Conformational Ensembles of Calmodulin Revealed by Nonperturbing Site-Specific Vibrational Probe Groups.

Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein-protein binding interfaces. The infrared absorption bands for the probe groups reported many dramatic changes in the probes' local environments as CaM went from apo- to calcium-saturated to target peptide-bound conditions, including large frequency shifts and a variety of line shapes from narrow (interpreted as a rigid and invariant local environment) to symmetric to broad and asymmetric (likely from multiple coexisting and dynamically exchanging structures). The fast intrinsic time scale of infrared spectroscopy means that the line shapes report directly on site-specific details of calmodulin's variable structural distribution. Though quantitative interpretation of the probe line shapes depends on a direct connection between simulated ensembles and experimental data that does not yet exist, formation of such a connection to data such as that reported here would provide a new way to evaluate conformational ensembles from data that directly contains the structural distribution. The calmodulin probe sites developed here will also be useful in evaluating the binding mode of calmodulin with many uncharacterized regulatory targets.

AMD-like retinopathy associated with intravenous iron.

Iron accumulation in the retina is associated with the development of age-related macular degeneration (AMD). IV iron is a common method to treat iron deficiency anemia in adults, and its retinal manifestations have not hitherto been identified. To assess whether IV iron formulations can be retina-toxic, we generated a mouse model for iron-induced retinal damage. Male C57BL/6J mice were randomized into groups receiving IV iron-sucrose (+Fe) or 30% sucrose (-Fe). Iron levels in neurosensory retina (NSR), retinal pigment epithelium (RPE), and choroid were assessed using immunofluorescence, quantitative PCR, and the Perls' iron stain. Iron levels were most increased in the RPE and choroid while levels in the NSR did not differ significantly in +Fe mice compared to controls. Eyes from +Fe mice shared histological features with AMD, including Bruch's membrane (BrM) thickening with complement C3 deposition, as well as RPE hypertrophy and vacuolization. This focal degeneration correlated with areas of high choroidal iron levels. Ultrastructural analysis provided further detail of the RPE/photoreceptor outer segment vacuolization and Bruch's membrane thickening. Findings were correlated with a clinical case of a 43-year-old patient who developed numerous retinal drusen, the hallmark of AMD, within 11 months of IV iron therapy. Our results suggest that IV iron therapy may have the potential to induce or exacerbate a form of retinal degeneration. This retinal degeneration shares features with AMD, indicating the need for further study of AMD risk in patients receiving IV iron treatment.