In allosteric proteins, the binding of a ligand stabilizes conformational rearrangements, resulting in a switch between inactive and active states. These movements can propagate long distances to affect activity tens of angstroms from the ligand-binding site. In HCN channels, the direct binding of cAMP to a cytoplasmic ligand-binding domain stabilizes the opening conformational changes in the channel pore located more than 50 Å away1
. However, like in most allosteric proteins, the direction, magnitude, and nature of conformational changes in the channel are unknown.
Fluorescence resonance energy transfer (FRET), in which light energy absorbed by a donor is transferred to a nearby acceptor, is a powerful tool for measuring changes in molecular distances2
. The efficiency of FRET falls off with the sixth power of the distance between the two molecules, making FRET exquisitely sensitive to changes in distance. However, as a consequence of this steep distance dependence, FRET can measure distances effectively only in a narrow window around R0
, the distance at which FRET efficiency is 50%2
. Classical FRET methods are not always well suited to study intramolecular movements in proteins. This is chiefly due to the long R0
values of most fluorescent dye or protein FRET pairs (30–60 Å), their large sizes (15–30 Å), and their long flexible attachment linkers (10–15 Å) 3,4
. Combined with the dependence of the FRET on the relative orientation of the fluorophores, these properties can complicate the interpretation of FRET results. While lanthanide resonance energy transfer (LRET) methods have less orientation dependence than FRET, LRET still utilizes large chelating groups and dyes with long linkers, has similarly long R0
values (~30–100 Å), and has the added complication of very long excited state lifetimes3,5
What is needed to map conformational rearrangements in proteins is a technique that can work over shorter distances than classical FRET. We have developed such a method called “transition metal ion FRET”, which has shorter R0 values (about 10 Å), uses small dyes with short linkers, and is not sensitive to the orientation problems usually associated with FRET. Unlike NMR, EPR, and crystallography, transition metal ion FRET could theoretically be applied to proteins of any size, in membranes, on extracellular domains of proteins in living cells, and potentially on single molecules. It can measure the kinetics of the conformational change in real time and can be paired with other functional measurements. Here, we utilize this method along with x-ray crystallography to explore the conformational motions of the carboxyl terminal region of HCN2 during ligand-activation. Our results demonstrate the ability of transition metal ion FRET to reveal structural rearrangements in proteins and provide a model for allosteric movements of HCN2.