No abstract

Electrostatic interactions in proteins play a crucial role in determining the structure-function relation in biomolecules. In recent years, fluorescent probes have been extensively employed to interrogate the polarity in biological cavities through dielectric constants or semiempirical polarity scales. A choice of multiple spectroscopic methods, not limited by fluorophores, along with a molecular level description of electrostatics involving solute-solvent interactions, would allow more flexibility to pick and choose the experimental technique to determine the local electrostatics within protein interiors. In this work we report that ultraviolet/visible-absorption, infrared-absorption, or (13)C NMR can be used to calibrate the local electric field in both hydrogen bonded and non-hydrogen bonded protein environments. The local electric field at the binding site of a serum protein has been determined using the absorption wavelength as well as the carbonyl stretching frequency of its natural steroid substrate, testosterone. Excellent agreement is observed in the results obtained from two independent spectroscopic techniques.

No abstract

The vertebrate retina harbors rod and cone photoreceptors. Human vision critically depends on cone photoreceptor function. In the phototransduction cascade, cGMP activates distinct rod and cone isoforms of the cyclic nucleotide-gated (CNG) channel. Excessive cGMP levels initiate a pathophysiological rollercoaster, which starts with CNG channel over-activation, typically in rod photoreceptors. This triggers cell death of rods first, and then cones, and is the root cause of many blinding retinal diseases, including Retinitis pigmentosa. While targeting of CNG channels has been proposed for therapeutic purposes, thus far, it has not been possible to inhibit rod CNG channels without compromising cone function. Here, we present a novel strategy, based on cGMP analogues with opposing actions on CNG channels, which enables the selective modulation of either rod or cone photoreceptor activity. The combined treatment with the weak rod-selective CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and the cone-selective CNG-channel activator (8-pCPT-cGMP) essentially normalized rod CNG-channel function while preserving cone functionality at physiological and pathological cGMP levels. Hence, combinations of cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies. Moreover, this strategy may allow for improvements in visual performance in certain light environments.

Cyclic nucleotide-gated (CNG) channels play an essential role within the phototransduction cascade in vertebrates. Although cone and rod light responses are mediated through similar pathways, each photoreceptor type relies on a specific CNG-channel isoform. In many forms of retinal degeneration, increased cGMP levels initiate a pathophysiological rollercoaster, which starts with an enhanced CNG-channel activation, often in rod photoreceptors. This causes cell death of both rods and cones, and eventually leads to complete blindness. Unfortunately, the targeting of the desired channel isoform still constitutes the bottleneck for many therapeutic schemes. Here, we present a novel strategy, based on cGMP analogues with opposite types of action, which allows for the selective modulation of either rod or cone photoreceptors. A combined treatment with a weak rod-selective CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and a cone-selective CNG-channel activator (8-pCPT-cGMP) preserved the normal CNG-channel function at physiological and pathological cGMP levels. The effectiveness of this approach was tested and confirmed in explanted mouse retina. Under physiological conditions, the inhibitor silenced the rods selectively and decreased the dependency of cone responses on light intensity. Remarkably, the activator, when applied together with the inhibitor, reinstated only the light responsiveness of cones. Yet, when applied alone, the activator dampened rod responses more strongly than those of cones. Hence, combinations of cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies. Beyond therapies for retinal degeneration diseases, treatments based on this strategy may allow modulation of visual performance in certain light environments or disease conditions.