Mapping the Structure and Conformational Movements of Proteins with Transition Metal Ion Fret

ArticleinNature Methods 6(7):532-7 · July 2009with39 Reads
DOI: 10.1038/nmeth.1341 · Source: PubMed
Visualizing conformational dynamics in proteins has been difficult, and the atomic-scale motions responsible for the behavior of most allosteric proteins are unknown. Here we report that fluorescence resonance energy transfer (FRET) between a small fluorescent dye and a nickel ion bound to a dihistidine motif can be used to monitor small structural rearrangements in proteins. This method provides several key advantages over classical FRET, including the ability to measure the dynamics of close-range interactions, the use of small probes with short linkers, a low orientation dependence, and the ability to add and remove unique tunable acceptors. We used this 'transition metal ion FRET' approach along with X-ray crystallography to determine the structural changes of the gating ring of the mouse hyperpolarization-activated cyclic nucleotide-regulated ion channel HCN2. Our results suggest a general model for the conformational switch in the cyclic nucleotide-binding site of cyclic nucleotide-regulated ion channels.
    • "While the structure of the whole channel is not known at the moment, several crystal structures are available for the intracellular C-terminal portion of HCN1 [209], HCN2210211 [209, 212], HCN4208209 213], and the sea urchin sperm SPIH channels [214]; this allowed detailed studies on the interaction of cyclic nucleotides and analogues, as outlined in the previous section. More important, in the cytosolic domain of the HCN4 channel an additional binding site has been recently discovered, which could be exploited for selective modulation. "
    [Show abstract] [Hide abstract] ABSTRACT: Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, the molecular correlate of the hyperpolarization-activated current (If/Ih), are membrane proteins which play an important role in several physiological processes and various pathological conditions. In the Sino Atrial Node (SAN) HCN4 is the target of ivabradine, a bradycardic agent that is, at the moment, the only drug which specifically blocks If. Nevertheless, several other pharmacological agents have been shown to modulate HCN channels, a property that may contribute to their therapeutic activity and/or to their side effects. HCN channels are considered potential targets for developing drugs to treat several important pathologies, but a major issue in this field is the discovery of isoform-selective compounds, owing to the wide distribution of these proteins into the central and peripheral nervous systems, heart and other peripheral tissues. This survey is focused on the compounds that have been shown, or have been designed, to interact with HCN channels and on their binding sites, with the aim to summarize current knowledge and possibly to unveil useful information to design new potent and selective modulators.
    Full-text · Article · Mar 2016
    • "Toxins are capable of promoting conformational changes on their target ion channels. Since distances within a single functional protein are small, methods such as FRET (Fluorescence Resonance Energy Transfer) are useful techniques for measuring distances within an ion channel molecule [34] and the spatial changes that may occur in channels [209] upon allosteric modification of function as that promoted by some toxin molecules [210] . One such molecule is Batrachotoxin (BtchTX), a potent toxin found in Central and South American poison dart frogs, beetles of the family melyridae, and some New Guinean birds. "
    [Show abstract] [Hide abstract] ABSTRACT: Ion channels constitute a varied class of membrane proteins with pivotal roles in cellular physiology and that are fundamental for neuronal signaling, hormone secretion and muscle contractility. Hence, it is not unanticipated that toxins from diverse organisms have evolved to modulate the activity of ion channels. For instance, animals such as cone snails, scorpions, spiders and snakes use toxins to immobilize and capture their prey by affecting ion channel function. This is a beautiful example of an evolutionary process that has led to the development of an injection apparatus from predators and to the existence of toxins with high affinity and specificity for a given target. Toxins have been used in the field of ion channel biophysics for several decades to gain insight into the gating mechanisms and the structure of ion channels. Through the use of these peptides, much has been learned about the ion conduction pathways, voltage-sensing mechanisms, pore sizes, kinetics, inactivation processes, etc. This review examines an assortment of toxins that have been used to study different ion channels and describes some key findings about the structure-function relationships in these proteins through the details of the toxin-ion channel interactions.
    Full-text · Article · Feb 2015
    • "al (2007) on the cyclic nucleotide modulated channel SpIH, a modulation mechanism was suggested in which the β-roll interactions were thought to play an important role in cyclic nucleotide binding affinity whereas interactions with the C-helix were described to be involved in efficiency of the allosteric transitions. In addition, using transition metal ion FRET it was demonstrated that agonist binding causes a stabilization of the C-helix in HCN2 [16, 28]. Additional evidence supporting an important dynamic role of the C-helix comes from X-ray crystal structures and NMR structures of the MlotiK1 CNBD. "
    [Show abstract] [Hide abstract] ABSTRACT: Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.
    Full-text · Article · Jan 2015
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