Figure 1 Site-directed spin labeling of calmodulin. (A) Labeled sites in calmodulin. The structure of calmodulin is shown as a grey ribbon, the labeled sites are black spheres, and calcium ions are grey spheres. (B) Reaction of the methanethiosulfonate reagent (more ...)
Figure 2 Calcium induced lineshape changes of spin-labeled calmodulin. A single domain of calcium-bound calmodulin is depicted as a ribbon structure with the side chain of the mutated residue shown as CPK models (arrows). Two bound calcium ions are depicted as (more ...)
The most sensitive mutant (I100R1) and its structural homologue in the N-terminal lobe (I27R1) were selected for further study (). In a series of titration experiments a constant concentration of calmodulin (100 µM) was mixed with different concentrations of calcium (0–100 mM). show the observed lineshapes for these two mutants as a function of increasing calcium concentration. As the calcium concentration is raised, a steady transition from the apo lineshape to the holo lineshape is observed in both mutants. Indeed, at each concentration of calcium, the lineshape can be described as a linear combination of the apo and holo lineshapes, indicating that calcium is binding to an increasing fraction of calmodulin with increasing concentration (data not shown). For further analysis of the structural transition, it is convenient to define a parameter based on the lineshape. The ratio between two prominent lineshape features (Aim/Ap-p, see inset of ) is plotted as a function of total calcium in . These graphs demonstrate the conformational change of calmodulin as sampled locally by the two structurally homologous spin probes. It is immediately apparent that the two mutants react quite differently to increasing calcium. This difference is due to the different calcium affinities of the two sites. shows the conformational change as a function of the estimated free calcium concentration in solution. The data demonstrate that the local calcium affinity of the N-terminal site is about one order of magnitude higher than that of the C-terminal site.
Figure 3 Titration of the I27R1 and I100R1 mutants with calcium. (A, B) Continuous wave EPR spectra of I27R1 (A) and I100R1 (B) in response to increasing concentrations of calcium. (C, D) Plot of the ratio of the lineshape features Aim (immobilized) and Ap-p (peak-to-peak) (more ...)
The local nature of the structural transition reported by calmodulin spin labels extends to the level of the individual EF-hand. demonstrates how the I100R1 probe reacts to the destruction of the two calcium binding sites located within the same lobe as the probe itself (). The mutant D93A removes a coordinating carboxyl group of the calcium binding site located in the same EF-hand as the I100R1 probe, whereas the D129A mutant does the same for the remote EF-hand of the lobe. The effect of these two mutants on the calcium-dependent signal of I100R1 is very different (). The binding affinity was only slightly reduced by the D129A mutant (circles), suggesting little cooperativity between the two calcium binding sites, whereas the destruction of the local calcium binding site had a dramatic impact on the calcium-dependence of the spectrum (squares). The immobilization of the I100R1 sidechain thus depends primarily on calcium binding to the site located in the same EF-hand as the probe. Indeed, a simple model with four independent calcium binding sites and strictly local immobilization fits the titration data of quite well (dotted lines). The data thus underscore the local nature of the conformational change reported by I100R1.
Figure 4 Spin-labeled calmodulin reports on local structural changes in proximity of the probe. (A) Close-up view of the two EF-hand domains in the C-terminal lobe of calmodulin (grey ribbons) with side chains draw in black and calcium ions shown as grey spheres. (more ...)
Having characterized the calcium-dependent properties of I100R1, we next used this mutant as a calcium sensor to demonstrate calcium release through photolysis of DM-nitrophen, a light-sensitive calcium chelator (, inset
). In absence of light, DM-nitrophen binds calcium with high affinity (Kd ~5 nM) (6
). In an initial test experiment, photolysis of DM-nitrophen was performed outside the EPR cavity, i.e. the sample was measured inside the cavity, illuminated with UV light outside the cavity, and then re-measured (). The concentrations of DM-nitrophen, calmodulin, and calcium were chosen so that the majority of calcium was bound to the chelator, thus generating a lineshape of the mixture before illumination that was essentially the same as in the complete absence of calcium (, left
). After a few seconds of exposure to UV light (near 350 nm) outside the cavity, the lineshape had immobilized substantially (, right
), indicating successful photolysis of DM-nitrophen and binding of released calcium to calmodulin. In order to illuminate the sample directly inside the cavity, the light output of a xenon flash lamp was directed to the single light port of the EPR cavity via a light guide. The left trace in shows the EPR signal of a fresh mixture at a fixed magnetic field (at the most sensitive point in the spectrum, see asterisk
) while a UV flash was administered to the cavity (arrow
). After a short light-induced instability of the EPR signal, the trace settled on a lower level, indicating successful photolysis of DM-nitrophen inside the cavity. Photolysis is far from complete after a single flash (, right
). This property can be used to improve the light-induced signal by averaging multiple traces. The light response is specific for calmodulin: a control sample (spin-labeled KcsA) showed only a transient light-induced instability, but no sustained signal change (data not shown). The data demonstrate that the calcium-induced structural transition of calmodulin was complete within 100 ms. Limiting the interaction between light and EPR cavity should help reduce or eliminate light-induced instabilities that prevent the transition from being observed. This could be achieved through the use of alternative cavities with a direct light path across the sample.
Figure 5 Flash photolysis of caged calcium using I100R1 as a calcium sensor. (A) Spectra of I100R1 before (left) and after (right) UV illumination outside of the EPR cavity. The chemical structure of DM-nitrophen is shown with the photo-cleavable bond indicated (more ...)