IndraLab
Statements
sparser
"Comparison of properties of GFP-L2-AgTx2 and its structural precursor His6-GFP-L2-AgTx2 shows that any of these ligands can be efficiently used as a selective fluorescent probe in the bioengineering analytical system on the basis of KcsA-Kv1.3 channel to search for Kv1.3 channel blockers (both among individual compounds and in complex mixtures) and to estimate their activity ( xref E,F, xref )."
sparser
"The chemical shifts of the destabilized state fit remarkably
well with the chemical shifts of NaK2K in the presence of Na + ions. xref In contrast, experiments on KcsA-Kv1.3
showed that 15 NH 4 + ions did not have
any destabilizing effects on this channel and that the behavior we
observed in NaK2K is not general for K + -selective ion channels."
sparser
"This enables the detailed analysis
of the structure and dynamics of membrane proteins under near-to-physiological
conditions. xref − xref We were able to detect 15 NH 4 + ions
bound in the SF of the non-selective ion channel NaK, its K + -selective mutant NaK2K, and KcsA-Kv1.3, xref a mutant of KcsA resembling the human voltage-gated channel Kv1.3."
sparser
"This cryptic binding pocket is not detectable in the absence of the drug ( xref ), which suggests that it has been induced in a similar manner to previous reports of toxin interactions with KcsA-Kv1.3 that induce conformational changes in both the toxin and the channel structure to generate a high-affinity binding site ( xref ; xref )."
sparser
"For example, variations in ssNMR cross-peak amplitudes are seen for Ser and Thr peaks (resonating
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388 A. Lange et al.
Figure 7: Two-dimensional 13C-13C spin diffusion (SD) spectra of uniformly [13C, 15N] labeled KcsA-Kv1.3 in complex with unlabeled KTX at different probe temperatures."
sparser
"Thermodynamic measurements of ion binding using isothermal titration calorimetry (ITC) report a K d of ∼0.43 mM for K + binding. xref Electrophysiological experiments on the KcsA-Kv1.3 chimera report two high affinity binding sites in the pore: an inner site that has a K d of 6.5 µM and the outer site has a K d of 0.9 mM."
sparser
"Because of limited resolution in the proton dimension, the (1H,13C) FSLG experiment does not allow for an unambiguous identification of extracellular domains of KcsA-Kv1.3 in the spectrum.
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Solid-State NMR of a Chimeric K+ Channel 385
Figure 4: Two-dimensional FSLG 1H-13C correlation spectrum of uniformly [13C, 15N] labeled KcsA-Kv1.3 in complex with unlabeled KTX."
sparser
"Solid-state NMR studies on a Kv1.3–KcsA chimera grafted with Kv1.3’s outer vestibule revealed that binding of kaliotoxin from scorpion venom caused profound conformational changes in the external selectivity filter of the channel, including a large shift in the position of Y78 (the residue equivalent to Y447 in Kv1.3) ( xref , xref ) Follow-up all-atom MDS studies coupled with solid-state NMR and electrophysiological measurements revealed that kaliotoxin-induced conformational changes in KcsA–Kv1.3 were structurally and functionally related to recovery from C-type inactivation ( xref )."
sparser
"Solid-state NMR has already been used successfully to investigate the structure and dynamics of several membrane proteins ( xref , xref ), including the ion channels KcsA-Kv1.3 ( xref ), wild-type KcsA ( xref , xref ), VDAC (voltage-dependent anion channel) ( xref ), M2 ( xref , xref ), and NaK and NaK2K ( xref )."
sparser
"As shown in Figure 6b–e, residues that are part of or close to the selectivity filter are, likewise, not significantly perturbed by variations in the K+ concentration.
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Solid-State NMR of a Chimeric K+ Channel 387
Figure 6: (a) Two-dimensional 13C–13C spin diffusion (SD) spectra of uniformly [13C, 15N] labeled KcsA-Kv1.3 (without KTX) at different K+ concentrations."
sparser
"A-AgTx2 was demonstrated to compete with peptide and low-molecular-weight blockers of the Kv1.3 channel for the binding to the Kv1.3 site, and it can be used as a component of the bioengineering analytical system based on the KcsA–Kv1.3 hybrid channel to study outer-pore blockers of the Kv1.3 channel."
sparser
"To examine the capability of the Nano-oscillator platform for measuring small molecule binding kinetics and demonstrate its potential application in drug screening, we studied the binding of a small molecule drug candidate 1 (4-(2-ethylpiperidin-1-yl)-2-methyl-6-phenyl-5H-pyrrolo[3,2-d] pyrimidine, MW = 320 Da) to a chimeric ion channel protein KcsA-Kv1.3 ( xref ), which plays an important role in autoimmune diseases. xref To stabilize the membrane protein in aqueous solution, KcsA-Kv1.3 was encapsulated in a Nanodisc consisting of a nano-scale lipid bilayer disc fastened by a membrane scaffold protein."
sparser
"We also measured a peptide blocker of KcsA-Kv1.3, ShK, which is more potent than 1 and has been considered as a model by pharmaceutical companies for developing immunosuppressant drugs. xref The binding curves and fittings are shown in xref , and k a , k d , and K D were determined to be 2.0×10 7 M −1 s −1 , 4.0×10 −3 s −1 , and 0.20 nM, respectively."
sparser
"To verify the specific binding of ShK, two additional experiments were carried out by adding ShK to empty Nanodisc and adding IgG to KcsA-Kv1.3 Nanodisc, and neither of them produced detectable oscillation amplitude changes ( xref , curves marked with control 1 and control 2, respectively)."
sparser
"In particular, we recently used high-resolution solid-state NMR to investigate the interaction of kaliotoxin (KTX), a 38-residue peptide found in the venom of the scorpion Androctonus mauretanicus mauretanicus, with the outer vestibule and the K+ conduction pore of purified KcsA-Kv1.3 channels (31)."
sparser
"According to a previous study that temperature would influence the binding kinetics between KcsA-Kv1.3 and small molecule due to the effect on the flexibility of lipid bilayer, we repeated the experiment under 30 °C and found the kinetic constants became, (2.7 ± 0.5) × 10 4 M −1 s −1 , (1.9 ± 0.4) × 10 −2 s −1 and 6.8 ± 1.2 × 10 2 nM (Res = 2.785), as shown in xref ."
sparser
"The results are close to the value in literature. xref , xref For PAP-1 and KcsA-Kv1.3 nanodisc binding, the k a , k d and K D are fitted to be (3.8 ± 0.6) × 10 2 M −1 s −1 , (2.3 ± 0.1) × 10 −2 s −1 and 65 ± 11 μM (Res = 3.785,) at 25 °C and (4.8 ± 0.4) × 10 2 M −1 s −1 , (9.6 ± 1.1) × 10 −2 s −1 and (2.1 ± 0.2) ×10 2 μM (Res = 2.614) at 30 °C, respectively ( xref and xref )."
sparser
"Results obtained by using (1H,13C) correlation experiments on fully labeled variants of KcsA-Kv1.3 suggest that further methodological and preparative work is necessary to make proton spectroscopy a viable tool to study membrane proteins of comparable size to our system by ssNMR."
sparser
"HTX is a new Kv1.3 channel blocker from the scorpion Heterometrus laoticus . xref MTX is a high affinity blocker of Kv1.2 and K Ca channels xref as well as a moderate affinity blocker of Kv1.3 channel. xref To confirm correct folding of recombinant HTX and MTX, we have tested their ability to bind to the Kv1.3-channel binding site formed in the hybrid KcsA-Kv1.3 channel."
sparser
"To do this, a bioengineering system was used, which was based on a KcsA-Kv1.3 channel embedded in the membrane of spheroplasts and fluorescently labeled peptide blocker agitoxin 2. xref This system is a reliable analytical tool to search for Kv1.3 blockers in complex mixtures and among individual compounds as well as to characterize their activities. xref Using this system as described elsewhere, xref we have observed displacement of fluorescent agitoxin 2 from the Kv1.3-channel binding site by HTX and MTX ( xref )."
sparser
"Rather high concentration of HTX or MTX was required to displace agitoxin 2 from the KcsA-Kv1.3 channel because affinity of agitoxin 2 to the Kv1.3 binding site was much higher xref than that of the tested peptides in accordance with the properties of wild type HTX xref and MTX. xref "
sparser
"Given the limited spectral interval in which 1H resonances in proteins are found (44), the resulting resolution is, however, insufficient to resolve individual correlations
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384 A. Lange et al.
Figure 3: Two-dimensional 1H-13C correlation spectrum of uniformly [13C, 15N] labeled KcsA-Kv1.3 in complex with unlabeled KTX."
sparser
"To estimate applicability of GFP–MgTx for recognition of Kv1.3 blockers, we studied displacement of GFP–MgTx from the complexes with KcsA–Kv1.3 by known pore blockers of Kv1.3 channel, namely, two peptide toxins, ChTx and HgTx1, and a low-molecular-weight blocker tetraethylammonium (TEA), which has millimolar affinity to Kv1.3 [ xref ] ( xref )."
sparser
"To characterize potential of GFP-L2-AgTx2 and His6-GFP-L2-AgTx2 as a component of the KcsA-Kv1.3-based bioengineering system for searching Kv1.3 channel ligands in complex mixtures, the response of complexes between KcsA-Kv1.3 and GFP-L2-AgTx2 or His6-GFP-L2-AgTx2 to addition of scorpion venoms was measured ( xref D)."