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1.
Increased triglyceride-rich lipoproteins (TGRLs) in the postprandial state are associated with atherosclerosis. We investigated whether the postprandial state induced structural changes at the apolipoprotein E4 (apoE4) C terminus, its principal lipid binding domain, using electron paramagnetic resonance (EPR) spectroscopy of a site-directed spin label attached to the cysteine of apoE4-W264C. Spin coupling between labels located in the C termini was followed after mixing with preprandial and postprandial human plasma samples. Our results indicate that postprandial plasma triggers a reorganization of the protein such that the dipolar broadening is diminished, indicating a reduction in C-terminal interaction. The loss of spectral broadening was directly correlated with an increase in postprandial plasma triglycerides and was reduced with delipidated plasma. The spin-labeled apoE4 displayed a lipid preference of VLDL > LDL > HDL in the preprandial and postprandial states. The apoE4 shift to VLDL during the postprandial state was accompanied by a loss in spectral broadening of the protein. These findings suggest that apoE4 associated with LDL maintains self-association via its C terminus and that this association is diminished in VLDL-associated protein. Lipolyzed TGRL reflected a depletion of the C-terminal interaction of apoE4. Addition of palmitate to VLDL gave a similar response as lipolyzed TGRL, suggesting that lipolysis products play a major role in reorganizing apoE4 during the postprandial state. 相似文献
2.
John F. Hess Madhu S. Budamagunta Atya Aziz Paul G. FitzGerald John C. Voss 《Protein science : a publication of the Protein Society》2013,22(1):47-55
Very little data have been reported that describe the structure of the tail domain of any cytoplasmic intermediate filament (IF) protein. We report here the results of studies using site directed spin labeling and electron paramagnetic resonance (SDSL‐EPR) to explore the structure and dynamics of the tail domain of human vimentin in tetramers (protofilaments) and filaments. The data demonstrate that in contrast to the vimentin head and rod domains, the tail domains are not closely apposed in protofilaments. However, upon assembly into intact IFs, several sites, including positions 445, 446, 451, and 452, the conserved “beta‐site,” become closely apposed, indicating dynamic changes in tail domain structure that accompany filament elongation. No evidence is seen for coiled‐coil structure within the region studied, in either protofilaments or assembled filaments. EPR analysis also establishes that more than half of the tail domain is very flexible in both the assembly intermediate and the intact IF. However, by positioning the spin label at distinct sites, EPR is able to identify both the rod proximal region and sites flanking the beta‐site motif as rigid locations within the tail. The rod proximal region is well assembled at the tetramer stage with only slight changes occurring during filament elongation. In contrast, at the beta site, the polypeptide backbone transitions from flexible in the assembly intermediate to much more rigid in the intact IF. These data support a model in which the distal tail domain structure undergoes significant conformational change during filament elongation and final assembly. 相似文献
3.
Multifrequency electron paramagnetic resonance (EPR), combined with site-directed spin labeling, is a powerful spectroscopic tool to characterize protein dynamics. The lineshape of an EPR spectrum reflects combined rotational dynamics of the spin probe's local motion within a protein, reorientations of protein domains, and overall protein tumbling. All these motions can be restricted and anisotropic, and separation of these motions is important for thorough characterization of protein dynamics. Multifrequency EPR distinguishes between different motions of a spin-labeled protein, due to the frequency dependence of EPR resolution to fast and slow motion of a spin probe. This gives multifrequency EPR its unique capability to characterize protein dynamics in great detail. In this review, we analyze what makes multifrequency EPR sensitive to different rates of spin probe motion and discuss several examples of its usage to separate spin probe dynamics and overall protein dynamics, to characterize protein backbone dynamics, and to resolve protein conformational states. 相似文献
4.
Marie‐Ange Do Cao Serge Crouzy Miyeon Kim Michel Becchi David S. Cafiso Attilio Di Pietro Jean‐Michel Jault 《Protein science : a publication of the Protein Society》2009,18(7):1507-1520
Previously published 3‐D structures of a prototypic ATP‐binding cassette (ABC) transporter, MsbA, have been recently corrected revealing large rigid‐body motions possibly linked to its catalytic cycle. Here, a closely related multidrug bacterial ABC transporter, BmrA, was studied using site‐directed spin labeling by focusing on a region connecting the transmembrane domain and the nucleotide‐binding domain (NBD). Electron paramagnetic resonance (EPR) spectra of single spin‐labeled cysteine mutants suggests that, in the resting state, this sub‐domain essentially adopts a partially extended conformation, which is consistent with the crystal structures of MsbA and Sav1866. Interestingly, one of the single point mutants (Q333C) yielded an immobilized EPR spectrum that could arise from a direct interaction with a vicinal tyrosine residue. Inspection of different BmrA models pointed to Y408, within the NBD, as the putative interacting partner, and its mutation to a Phe residue indeed dramatically modified the EPR spectra of the spin labeled Q333C. Moreover, unlike the Y408F mutation, the Y408A mutation abolished both ATPase activity and drug transport of BmrA, suggesting that a nonpolar bulky residue is required at this position. The spatial proximity of Q333 and Y408 was also confirmed by formation of a disulfide bond when both Q333 and T407 (or S409) were replaced jointly by a cysteine residue. Overall, these results indicate that the two regions surrounding Q333 and Y408 are close together in the 3‐D structure of BmrA and that residues within these two sub‐domains are essential for proper functioning of this transporter. 相似文献
5.
We investigated aqueous solutions containing nitrite ions and DMPO (5,5-dimethyl-1-pyrroline-N-oxide) by electron spin resonance (ESR) in the pH range from 1 to 6. A DMPO-OH signal was observed below pH 3.0 in the presence of nitrite ions, whereas in the absence of nitrite ion, an extremely weak signal was observed below pH 1.5. Addition of methanol, a hydroxyl radical scavenger, to this system did not lead to the appearance of a detectable DMPO-CH2OH signal. The possibility of this DMPO-OH signal being due to a genuine spin trapping process with hydroxyl radical was, therefore, ruled out. The reactivities of reactive nitrogen species (RNS) in this system with DMPO have also been investigated by density functional theory (DFT) at the IEFPCM (water)/B3LYP/6–311?+?G ** level of theory. On the basis of the pH dependence of the signal intensity and the redox potential E° (versus SHE) calculated by DFT theory, we propose that the origin of this signal is “inverted spin trapping” via one-electron oxidation of DMPO by H2ONO+, followed by the nucleophilic addition of water. Prevention of these false-positive results when detecting hydroxyl radical using ESR spin trapping requires an awareness of both the presence of nitrite ions in the solution and the solution pH. 相似文献
6.
Jeffrey D. Carter Estrella G. Gonzales Xi Huang Adam N. Smith Ian Mitchelle S. de Vera Peter W. D’Amore James R. Rocca Maureen M. Goodenow Ben M. Dunn Gail E. Fanucci 《FEBS letters》2014
Conformational sampling of pre- and post-therapy subtype B HIV-1 protease sequences derived from a pediatric subject infected via maternal transmission with HIV-1 were characterized by double electron–electron resonance spectroscopy. The conformational ensemble of the PRE construct resembles native-like inhibitor bound states. In contrast, the POST construct, which contains accumulated drug-pressure selected mutations, has a predominantly semi-open conformational ensemble, with increased populations of open-like states. The single point mutant L63P, which is contained in PRE and POST, has decreased dynamics, particularly in the flap region, and also displays a closed-like conformation of inhibitor-bound states. These findings support our hypothesis that secondary mutations accumulate in HIV-1 protease to shift conformational sampling to stabilize open-like conformations, while maintaining the predominant semi-open conformation for activity. 相似文献
7.
Pirman NL Milshteyn E Galiano L Hewlett JC Fanucci GE 《Protein science : a publication of the Protein Society》2011,20(1):150-159
Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA3 is used as a model system demonstrating SDSL-EPR characterization of conformational changes in small IDP systems. IA3 has 68 amino acids, is unstructured in solution, and becomes α-helical upon addition of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE). Two single cysteine substitutions, one in the N-terminus (S14C) and one in the C-terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h(+1)/h(0), the local tumbling volume (VL), and the percent change of the h(−1) intensity were utilized to quantitatively monitor TFE-induced conformational changes. The values of h(+1)/h(0) as a function of TFE titration varied in a sigmoidal manner and were fit to a two-state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA3. The other parameters provide site-specific information and show that S14C-SL undergoes a conformational change resulting in more restricted motion than N58C-SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α-helical propensity of the N-terminal segment of IA3. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured-to-helical conformations in IDPs by SDSL. 相似文献
8.
Hanson SM Cleghorn WM Francis DJ Vishnivetskiy SA Raman D Song X Nair KS Slepak VZ Klug CS Gurevich VV 《Journal of molecular biology》2007,368(2):375-387
Arrestins regulate the activity and subcellular localization of G protein-coupled receptors and other signaling molecules. Here, we demonstrate that arrestins bind microtubules (MTs) in vitro and in vivo. The MT-binding site on arrestins overlaps significantly with the receptor-binding site, but the conformations of MT-bound and receptor-bound arrestin are different. Arrestins recruit ERK1/2 and the E3 ubiquitin ligase Mdm2 to MTs in cells, similar to the arrestin-dependent mobilization of these proteins to the receptor. Arrestin-mediated sequestration of ERK to MTs reduces the level of ERK activation. In contrast, recruitment of Mdm2 to MTs by arrestin channels Mdm2 activity toward cytoskeleton-associated proteins, increasing their ubiquitination dramatically. The mobilization of signaling molecules to MTs is a novel biological function of arrestin proteins. 相似文献
9.
Wu N Hanson SM Francis DJ Vishnivetskiy SA Thibonnier M Klug CS Shoham M Gurevich VV 《Journal of molecular biology》2006,364(5):955-963
Arrestins serve as multi-functional regulators of G-protein coupled receptors, interacting with hundreds of different receptor subtypes and a variety of other signaling proteins. Here we identify calmodulin as a novel arrestin interaction partner using three independent methods in vitro and in cells. Arrestin preferentially binds calcium-loaded calmodulin with a Kd value of approximately 7 microM, which is within range of endogenous calmodulin concentrations. The calmodulin binding site is localized on the concave side of the C-domain and a loop in the center of the arrestin molecule, significantly overlapping with receptor and microtubule-binding sites. Using purified proteins, we found that arrestins sequester calmodulin, preventing its binding to microtubules. Nanomolar affinity of arrestins for their cognate receptors makes calmodulin an ineffective competitor for arrestin binding at relatively high receptor concentrations. The arrestin-calmodulin interaction likely regulates the localization of both proteins and their availability for other interaction partners. 相似文献
10.
Timothy F. Cunningham Soraya Pornsuwan W. Seth Horne Sunil Saxena 《Protein science : a publication of the Protein Society》2016,25(5):1049-1060
Protein spin labeling to yield the nitroxide‐based R1 side chain is a powerful method to measure protein dynamics and structure by electron spin resonance. However, R1 measurements are complicated by the flexibility of the side chain. While analysis approaches for solvent‐exposed α‐helical environment have been developed to partially account for flexibility, similar work in β‐sheets is lacking. The goal of this study is to provide the first essential steps for understanding the conformational preferences of R1 within edge β‐strands using X‐ray crystallography and double electron electron resonance (DEER) distance measurements. Crystal structures yielded seven rotamers for a non‐hydrogen‐bonded site and three rotamers for a hydrogen‐bonded site. The observed rotamers indicate contextual differences in R1 conformational preferences compared to other solvent‐exposed environments. For the DEER measurements, each strand site was paired with the same α‐helical site elsewhere on the protein. The most probable distance observed by DEER is rationalized based on the rotamers observed in the crystal structure. Additionally, the appropriateness of common molecular modeling methods that account for R1 conformational preferences are assessed for the β‐sheet environment. These results show that interpretation of R1 behavior in β‐sheets is difficult and indicate further development is needed for these computational methods to correctly relate DEER distances to protein structure at edge β‐strand sites. 相似文献
11.
14N-ENDOR evidence for imidazole coordination in copper proteins 总被引:1,自引:0,他引:1
H Yokoi 《Biochemical and biophysical research communications》1982,108(3):1278-1284
14N-ENDOR studies of simple nitrogen-coordinated copper(II) complexes in frozen aqueous solutions show that the nitrogen hyperfine constants, A″ and A⊥, of imidazole are much more isotropic () than those of the other biologically-related ligand nitrogens. From this result, combined with 14N-ENDOR results of some copper proteins containing imidazoles as ligands, it is concluded that R < 1.10 for nitrogen hyperfine constants can be employed as an empirical criterion for demonstration of the existence of imidazole coordination in copper proteins. 相似文献
12.
Crystal structures of thrombin complexed with two spin labels called para-V, 4-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-p-(fluorosulfonyl) benzamidine, and meta-V, 3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-m-(fluorosulfonyl) benzamidine, have been completed at 2.0 and 3.0 Å resolution, respectively. Previous electron spin resonance studies with these labels gave rise to a low-resolution topography map of thrombin's extended active site. These labels monitor two distinct areas of the thrombin active site: (1) an apolar binding site which manifests itself in an biphasic activation/inhibition effect on thrombin activity and (2) a region sensitive to -thrombin autoproteolytic cleavage(s) to -thrombin (Arg75-Tyr76 and/or Arg77A-Asn78, and Lys149E-Gly150, chymotrypsin numbering). Para-V was found to bind along the substrate binding cleft, while meta-V was found to bind both at the substrate primary specificity pocket and at a site which interacts with the -cleavage loop. These studies reaffirm that accurate information may be gained from solution studies and indicates the complementarity of solid-state studies. 相似文献
13.
Ricardo H. Flores Jiménez Marie‐Ange Do Cao Miyeon Kim David S. Cafiso 《Protein science : a publication of the Protein Society》2010,19(2):269-278
Site‐directed spin labeling (SDSL) was used to investigate local structure and conformational exchange in two bacterial outer‐membrane TonB‐dependent transporters, BtuB and FecA. Protecting osmolytes, such as polyethylene glycols (PEGs) are known to modulate a substrate‐dependent conformational equilibrium in the energy coupling motif (Ton box) of BtuB. Here, we demonstrate that a segment that is N‐terminal to the Ton box in BtuB, is in conformational exchange between ordered and disordered states with or without substrate. Protecting osmolytes shift this equilibrium to favor the more ordered, folded state. However, a segment of BtuB that is C‐terminal to the Ton box that is not solvent exposed is insensitive to PEGs. Protecting osmolytes also modulate a conformational equilibrium in the Ton box of FecA, with larger molecular weight PEGs producing the largest shifts in the conformational free energy. These data indicate that solvent‐exposed regions of these transporters undergo conformational exchange and that regions of these transporters that are involved in protein–protein interactions sample multiple conformational substates. The sensitivity to solute provides an explanation for differences seen between two high‐resolution structures of BtuB, which each likely represent one conformation from a subset of states that are normally sampled by the protein. This work also illustrates how SDSL and osmolytes may be used to characterize and quantitate conformational equilibria in membrane proteins. 相似文献
14.
Megan Thompson Richard Chen Jessica Thomaston William F. DeGrado Kathleen P. Howard 《Protein science : a publication of the Protein Society》2015,24(3):426-429
The influenza A M2 protein is a 97‐residue integral membrane protein involved in viral budding and proton conductance. Although crystal and NMR structures exist of truncated constructs of the protein, there is disagreement between models and only limited structural data are available for the full‐length protein. Here, the structure of the C‐terminal juxtamembrane region (sites 50–60) is investigated in the full‐length M2 protein using site‐directed spin‐labeling electron paramagnetic resonance (EPR) spectroscopy in lipid bilayers. Sites 50–60 were chosen for study because this region has been shown to be critical to the role the M2 protein plays in viral budding. Continuous wave EPR spectra and power saturation data in the presence of paramagnetic membrane soluble oxygen are consistent with a membrane surface associated amphipathic helix. Comparison between data from the C‐terminal juxtamembrane region in full‐length M2 protein with data from a truncated M2 construct demonstrates that the line shapes and oxygen accessibilities are remarkably similar between the full‐length and truncated form of the protein. 相似文献
15.
Swanson MA Kathirvelu V Majtan T Frerman FE Eaton GR Eaton SS 《Protein science : a publication of the Protein Society》2011,20(3):610-620
Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid β-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for βIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open). 相似文献
16.
Urška Batista Maja Garvas Marjana Nemec Milan Schara Peter Veranič Tilen Koklic 《Cell biology international》2010,34(6):663-668
Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged. 相似文献
17.
Blokesch M Albracht SP Matzanke BF Drapal NM Jacobi A Böck A 《Journal of molecular biology》2004,344(1):155-167
Carbamoylphosphate has been shown to be the educt for the synthesis of the CN ligands of the NiFe metal centre of hydrogenases from Escherichia coli. In the absence of carbamoylphosphate, cells accumulate a complex of two hydrogenase maturation proteins, namely HypC and HypD for the synthesis of hydrogenase 3. A procedure for the purification of wild-type HypD protein or of a biologically active derivative carrying the Strep-tagII((R)) at the N terminus has been developed. HypD is a monomeric protein possessing about 4 mol of iron per mol of protein. Electron paramagnetic resonance (EPR) and Mossbauer spectroscopy demonstrated that the iron is present as a diamagnetic [4Fe-4S](2+) cluster. The complex between HypC and HypD can be cross-linked by a number of thiol and primary amine-specific linkers. When HypD and HypC were overproduced side-by-side with HypE, the HypC-HypD complex contained substoichiometric amounts of HypE whose proportion in the complex could be augmented when HypF was also overproduced. HypE trapped in this complex could be carbamoylated by protein HypF and after dehydration transferred the cyano group to the HypC-HypD part of the complex. Free HypC and HypD were not cyanated by HypE-CN. An active HypC-HypD complex from anaerobic cells was inactivated by incubation with K(3)[Fe(CN)(6)] but not with K(4)[Fe(CN)(6)]. The results suggest the existence of a dynamic complex between the hydrogenase maturation proteins HypD, HypC, HypE and HypF, which is the site of ligand biosynthesis and attachment to the iron atom of the NiFe site in hydrogenase 3. 相似文献
18.
Guo Z Cascio D Hideg K Hubbell WL 《Protein science : a publication of the Protein Society》2008,17(2):228-239
Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain. 相似文献
19.
Januszyk K Fleissner MR Atchabahian L Shieh FK Altenbach C Martin SL Guo F Hubbell WL Clubb RT 《Protein science : a publication of the Protein Society》2011,20(7):1231-1243
Long interspersed nuclear element-1 is a highly abundant mammalian retrotransposon that comprises 17% of the human genome. L1 retrotransposition requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. ORF1p has been shown to adopt a homo-trimeric, asymmetric dumbbell-shaped structure. However, its atomic-level structure and mechanism of RNA binding remains poorly understood. Here, we report the results of a site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) study of 27 residues within the RNA binding region of the full-length protein. The EPR data are compatible with the large RNA binding lobe of ORF1p containing a RNA recognition motif (RRM) domain and a carboxyl-terminal domain (CTD) that are predicted from crystallographic and NMR studies of smaller fragments of the protein. Interestingly, the EPR data indicate that residues in strands β3 and β4 of the RRM are structurally unstable, compatible with the previously observed sensitivity of this region to proteolysis. Affinity measurements and RNA-dependent EPR spectral changes map the RNA binding site on ORF1p to residues located in strands β3 and β4 of the RRM domain and to helix α1 of the CTD. Complementary in vivo studies also identify residues within the RRM domain that are required for retrotransposition. We propose that in the context of the full-length trimeric protein these distinct surfaces are positioned adjacent to one another providing a continuous surface that may interact with nucleic acids. 相似文献
20.
Hannah A. DeBerg Peter S. Brzovic Galen E. Flynn William N. Zagotta Stefan Stoll 《The Journal of biological chemistry》2016,291(1):371-381
Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating electrical activity in the heart and brain. They are gated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD), which is connected to the channel pore by a C-linker region. Binding of cyclic nucleotides increases the rate and extent of channel activation and shifts it to less hyperpolarized voltages. We probed the allosteric mechanism of different cyclic nucleotides on the CNBD and on channel gating. Electrophysiology experiments showed that cAMP, cGMP, and cCMP were effective agonists of the channel and produced similar increases in the extent of channel activation. In contrast, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) on the isolated CNBD indicated that the induced conformational changes and the degrees of stabilization of the active conformation differed for the three cyclic nucleotides. We explain these results with a model where different allosteric mechanisms in the CNBD all converge to have the same effect on the C-linker and render all three cyclic nucleotides similarly potent activators of the channel. 相似文献