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PubMed1989

Behavior of spin labels in a variety of interdigitated lipid bilayers.

The behavior of a number of spin labels in several lipid bilayers, shown by X-ray diffraction to be interdigitated, has been compared in order to evaluate the ability of the spin label technique to detect and diagnose the structure of lipid bilayers. The main difference between interdigitated and non-interdigitated gel phase bilayers which can be exploited for determination of their structure using spin labels, is that the former have a much less steep fluidity gradient. Thus long chain spin labels with the nitroxide group near the terminal methyl of the chain, such as 16-doxylstearic acid, its methyl ester, or a phosphatidylglycerol spin label containing 16-doxylstearic acid (PG-SL), are more motionally restricted and/or ordered in the interdigitated bilayer than in the non-interdigitated bilayer. This difference is large enough to be of diagnostic value for all three spin labels in the interdigitated bilayers of dihexadecylphosphatidylcholine, dipalmitoylphosphatidylcholine/ethanol, and 1,3-dipalmitoylphosphatidylcholine. However, it is not large enough to be of diagnostic value at low temperatures. Use of probes with the nitroxide group closer to the apolar/polar interface reveals that these latter interdigitated bilayers are more disordered or less closely packed. As the temperature is increased, however, the motion of the PG-SL does not increase as much in these interdigitated bilayers as in non-interdigitated bilayers. The difference in the motion and/or order of PG-SL between interdigitated and non-interdigitated bilayers is large enough at higher temperatures to be of value in diagnosing the structure of the bilayers. Thus by choice of a suitable spin label and a suitable temperature, this technique should prove useful for detection and diagnosis of lipid bilayer structure with a good degree of reliability. Caution must, of course be exercised, as with any spectroscopic technique. Spin labels will also be invaluable for more detailed studies of known interdigitated bilayers, which would be time- and material-consuming, if carried out using X-ray diffraction solely.

Electron Spin Resonance Spectroscopy↗
PubMed1999

Perfusion of the rat ovary: application of pulsed arterial spin labeling MRI.

Pulsed arterial spin labeling was used for mapping ovarian perfusion and measurement of blood velocity in the ovarian artery. Arterial blood was tagged upstream by pulsed slice selective saturation, and saturation transfer due to perfusion was monitored within the rat ovary. The velocity of arterial blood was determined from the dependence of the saturation transfer on the thickness of the saturation slice and the delay between successive saturation pulses. This method allows for determination of arterial velocity, even when the artery itself is not identified in the images. The arterial velocity of blood to the ovary was 3.6+/-0.6 cm x s(-1). The mean ovarian perfusion was 8.7+/-3.5 ml x min(-1) x g(-1) during the surge of luteinizing hormone and 5.9+/-3.0 ml min(-1) x g(-1) during the luteal phase. Arterial labeling can thus be used for following vascular remodeling and angiogenesis during the ovarian cycle by MRI.

Animals↗
PubMed2006

Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy.

We used site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy to study the induced folding of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)). Four single-site N(TAIL) mutants (S407C, S488C, L496C, and V517C), located in three conserved regions, were prepared and labeled with a nitroxide paramagnetic probe. We could monitor the gain of rigidity that N(TAIL) undergoes in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) or one of its physiological partners, namely, the C-terminal domain (XD) of the viral phosphoprotein. The mobility of the spin label grafted at positions 488, 496, and 517 was significantly reduced upon addition of XD, contrary to that of the spin label bound to position 407, which was unaffected. Furthermore, the EPR spectra of spin-labeled S488C and L496C bound to XD in the presence of 30% sucrose are indicative of the formation of an alpha-helix in the proximity of the spin labels. Such an alpha-helix had been already identified by previous biochemical and structural studies. Using TFE we unveiled a previously undetected structural propensity within the N-terminal region of N(TAIL) and showed that its C-terminal region "resists" gaining structure even at high TFE concentrations. Finally, we for the first time showed the reversibility of the induced folding process that N(TAIL) undergoes in the presence of XD. These results highlight the suitability of site-directed spin-labeling EPR spectroscopy to identify protein regions involved in binding and folding events, while providing insights at the residue level.

Biophysics↗
PubMed1984

Molecular dynamics of 3,3',5-triiodo-L-thyronine in model membranes: a spin label study.

A spin-labeled derivative of 3,3',5-triiodo-L-thyronine, 3-[( alpha-carboxy-4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenethyl++ +] carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy (SL-T3) has been synthesized. Evaluation of its binding to nuclei after incubation with rat pituitary tumor GH3 cells at 37 degrees C showed that it bound to nuclei with a 18% potency of that of T3. The dynamic interaction of SL-T3 with multilamellar vesicles prepared from dimyristoylphosphatidylcholine (DMPC) was investigated using electron spin resonance techniques. At 31 degrees C, the lateral diffusion constant of SL-T3 in DMPC membranes was found to be 3.0 X 10(-8) cm2/s as determined by the ESR line-broadening method. The temperature dependency of the ESR spectrum of SL-T3 in DMPC multilamellar vesicles showed a break at 23.5 degrees C, which is close to the main phase-transition temperature, 23.7 degrees C, of DMPC membranes. This suggests that the motion of the probe reflects the motion of phospholipids in DMPC membranes, and that the probe itself does not perturb the membrane structure. SL-T3 appears to be a useful probe for studying the motion of thyroid hormone in the plasma membrane of responsive cells.

Animals↗
PubMed1979

Spin label studies on the interactions of bovine adrenodoxin with NADPH-adrenodoxin reductase and with cytochrome P-450scc.

Adrenodoxin of bovine adrenocortical mitochondria was spin-labeled with two different spin-labeling reagents, N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)imidazole (I) and N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (II), without major loss of its activity for electron transport from NADPH to cytochrome c. The EPR spectrum of adrenodoxin spin-labeled with either of the reagents showed a pattern typical of a moderately immobilized spin label. When adrenodoxin was treated with (I), approximately two amino acid residues per molecule were spin-labeled, whereas a single residue was labeled by (II). While assition of NADPH to adrenodoxin spin-labeled with (I) did not diminish the EPR signal intensity, addition of the reductant to the labeled adrenodoxin in the presence of adrenodoxin reductase caused slow reduction of the spin label, the rate of which was dependent on the aerobicity. Addition of adrenodoxin reductase to adrenodoxin spin-labeled with (I) or (II) resulted in the appearance of a more immobilized component in the EPR spectrum. The ratio of the more immobilized component to the less immobilized component was saturated at a molar ratio of one to one. Addition of cytochrome P-450scc to adrenodoxin labeled with (I) had similar effects on the EPR spectrum.

Adrenal Cortex↗
PubMed1996

Watching proteins move using site-directed spin labeling.

Site-directed spin labeling of proteins has proven to be a practical means for determining secondary structure and its orientation; surfaces of tertiary interactions; inter-residue distances; chain topology and depth of a given side chain from the membrane/aqueous surface in membrane proteins; and local electrostatic potentials at solvent-exposed sites. Moreover, the mobility of a side chain together with its solvent-accessibility may serve to uniquely identify the topographical location of specific residues in the protein fold. Future spectral analysis should permit a quantitative estimation of the contribution of backbone flexibility to the overall side-chain dynamics. The ability to time-resolve the structural features mentioned above makes SDSL a powerful approach for exploring the evolution of structure on the millisecond time scale. We anticipate future applications to the study of protein folding both in solution and in chaperone-mediated systems.

Electron Spin Resonance Spectroscopy↗
PubMed2001

Theoretical analysis of the effect of imperfect slice profiles on tagging schemes for pulsed arterial spin labeling MRI.

Pulsed arterial spin labeling (ASL) techniques provide a noninvasive method of obtaining qualitative and quantitative perfusion images with MRI. ASL techniques employ inversion recovery and/or saturation recovery to induce perfusion weighting, and thus the performance of these techniques is dependent on the slice profiles of the inversion or saturation pulses. This article systematically examines through simulations the effects of slice profile imperfections on the perfusion signal for nine labeling schemes, including FAIR, FAIRER, and EST (UNFAIR). Each sequence is evaluated for quantitative accuracy, suppression of stationary signal, and magnitude of perfusion signal. Perfusion effects are modeled from a modified Bloch equation and experimentally determined slice profiles. The results show that FAIR, FAIRER, and EST have excellent tissue suppression. The magnitude of the perfusion signal is comparable for FAIR and FAIRER, with EST providing a slightly weaker signal. For quantitative measurements, all three methods underestimate the perfusion signal by more than 20%. Of the additional six ASL techniques examined, only one performed well in this model. This method, which combines inversion and saturation recovery, yields improved signal accuracy (<15% difference from the theoretical value) and tissue suppression similar to that of FAIR and its variants, but has only half the signal. Magn Reson Med 46:141-148, 2001.

Humans↗
PubMed2006

Noninvasive measurements of regional cerebral perfusion in preterm and term neonates by magnetic resonance arterial spin labeling.

Magnetic resonance arterial spin labeling (ASL) at 3 Tesla has been investigated as a quantitative technique for measuring regional cerebral perfusion (RCP) in newborn infants. RCP values were measured in 49 healthy neonates: 32 preterm infants born before 34 wk of gestation and 17 term-born neonates. Examinations were performed on unsedated infants at postmenstrual age of 39-40 wk in both groups. Due to motion, reliable data were obtained from 23 preterm and 6 term infants. Perfusion in the basal ganglia (39 and 30 mL/100 g/min for preterm and term neonates, respectively) was significantly higher (p < 0.0001) than in cortical gray matter (19 and 16 mL/100 g/min) and white matter (15 and 10 mL/100 g/min), both in preterm neonates at term-equivalent age and in term neonates. Perfusion was significantly higher (p = 0.01) in the preterm group than in the term infants, indicating that RCP may be influenced by developmental and postnatal ages. This study demonstrates, for the first time, that noninvasive ASL at 3T may be used to measure RCP in healthy unsedated preterm and term neonates. ASL is, therefore, a viable tool that will allow serial studies of RCP in high-risk neonates.

Cerebrovascular Circulation↗
PubMed2006

Hypoperfusion in frontotemporal dementia and Alzheimer disease by arterial spin labeling MRI.

OBJECTIVES: To test if arterial spin labeling (ASL) MRI could detect a pattern of hypoperfusion in frontotemporal dementia (FTD) vs cognitively normal (CN) control subjects; to determine the regional difference of perfusion between FTD and Alzheimer disease (AD); and to determine whether hypoperfusion in FTD correlates with cognitive impairment. METHODS: We included 21 patients with FTD, 24 patients with AD, and 25 CN subjects in this cross-sectional MRI study. All subjects had MRI scans including T1-weighted structural images and ASL-MR images. RESULTS: ASL-MRI detected a pattern of hypoperfusion in right frontal regions in patients with FTD vs CN subjects, similar to PET and SPECT. FTD had higher perfusion than AD in the parietal regions and posterior cingulate. Frontal hypoperfusion in FTD correlated with deficits in judgment and problem solving. Adding frontal perfusion to gray matter (GM) atrophy significantly improved the classification of FTD from normal aging to 74%, and adding parietal perfusion to GM atrophy significantly improved the classification of FTD from AD to 75%. Combining frontal and parietal lobe perfusion further improved the classification of FTD from AD to 87%. CONCLUSION: Frontotemporal dementia and Alzheimer disease display different spatial distributions of hypoperfusion on arterial spin labeling MRI. With further development and evaluation, arterial spin labeling MRI could contribute to the differential diagnosis between frontotemporal dementia and Alzheimer disease.

Adult↗
PubMed2006

Regional variation of cerebral blood flow and arterial transit time in the normal and hypoperfused rat brain measured using continuous arterial spin labeling MRI.

Continuous arterial spin labeling (CASL) is a noninvasive magnetic resonance (MR) method for measuring cerebral perfusion. In its most widely used form, CASL incorporates a postlabeling delay to minimize the sensitivity of the technique to transit time effects, which otherwise corrupt cerebral blood flow (CBF) quantification. For this delay to work effectively, it must be longer than the longest transit time present in the system. In this work, CASL measurements were made in four coronal slices in the rat brain using a range of postlabeling delays. By doing this, direct estimation of both CBF and arterial transit time (delta(a)) was possible. These measurements were performed in the normal brain and during hypoperfusion induced by occlusion of the common carotid arteries. It was found that, in the normal rat brain, significant regional variation exists for both CBF and delta(a). Mean values of CBF and delta(a) in the selected gray matter regions of interest were 233 mL/100 g min and 266 ms, respectively, with the latter ranging from 100 to 500 ms. Therefore, use of a 500-ms postlabeling delay is suitable for any location in the normal rat brain. After common carotid artery occlusion, CBF decreased and delta(a) increased by regionally dependent amounts. In the sensory cortex, delta(a) increased to a mean value of 740 ms, significantly greater than 500 ms. These results highlight the importance of either (a) determining delta(a) as part of the CASL measurement or (b) knowing the approximate range of values delta(a) is likely to take for a given application, so that the parameters of the CASL sequence can be chosen appropriately.

Animals↗
PubMed1984

An electron spin resonance study of temperature-induced structural changes of the spin-labeled myosin head: an evidence for two conformational states of myosin head.

Heavy meromyosin labeled at the SH1 thiol group with an iodoacetamide spin label was studied by electron spin resonance spectroscopy at various temperatures in the presence and absence of nucleotides and PPi. The electron spin resonance spectra of the spin label bound to myosin head showed temperature-dependent changes indicating changes of the structure around the SH1 thiol group of the myosin head. As the temperature was elevated, the bound spin label was more mobilized in all the systems examined. The mobilization of the bound spin label by the elevation of temperature was enhanced in the presence of nucleotides or PPi. The temperature-dependent spectral changes had isosbestic points indicating that the structural changes around the SH1 thiol group took place between two states of the bound spin label, a weakly immobilized and a strongly immobilized state.

Animals↗
PubMed2006

Modeling a spin-labeled fusion peptide in a membrane: implications for the interpretation of EPR experiments.

Site-directed spin-labeling and electron paramagnetic resonance are powerful tools for studying structure and conformational dynamics of proteins, especially in membranes. The position of the spin label is used as an indicator of the position of the site to which it is attached. The interpretation of these experiments is based on the assumptions that the spin label does not affect the peptide configuration and that it has a fixed orientation and distance with respect to the protein backbone. Here, the validity of these assumptions is examined through implicit membrane molecular dynamics simulations of the influenza hemagglutinin fusion peptide that has been labeled with methanethiosulfonate spin label. We find that the methanethiosulfonate spin label can occasionally induce peptide orientations that differ from those adopted by the wild-type peptide. Furthermore, the spin-label resides, on average, several Angstroms deeper in the membrane than the corresponding backbone C(alpha)-atom even at sites pointing toward the solvent. The nitroxide spin label exhibits flexibility and adopts various configurations depending on the surrounding residues.

Amino Acid Sequence↗
PubMed1976

Spin-labeled stearates as probes for microenvironment of murine thymocyte adenylate cyclase-cyclic adenosine 3':5'-monophosphate system.

The interaction of various spin-labeled compounds with the murine thymocyte adenylate cyclase-cyclic AMP system was investigated. Electron paramagnetic resonance spectra from spin-labeled compounds were used to calculate the order parameter, S, and indicated that the thymocyte plasma membrane is a relatively rigid structure. Increasing concentrations of spin-labeled stearates, but not their corresponding methyl esters, resulted in increased membrane fluidity, partial lysis, and concomitant complete inhibition of cholera toxin-mediated increases in cyclic AMP content. Upon subsequent isolation of plasma membranes from these cells, cholera toxin-stimulated adenylate cyclase activity was also completely inhibited. Direct addition of spin-labeled stearates, but not spin-labeled methyl stearates, to thymocyte homogenates caused a dramatic reduction of basal, cholera toxin-, isoproterenol-, NaF-, and prostaglandin E1-stimulated adenylate cyclase activity. Inhibition was complete within the first minute of addition to homogenates and required approximately 0.2 mM spin-labeled stearate I(12,3) for half-maximal inhibition. This inhibition occurred in the presence or absence of an ATP-regenerating system and was not readily reversible. Furthermore, since the membrane cyclic phosphodiesterase activity was not altered by spin-labeled stearates, their inhibition was attributed to a direct action of stearate spin labels on adenylate cyclase. Neither stearate, methyl stearate, spin-labeled methyl stearates nor 2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempo) altered cell viability or enzyme activities at the concentrations studied. Spin-labeled stearates seemed to intercalate into different areas of the plasma membrane than their corresponding methyl esters. Furthermore, the action of spin-labeled stearates appeared to be on the exterior of the plasma membrane rather than the interior. These results illustrate the presence of multilipid domains and the importance of selected lipids and lipid-protein interactions in the adenylate cyclase-cyclic AMP system. Thymocyte adenylate cyclase is described in terms of a current model for membrane proteins.

Adenylyl Cyclases↗
PubMed1979

Synthesis and use of a new spin-labeled analogue of ADP with platelet-aggregating activity.

A new spin-labeled derivative of ADP, 2-(4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl)thioadenosine-5'-diphosphate, has been synthesized. The compound causes both the reversible and irreversible phases of aggregation of human blood platelets at concentrations similar to those required for similar phases of aggregation by ADP itself. The spin-labeled ADP also rivals ADP as a substrate for pyruvate kinase. The interaction of intact human blood platelets and of isolated platelet membranes with the platelet-aggregating spin-labeled derivatives of ADP has been studied. The dramatic decrease in the ESR signal of the spin label is primarily due to chemical reduction of the nitroxide, rather than immobilization of the label. When platelets and spin-labeled ADP are mixed, a rapid burst of nitroxide reduction occurs, followed by a much slower reduction similar in time course to that seen for other spin labels. The rapid burst of reduction, but not the slow reduction, is inhibited by adenosine, an inhibitor of ADP-induced platelet aggregation, and by sulfhydryl-blocking agents. Experiments conducted with Ellman's reagent and platelet membranes or washed platelets revealed a 10 to 30% increase in the number of reactive membrane sulfhydryl groups when ADP was present. These results indicate that there is an increase in the number of reactive sulfhydryl groups on the platelet surface when platelets or membranes are stimulated by ADP.

Adenosine Diphosphate↗
PubMed1997

Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels.

Electron paramagnetic resonance (EPR) spectra of the two-atom-tethered six-membered ring thymidylate spin label (DUMTA) incorporated into duplexes of different sizes were found to display a helix length dependence and a local-order parameter S = 0.32 +/- 0.01 for B-DNA based on the dynamic cylinder model (Keyes, R. S., and A. M. Bobst. 1995. Detection of internal and overall dynamics of a two-atom-tethered spin-labeled DNA. Biochemistry. 34:9265-9276). This sensitivity to size, which reflects global tumbling, is now reported for the more flexible five-atom-tethered five-membered ring thymidylate spin label (DUAP) that can be readily incorporated enzymatically and sequence specifically into nucleic acids of different sizes. The DUAPs containing B-DNA systems were simulated with the same dynamic cylinder model, giving S = 0.20 +/- 0.01 for the more flexibly tethered spin label. This shows that S is dependent on tether length but not on global motion. An analysis with the same motional model of the B-Z transition in a (dG-dC)n polymer containing the five-atom-tethered six-membered ring cytidylate spin label (DCAT) (Strobel, O. K., R. S. Keyes, and A. M. Bobst. 1990b. Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues. Biochemistry. 29:8522-8528) revealed an increase in S from 0.15 +/- 0.01 to 0.26 +/- 0.01 in response to the B- to Z-DNA transition. This indicates that S is not only sensitive to tether length, but also to conformational changes in DNA. Both the DUAP- and the DCAT-labeled systems were also simulated with a base disk model. From the DUAP spectral series, the perpendicular component of the correlation time tau perpendicular describing the spin-labeled base diffusion was found to be sensitive to global tumbling, confirming earlier results obtained with DUMTA. The DCAT polymer results demonstrated that tau perpendicular monitors a conformational change from B- to Z-DNA, indicating that tau perpendicular is also sensitive to local base dynamics. These results confirm that the dynamics of five-atom-tethered nitroxides are coupled to the nucleic acid dynamics and, as with two-atom-tethered spin labels, can be characterized by S and tau perpendicular. The analyses of both spin-labeled systems provide good evidence for spin-labeled base motions within double-stranded DNA occurring on the nanosecond time scale, and establish that both labels can be used to monitor changes in global tumbling and local order parameter due to variations in DNA conformation and protein-DNA interactions.

Base Sequence↗
PubMed1985

Anti-nitroxide immunoglobulin G: analysis of antibody specificity and their application as probes for spin-labeled proteins.

Antibodies have been elicited to the nitroxide spin-label 4-maleimido-2,2,6,6-tetramethyl-piperidinyl-1-oxy conjugated, via protein sulfhydryl groups, to bovine serum albumin. Antibody-hapten cross-reactivity was demonstrated by double immunodiffusion and by a broadening of the nitroxide electron paramagnetic resonance spectrum. The specificity of the antibodies with respect to hapten structure was examined by means of a simple filter binding assay. Under these conditions, antibodies were shown to distinguish between the nitroxide and hydroxylamine derivatives and between spin-labels comprising either five- or six-membered ring structures. In addition, protein-bound nitroxide spin-labels were detected at the nanogram level by immunoblotting. By use of this method, the specificity of the antibody-hapten reaction predicted by the filter binding assay procedure was utilized to differentially detect various types of bound spin-label. Finally, antibodies were used to identify protein-bound nitroxide spin-label of protein fractionated by gel electrophoresis.

Animals↗
PubMed1981

Dynamic states of phospholipids in Escherichia coli B membrane. Electron spin resonance studies with biosynthetically generated phospholipid spin labels.

Mobility of phospholipid hydrocarbons in the Escherichia coli B membrane fractions was studied by labeling phosphatidylethanolamine or phosphatidylglycerol in situ by biosynthetic incorporation of the spin label. For this purpose, CDP-diacylglycerol spin label was synthesized from phosphatidic acid spin label and cytidine 5'-phosphoromorpholidate and purified by thin-layer chromatography. DCP-diacylglycerol spin label was then incorporated into phospholipids biosynthetically. ESR spectra of these E. coli B membrane fractions showed that phosphatidylglycerol tended to interact with membrane proteins through the mediation of Mg2+, whereas phosphatidylethanolamine had less of this tendency and was more involved in the formation of the bulk of the bilayer continuum of the membrane. These conclusions were also supported by labeling membranes with exogenous spin-labeled phospholipids, although there was some indication that exogenous phospholipids were incorporated into sites different from the sites of incorporation of phospholipids newly synthesized in situ.

Cell Membrane↗
PubMed2004

Spin-labelled vacuolar-ATPase inhibitors in lipid membranes.

Two spin-labelled derivatives of the 5-(2-indolyl)-2,4-pentadienoyl class of inhibitors of the vacuolar ATPase have been synthesised and their EPR properties characterised in phospholipid membranes. One spin-labelled inhibitor is the amide derivative of pentadienic acid and 4-amino-TEMPO (INDOL6), and the other is the 3-hydroxymethyl-PROXYL ester (INDOL5). The response of the EPR spectra to the chain-melting transition of dimyristoyl phosphatidylcholine (DMPC) bilayers demonstrates that both derivatives incorporate in phospholipid membranes. The axially anisotropic EPR spectra of INDOL6 in fluid DMPC membranes indicate that the indolyl-pentadienoyl inhibitors intercalate between the lipid chains, in the membrane. INDOL5, designed to possess additional internal segmental mobility, exhibits more nearly isotropic motion of the spin-label moiety in fluid membranes than does INDOL6. The EPR characteristics of INDOL5 are therefore well suited to detecting specific ligand-protein interactions. Progressive saturation EPR experiments with polar and hydrophobic relaxation agents (aqueous Ni2+ and oxygen) show that the nitroxide group is buried in the membrane, with the indole moiety providing the anchor at the membrane polar-apolar interface. Rates of spin-label reduction by externally added ascorbate confirm this assignment. These two spin-labelled derivatives provide complementary EPR probes of the lipid environment (INDOL6), and of ligand-protein interactions (INDOL5), for this class of V-ATPase inhibitor.

Anisotropy↗