19F NMR Probe Research Articles

Incorporation of tryptophan analogues into staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment: spectroscopic studies.

We have biosynthetically incorporated several tryptophan analogues into three forms of Staphylococcal nuclease to investigate the spectroscopic characteristics of these "intrinsic" probes and their effect on the structure of the proteins. The set of tryptophan analogues includes 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan. 5-Hydroxytryptophan and 7-azatryptophan have red-shifted absorbance spectra, and the latter has a red-shifted fluorescence, which is very sensitive to its environment (being heavily quenched in water). The fluorotryptophans can serve as 19F NMR probes, and 4-fluorotryptophan has a very low fluorescence quantum yield, thus making it a "knock-out" fluorescence analogue. The set of proteins studied includes wild-type nuclease, which has a single tryptophan site at position 140; its V66W mutant, which has a second tryptophan at position 66; and the Delta 137-149 fragment, V66W', which only has a tryptophan at position 66. The environments of positions 66 and 140 are significantly different; position 140 is near the end of the long C-terminal alpha-helix and is moderately solvent-exposed, whereas position 66 is in the beta-barrel core region of the protein and is surrounded by apolar side chains. Absorbance and 19F NMR spectra are used to estimate the extent of analogue incorporation for each protein. Steady-state and time-resolved fluorescence data are reported to characterize the emission of the analogues in these positions in the three proteins and to develop the use of the analogues as probes of protein structure and dynamics. Circular dichroism spectra are reported to show that, in all but a couple of cases, the secondary structure of the proteins containing the analogues is not significantly perturbed by the probes. Additionally, fluorescence anisotropy decay data show the variants of wild-type nuclease to have a rotational correlation time similar to that of tryptophan-containing nuclease.

NMR measurements of Ca2+ and H+ transport mediated by A23187 and reconstituted plasma membrane Ca(2+)-ATPase.

NMR-based assays for measuring the fluxes of Ca2+, H+, and ATP in liposomal systems are presented. The 19F NMR Ca(2+)-chelating molecule 5,5-difluoro-1,2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) was trapped inside large unilamellar vesicles and used to monitor passive and A23187-mediated Ca2+ transport into them. The data were analyzed using progress curves of the transport reaction. They demonstrated the general applicability of 5FBAPTA as a 19F NMR probe of active Ca2+ transport. 31P NMR time-courses were used to monitor simultaneously the ATP hydrolysing activity of the reconstituted human erythrocyte Ca(2+)-ATPase and the concomitant acidification of the reaction medium in a suspension of small unilamellar vesicles. Using an estimate of the extraliposomal buffering capacity, the H+/ATP coupling stoichiometry, in the presence of A23187, was estimated from the NMR-derived data at steady state; it amounted to 1.4 +/- 0.3. This result is discussed with respect to the issue of molecular 'slip' in the context of a non-equilibrium thermodynamics model of the pump (accompanying paper in this issue). Importantly, NMR, in contrast to optical detection methods, can potentially register all fluxes and (electro)chemical gradients involved in the Ca(2+)-ATPase-mediated H+/Ca2+ counterport, in a single experiment.

Functional Porphyrins as Receptor Models.

A series of functionalized porphyrins were prepared to show their potential for the models of various types of receptors. Atropisomerization was controlled by the combination of meso substituents and β-substituents. Chiral porphyrins were also prepared according to several different approaches. Molecular recognition functions of these functional porphyrins are also described. Perfluoroalkylporphyrins showed characteristic oxidation potential and can serve as a 19F NMR probe. They are applied to anovel catalyst and to the study of heme-protein interactions.

A Comparison of Three Commercial Perfluorocarbon Emulsions as High-Field 19F NMR Probes of Oxygen Tension and Temperature

The increasing use of perfluorocarbons (PFCs) as probes of tissue oxygenation on the basis of the 19F spin-lattice relaxation rate ( R 1) prompts comparison between several commercially available PFC blood-substitute emulsions, The relative sensitivities to both oxygen and temperature of the emulsions Oxypherol, Oxygent, and Fluosol at 7 T have been investigated, The linear response of R 1 with pO 2 was confirmed for each resonance in the range 4-50°C, In general, R 1 is not a linear function of temperature, and we demonstrate this for perfluorotributylamine (FTBA) over an extended range of temperatures, 239-343 K. Theoretical R 1 values of FTBA were compared with experimental data by computer modeling of relaxation mechanisms, To further understand the structure-function relationships of PFCs, 2D COSY experiments were used for the assignment of resonances and as an aid in interpretation of relaxation mechanisms, Finally, the utility of Oxygent and Oxypherol for measuring tissue pO 2 in perfused rat hearts was compared before, during, and after eight minutes of global ischemia.

(Trifluoromethyl)lumazine derivatives as 19F NMR probes for lumazine protein.

Lumazine protein acts as an electronic excited state transducer in bioluminescence of Photobacterium species. The protein binds 6,7-dimethyl-8-(D-ribityl)lumazine (1) which serves as the fluorophore. This compound also serves as a biosynthetic precursor of riboflavin and is the substrate of the enzyme riboflavin synthase. This enzyme and lumazine protein show considerable sequence homology. The interaction of lumazine apoprotein with several trifluoromethyl analogs of 6,7-dimethyl-8-ribityllumazine was investigated by 19F NMR spectroscopy. Upon binding to the protein, the 19F NMR resonances of the ligand shift to lower field with broadened line widths to around 30 Hz. By comparison, all ligands studied show more complex NMR spectra when bound to riboflavin synthase. Only one position 7 epimer (designated epimer A) of 6,7-bis(trifluoromethyl)-7-hydroxy-8-(D-ribityl)lumazine binds to lumazine apoprotein and riboflavin synthase. The apoprotein can also bind lumazine derivatives with a quarternary C-7. It is suggested that the binding site of lumazine protein corresponds to the donor binding site of riboflavin synthase.

Difluorophosphate as a 19F NMR probe of erythrocyte membrane potential

Erythrocyte membrane potential can be estimated by measuring the transmembrane concentration (activity) distribution of a membrane-permeable ion. We present here the study of difluorophosphate (DFP) as a 19F NMR probe of membrane potential. This bicarbonate and phosphate analogue has a pKa of 3.7 +/- 0.2 (SD, n = 4) and therefore exists almost entirely as a monovalent anion at physiological pH. When it is incorporated into red cell suspensions, it gives two well resolved resonances that arise from the intra- and extracellular populations; the intracellular resonance is shifted approximately 130 Hz to higher frequency from that of the extracellular resonance. Hence the transmembrane distribution of DFP is readily assessed from a single 19F NMR spectrum and the membrane potential can be calculated using the Nernst equation. The membrane potential was independent of, DFP concentration in the range 4 to 59 mM, and haematocrit of the cell suspensions of 31.0 to 61.4%. The membrane potential determined by using DFP was 0.94 +/- 0.26 of that estimated from the transmembrane pH difference. The distribution ratios of intracellular/extracellular DFP were similar to those of the membrane potential probes, hypophosphite and trifluoroacetate. DFP was found to be transported across the membranes predominantly via the electrically-silent pathway mediated by capnophorin. Using magnetization transfer techniques, the membrane influx permeability-coefficient of cells suspended in physiological medium was determined to be 7.2 +/- 2.5 x 10(-6) cm s-1 (SD, n = 4).

19F nuclear magnetic resonance as a probe of structural transitions and cooperative interactions in heavy meromyosin.

An 19F NMR probe has been attached to the reactive sulfhydryl SH1 of the globular heads of rabbit skeletal heavy meromyosin. It serves as a sensitive monitor of the conformational state of the heads of heavy meromyosin in a manner similar to that seen for subfragment-1 (Shriver, J.W., and Sykes, B.D. (1982) Biochemistry 21, 3022-3028; Tollemar, U., Cunningham, K., and Shriver, J.W. (1986) Biochim. Biophys. Acta 873, 243-251). The NMR spectra indicate that there are at least two states for the heads in the SH1 region. The energetics of the interconversion of the two states of heavy meromyosin (HMM) differs significantly from that of S-1. In HMM in the absence of divalent cations, there are two reversible paths between the low temperature and high temperature states with a hysteresis-like behavior. One path is consistent with the head groups behaving independently and similar to S-1 alone. The second path indicates a coupling of the globular head region observed in S-1 with a second region forming a distinctly different cooperative unit. Upon addition of Ca(II) the hysteresis effect is lost and only the second cooperative unit is observed. Two explanations are offered for these results: the globular heads in HMM may couple with the S-2 segment, or the two globular heads of HMM may couple to form a larger cooperative unit. The ability to stabilize the larger cooperative unit with a divalent metal ion implicates a role for the LC2 light chain in coupling regions of the myosin molecule.

Properties of bound trifluoroethanol complexes with horse liver alcohol dehydrogenase

The substrate analogue 2,2,2-trifluoroethanol (TFE) has been used as a 19F NMR probe of the active site of alcohol dehydrogenase from horse liver (LADH). We are able to directly observe a single resonance assigned to TFE in its ternary complex with LADH and nicotinamide adenine dinucleotide (NAD). The chemical shift of this resonance is independent of pH between values of 6.2 and 8.9, suggesting that bound TFE does not change ionization state in this range. Both by 19F NMR self-exchange measurements and by ligand-displacement studies with pyrazole, we also find that displacement of TFE from its ternary complex with NAD is a linear function of proton concentration over a similar pH range, with more rapid desorption occurring at lower pH values. This suggests that the pK of 6.4 for this process seen previously by Kvassman and Pettersson [Kvassman, J., & Pettersson, G. (1980) Eur. J. Biochem. 103, 557] is not due to the ionization of bound TFE. These studies also show that the bound lifetime of TFE in its ternary complex with LADH and NAD is quite long (400s) at pH 8.7, suggesting the use of TFE as a kinetic trapping reagent in single-turnover stopped-flow experiments. Binding isotherms of NAD to LADH saturated with TFE at pH 8.7 or with pyrazole at pH 7.5 reveal essentially no cooperative behavior. The displacement time courses described above are all adequately fit as first-order processes, thus giving no evidence for site heterogeneity or site-site interaction in the binding of these ligands to dimeric LADH. TFE and pyrazole are used as reagents to further explore the question of site-site interaction from a kinetic point of view in the following paper [Anderson, D. C., & Dahlquist, F.W. (1982) Biochemistry (following paper in this issue)].

A study of conformational changes in two β-93 modified hemoglobin A's using a triphosphate spin label

The binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (spin-labeled triphosphate) to normal adult human hemoglobin (HbA) covalently labeled at the beta-93 sulfhydryl groups with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (I) was studied. HbA-I was used as a model for HbA labeled at the beta-93 SH groups with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (II) since the binding of SLTP to HbA-II could not be measured conveniently, in the presence of the paramagnetic resonance signal of II. Both HbA-I and HbA-II can be treated as variant hemoglobins with abnormal beta chains. The oxygen and SLTP binding data from HbA-I and oxygen binding data from HbA-II are consistent with a concerted transition model for cooperativity which assumes nonequivalence between alpha and beta subunits (GCT model). The distribution of environments "seen" by conformation sensitive probes such as II and trifluoracetone (19F NMR probe) attached to the beta-93 sulfhydryl groups of HbA can also be accounted for by the GCT model. It is proposed that the beta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group. Structural changes caused by ligation of the alpha-hemes are not discussed.

19F NMR probes for proteins; S-trifluoromethylmercuri-papain

19F NMR probes for proteins; S-trifluoromethylmercuri-papain

Fluorine-19 as a covalent active site-directed magnetic resonance probe in aspartate transaminase.

Phosphypyridoxyl trifluoroethylamine has been synthesized as an active site-directed 19F NMR probe for aspartate transaminase. This coenzyme derivative adds stoichiometrically to the apotransaminase as observed by both fluorescence and circular dichroism measurements. The fluorinated phosphypyridoxamine derivative, when bound to the apotransaminase, will not dissociate upon extensive dialysis or passage through Sephadex G-25. The compound behaves as a pyridoxamine phosphate derivative and not as a coenzyme-substrate complex, since both competing anions and dicarboxylic acid inhibitors still bind to the phosphopyridoxyl trifluoroethylamine enzyme. The 19F NMR spectra of the enzyme-bound phosphopyridoxyl trifluoroethylamine were measured as a function of pH, ionic strength, and temperature. The 19F MNR of the enzyme-bound coenzyme derivative revealed no predetermined asymmetry in the subunits of aspartate transaminase insolution in terms of differences in chemical shift or resonance line shape between the two environments. A pH-dependent chemical shift change of the single 19F resonance was observed, which is consistent with the influence of a single ionization with an apparent pKa of 8.4 in 0.10 M KCl at 30 degrees. Increasing the ionic strength resulted in increasing values for the observed pKa, the highest recorded value was 9.1 in 3.0 M KCl. The temperature dependence of the pH titration of the chemical shift gives deltaH' of ionization of 10.5 kcal/mol. The evidence suggests a possible epsilon-amino group, electrostatically affected by positive charges, being responsible for the titration effect of the active site-bound fluorine derivative of pyridoxamine phosphate.