Determining Nitric Oxide‐induced Conformational Changes in Soluble Guanylate Cyclase using Lanthanide‐based Resonance Energy Transfer

Abstract

Nitric Oxide (NO) is a crucial signaling molecule involved in vasodilation and regulation of blood pressure. A key target for NO signaling is soluble guanylate cyclase (sGC), a heterodimeric protein that upon NO binding converts GTP to a secondary messenger, cGMP. NO binding at the ferrous heme of the beta subunit of the sGC dimer induces a conformational change in the protein resulting in increased catalysis and increased production of cGMP. Attempts to enhance the enzymatic activity of sGC by drugs tartegeting cardiovascular disease are hindered by a lack of information on the mechanism of this allosteric regulation and the structure of the protein. One method that can provide a better understanding of conformational changes in sGC is lanthanide‐based resonance energy transfer (LRET). This technique monitors luminescence of terbium ion (Tb3+) bound to a lanthanide‐binding tag (LBT) inserted into a protein sequence. We have designed several sGC constructs with an LBT tag at different locations in the protein in addition to 6‐His‐tags, which can bind transition metals and quench luminescence of the LBT‐bound Tb3+. Rates for Tb3+ luminescence decay upon quenching by Cu2+ bound to the 6‐His‐tag were used to calculate the distance between the metal ions using the Förster equation. Our preliminary conclusions from the LRET experiments are: (i) Tb luminescence decays with a bi‐exponential time course and a rate constant for the slow phase of ~2.3 ms; (ii) significant quenching of Tb luminescence is observed in the presence of millimolar concentrations of Cu2+; (iii) a calculated distance of ~23 Å between Tb and Cu ions in construct NT19_LBT2 suggests the C and N termini lie close to one another in the protein. Future experiments will probe how these distances change upon ligand binding and the distances between the LBT tags and a candidate drug with quenching fluorophore. These decay times and distances will lead to greater understanding of sGC structure and dynamics and will aid in the design of new drugs to treat cardiovascular disease through allosteric stimulation of sGC.Support or Funding InformationNIH MARC Training Grant T34 GM08718, AHA 14GRNT2008006, NIH R01 GM117357, Ironwood Pharmaceuticals