Hexacyanochromate ion as a paramagnetic anion probe for active sites of enzymes

Hexacyanochromate ion, (Cr(CN)6)3-, was applied to ribonuclease T1 (RNase T1), which specifically cleaves RNA chains at guanylic acid residues. From kinetic studies, this anion was shown to bind to the active site of RNase T1 as a competitive inhibitor. Therefore, the line broadening effect of NMR resonances due to binding of (Cr(CN)6)3- was analyzed for the mapping of the active site of RNase T1. His-40 C2 proton resonance was significantly broadened, following His-92 C2 proton resonance upon binding of (Cr(CN)6)3-, while His-27 C2 proton resonance did not show any appreciable line broadening. Moreover, from the pH dependence of the line broadening effect, the binding of (Cr(CN)6)3- was shown to be controlled by the ionic state of Glu-58. Based on the present NMR results and x-ray crystal structure, the active site structure of RNase T1 is discussed.     

Protein Linewidth and Solvent Dynamics in Frozen Solution NMR

Solid-state NMR of proteins in frozen aqueous solution is a potentially powerful technique in structural biology, especially if it is combined with dynamic nuclear polarization signal enhancement strategies. One concern regarding NMR studies of frozen solution protein samples at low temperatures is that they may have poor linewidths, thus preventing high-resolution studies. To learn more about how the solvent shell composition and temperature affects the protein linewidth, we recorded ¹H, ²H, and ¹³C spectra of ubiquitin in frozen water and frozen glycerol-water solutions at different temperatures. We found that the ¹³C protein linewidths generally increase with decreasing temperature. This line broadening was found to be inhomogeneous and independent of proton decoupling. In pure water, we observe an abrupt line broadening with the freezing of the bulk solvent, followed by continuous line broadening at lower temperatures. In frozen glycerol-water, we did not observe an abrupt line broadening and the NMR lines were generally narrower than for pure water at the same temperature. ¹H and ²H measurements characterizing the dynamics of water that is in exchange with the protein showed that the ¹³C line broadening is relatively independent from the arrest of isotropic water motions.     

Monoanion inhibition and 35Cl nuclear magnetic resonance studies of renal dipeptidase.

Kinetic analyses of monoanion inhibition and 15Cl nuclear magnetic resonance at 5.88 MHz were employed to study monoanion interactions with the zinc metalloenzyme, renal dipeptidase. The enzyme-catalyzed hydrolysis of glycyldehydrophenylalanine exhibited competitive inhibition when the reaction rate was determined in the presence of the monovalent anions fluoride, chloride, bromide, iodide, azide, nitrate, or thiocyanate or upon the addition of the divalent anion, sulfate. Competitive inhibition was produced by these anions. One anion was bound per enzyme molecule, and except in the case of fluoride all of the anions appeared to bind at the same site. Cyanide ion produced a much more effective inhibition of renal dipeptidase than the other monoanions, and it was shown that two cyanide ions were bound per enzyme molecule. An investigation of the effect of pH upon monoanion inhibition suggested that the anion inhibitors bind to the group with a pK of approximately 7.8. Complete dissociation of this group (approximately pH 8.4) eliminates the inhibitory effect of anions. The 35Cl line broadening produced by renal dipeptidase in 0.5 M NaCl solutions was 100 times more effective than that produced by equivalent concentrations of aquozinc(II). The line broadening was dependent upon the concentration of the metalloenzyme and independent of the frequency of the exciting radiation. When zinc ion was removed from the metalloenzyme by dialysis or when chloride was titrated from the metalloenzyme by cyanide, line broadening was decreased. Treatment of renal dipeptidase with saturating concentrations of the competitive inhibitor, guanosine triphosphate, in the presence of 0.5 M NaCl also produced a significant decrease in the 35Cl line width. The 35Cl line broadening produced by renal dipeptidase was shown to decrease with increasing pH through the range pH 5.8-10.8. This line-width variation with pH appeared to result from the titration of a site on the metalloprotein with an approximate pK of 7.4. Temperature studies of 35Cl line broadening by the metalloenzyme in the presence of chloride and cyanide inhibitors suggest that the fast exchange process pertains and that the dominant relaxation mechanism is quadrupolar in nature.     

Methionine-90-spin-labeled bovine alpha-lactalbumin: electron spin resonance and NMR distance measurements

The unique methionine residue of bovine alpha-lactalbumin was modified by irreversible alkylation with the bromoacetamido nitroxide spin-label 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-N-oxyl. The line shape of the electron spin resonance (ESR) spectrum was indicative of a fairly mobile spin-label and was sensitive to the calcium-induced conformational change. Paramagnetic broadening of the spin-label ESR lines by a Gd(III) ion substituted at the high-affinity calcium site of the protein yielded a distance between the spin-label and the metal-binding site of 8.0 +/- 1.0 A. The extent of the paramagnetic line broadening by the covalently attached nitroxide spin-label on the proton resonances of several amino acid residues of the protein at 500 MHz allowed estimation of intramolecular distances between the methionine-90 residue and several resolvable protons.     

13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase

Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe 13C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO2 in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the 13CO NMR resonance; however, the chemical shift of the 13CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The 13CO line broadening occurs with a rate constant (1080 s-1 at 20 degrees C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between 13CO and CODH-bound 13CO2 because 13CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the 13CO2 line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between 12CO and 13CO2 in solution was shown to occur at the same rate as that of CO2 reduction, which is approximately 750-fold slower than the rate of 13CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN-) was also probed by 13C NMR. A functional C-cluster is not required for 13CN- broadening (unlike for 13CO), and its exchange rate constant is 30-fold faster than that for 13CO. The combined results indicate that the 13CO exchange includes migration of CO to the C-cluster, and CO oxidation to CO2, but not release of CO2 or protons into the solvent. They also provide strong evidence of a CO2 binding site and of an internal proton transfer network in CODH. 13CN- exchange appears to monitor only movement of CN- between solution and its binding to and release from CODH.     

Chloride binding to photosystem II in the dark is in slow exchange

We have studied the 35Cl- NMR line broadening in the presence of photosystem II (PS II) membranes from spinach in the dark. In the presence of NH3 (which other work has shown to competitively inhibit chloride binding to PS II) we observed no decrease in 35 Cl- linewidths. We conclude that binding of Cl- to the O2 evolving center in PS II in the dark (previously demonstrated by EPR) is in slow exchange on the NMR timescale. We assign the observed line broadening to interaction with non-specific binding sites and with free paramagnetics.     

Heterogeneity of Protein Substates Visualized by Spin-label EPR

The energy landscape of proteins is characterized by a hierarchy of substates, which give rise to conformational heterogeneity at low temperatures. In multiply spin-labeled membranous Na,K-ATPase, this heterogeneous population of conformations is manifest by strong inhomogeneous broadening of the electron paramagnetic resonance (EPR) line shapes and nonexponential spin-echo decays, which undergo a transition to homogeneous broadening and exponential relaxation at higher temperatures (previous study). In this study, we apply these EPR methods to small water-soluble proteins, of the type for which the existence of conformational substates is well established. Both α-helical and β-sheet aqueous proteins that are spin-labeled on a single cysteine residue display spin-echo decays with a single phase-memory time T_2M and conventional EPR line shapes with predominantly homogeneous broadening, over a broad range of temperatures from 77 K to ∼ 250 K or higher. Above ∼ 200 K, the residual inhomogeneous broadening is reduced almost to zero. In contrast, both the proteins and the spin label alone, when in a glycerol-water mixture below the glass transition, display heterogeneity in spin-echo phase-memory time and a stronger inhomogeneous broadening of the conventional line shapes, similar to multiply spin-labeled membranous Na,K-ATPase below 200 K. Above 200 K (or the glass transition), a single phase-memory time and predominantly homogeneous broadening are found in both spin-label systems. The results are discussed in terms of solvent-mediated protein transitions, the ability of single spin-label sites to detect conformational heterogeneity, and the desirability of exploring multiple sites for proteins with the size and complexity of the Na,K-ATPase.     

Heterogeneity of Protein Substates Visualized by Spin-label EPR

The energy landscape of proteins is characterized by a hierarchy of substates, which give rise to conformational heterogeneity at low temperatures. In multiply spin-labeled membranous Na,K-ATPase, this heterogeneous population of conformations is manifest by strong inhomogeneous broadening of the electron paramagnetic resonance (EPR) line shapes and nonexponential spin-echo decays, which undergo a transition to homogeneous broadening and exponential relaxation at higher temperatures (previous study). In this study, we apply these EPR methods to small water-soluble proteins, of the type for which the existence of conformational substates is well established. Both α-helical and β-sheet aqueous proteins that are spin-labeled on a single cysteine residue display spin-echo decays with a single phase-memory time T_2M and conventional EPR line shapes with predominantly homogeneous broadening, over a broad range of temperatures from 77 K to ∼ 250 K or higher. Above ∼ 200 K, the residual inhomogeneous broadening is reduced almost to zero. In contrast, both the proteins and the spin label alone, when in a glycerol-water mixture below the glass transition, display heterogeneity in spin-echo phase-memory time and a stronger inhomogeneous broadening of the conventional line shapes, similar to multiply spin-labeled membranous Na,K-ATPase below 200 K. Above 200 K (or the glass transition), a single phase-memory time and predominantly homogeneous broadening are found in both spin-label systems. The results are discussed in terms of solvent-mediated protein transitions, the ability of single spin-label sites to detect conformational heterogeneity, and the desirability of exploring multiple sites for proteins with the size and complexity of the Na,K-ATPase.     

Ferrocyanide carbon-13 NMR line broadening as a probe of electron transfer reactions

The electron transfer reaction between ferrocyanide ion and the blue copper protein, stellacyanin, has been investigated by means of 13C NMR line broadening of the inorganic oxidant. The temperature dependence of the ferrocyanide line broadening gives an activation energy for the electron transfer reaction of 17 +/- 3 kJ. The apparent rate constant decreases with increasing concentration of K4Fe(CN)6, a result which can be explained either by formation of a strong precursor ferrocyanide--stellacyanin [Cu(II)] complex or by increased formation of KFe(CN)3-6 ion pairs. The direct electron transfer between ferrocyanide and ferricyanide has also been studied by 13C NMR line broadening of the former species. The ferricyanide concentration dependence of the exchange line broadening yields a value for the apparent second-order rate constant at 25 degrees C of k = 1.65 . 10(3) M-1 . s-1, in agreement with previously reported values derived from 14N NMR and isotope exchange studies. This rate constant shows a linear dependence on the K+ concentration, independent of ionic strength, a result which confirms the importance of ion pair species such as KFe(CN)3-6 and KFe(CN)2-6 in the direct electron transfer mechanism. The general applications of the method are discussed, including the considerations which suggest that a wide range of electron transfer rates, from about 1 s-1 to 4 . 10(3) s-1, are, in principle, accessible to this technique. The potential utility of ferrocyanide 13C spin--lattice relaxation time measurements is decreasing the lower limit of this range is also discussed.     

Proton magnetic resonance study of cycloheximide-ribosome interactions

Cycloheximide-ribosome interactions from sensitive and resistant organisms were studied by proton magnetic resonance spectroscopic techniques. The two methyl resonances of cycloheximide upon interaction with ribosomes from Saccharomyces cerevisiae showed preferential broadening. Comparison of cycloheximide line broadening as effected by ribosomes from S. cerevisiae (sensitive) and Microsporum canis (resistant) revealed that less cycloheximide is bound to the M. canis ribosomes. From the decrease in line broadening observed with increasing temperature it may be concluded that cycloheximide-ribosome interaction is a fast exchange reaction. Tetracycline did not compete with cycloheximide for binding site(s) on the ribosomes of S. cerevisiae.     

Molecular motion at the critical point in lipid membranes

It is shown that the calculated temperature dependence of the deuterium nuclear magnetic resonance (NMR) of labeled cholesterol-phospholipid bilayers is changed by magic-angle spinning. This makes it possible to distinguish different molecular motions at temperatures near a miscibility critical point, for motions with distinct temperature-dependent correlation times. In this way one finds that NMR line broadening due to critical point composition fluctuations can be distinguished from broadening due to the formation and dissociation of complexes.     

Analytic approximations for the broadening of the spectral lines of hydrogen-like ions

Broadband approximate expressions for calculating the broadening of the spectral lines of hydrogen-like ions in a multicomponent plasma are derived taking into account both the influence of the interaction between plasma particles on the distribution function of the plasma microfield and the effect of the microfield dynamics on the broadening of the central component of the spectral line. With the approximate expressions proposed, the calculation of the shape of a given spectral line of a certain ion in a plasma with a given ion composition requires only a few seconds of computer time. The approximate expressions provide a good computational accuracy not only for the central component of the spectral line but also for the spectral line wings.     

Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.     

Conformational relaxation of a low-temperature protein as probed by photochemical hole burning. Horseradish peroxidase.

For the first time, conformational relaxation processes have been measured in a small protein, mesoporphyrin-horseradish peroxidase via their influence on spectral diffusion broadening of holes burnt in the fluorescence excitation spectrum of free base mesoporphyrin. Holes were burnt in three 0----0 bands of different tautomeric forms of the chromophore at 1.5 and 4 K, and the spectral diffusion broadening was measured in temperature cycling experiments between 4 and 30 K. The inhomogeneous linewidth for the tautomeric 0----0 bands was estimated to be 60-70 cm-1; the hole width was found narrow, being in the order of 350 MHz (10(-2) cm-1) at 1.5 K what allowed for an extremely sensitive detection of the conformational changes. Though proteins have many features in common with glasses, the spectral diffusion broadening of photochemical holes under temperature cycling conditions in mesoporphyrin horseradish peroxidase has a very different pattern as a function of temperature. Up to 12 K, the linewidth did not significantly change, then around 14 K; a steplike broadening was observed for all three tautomers, although to a different extent. The total magnitude of line broadening up to 30 K was large and also different for the tautomers. We argue that the difference between the behavior of this protein and that of glassy matrices originate from finite size effects; the protein may be characterized by a small number of TLS, and their distribution may bear discrete features.     

Thermal broadening of the Soret band in heme complexes and in heme-proteins: role of iron dynamics

We report the thermal broadening of the Soret band in heme-CO, heme-OH and protoporphyrin IX in the temperature range 300–20 K. For protoporphyrin IX the temperature dependent Gaussian line broadening follows the behavior predicted by the harmonic approximation in the entire temperature range investigated. In contrast, for heme-CO and heme-OH the harmonic behavior is obeyed only up to about 180 K and an anomalous line broadening increase is observed at higher temperatures. This effect is attributed to the onset of anharmonic motions of the iron atom with respect to the porphyrin plane. Comparison with previously reported analogous data for heme proteins enables us to suggest that the onset of substate interconversions in these latter systems can be reflected in motions of the iron atom with respect to the porphyrin plane. Correspondence to: L. Cordone     

81 Br-NMR studies of carbonic anhydrase

⁸¹Br nmr measurements have been made on high (bovine; BCA) and low (human-B; HCAB) specific activity forms of carbonic anhydrase and on a chemically modified form of the human enzyme (carboxyamidomethyl; CAM-HCAB). The high specific activity form of the enzyme, BCA, exhibits a ⁸¹Br line broadening which is determined by the lifetime of Br^? bound to the zinc ion of the enzyme. The low specific activity form of the enzyme, HCAB, under similar conditions of concentration, pH, etc., does not exhibit a ⁸¹Br nmr line broadening. $\text{Cl}{}^{\mathrm{?}}\text{Br}{}^{\mathrm{?}}$ competitive binding studies, using ³⁵Cl nmr, suggests that the failure to observe ⁸¹Br broadening is due to an increase in the lifetime of a zinc bound Br^?. An increase in this lifetime by a factor of 10–100 over that exhibited by BCA is sufficient to abolish the line broadening. A modified form of HCAB, CAM-HCAB, does, however, exhibit a ⁸¹Br nmr line broadening. Estimates of the lifetime of zinc bound Br^?, τ_M, are 4 × 10^?7 sec. for CAM-HCAB at pH 8 and 1 × 10^?7 sec. for BCA at pH 7. The lifetime for Br^? bound to HCAB is estimated to be ≥10^?6 sec.     

Cryogenic solid state NMR studies of fibrils of the Alzheimer’s disease amyloid-β peptide: perspectives for DNP

Dynamic Nuclear Polarization solid-state NMR holds the potential to enable a dramatic increase in sensitivity by exploiting the large magnetic moment of the electron. However, applications to biological solids are hampered in uniformly isotopically enriched biomacromolecules due to line broadening which yields a limited spectral resolution at cryogenic temperatures. We show here that high magnetic fields allow to overcome the broadening of resonance lines often experienced at liquid nitrogen temperatures. For a fibril sample of the Alzheimer’s disease β-amyloid peptide, we find similar line widths at low temperature and at room temperature. The presented results open new perspectives for structural investigations in the solid-state.     

Dynamic NMR studies of ligand-receptor interactions: design and analysis of a rapidly exchanging complex of FKBP-12/FK506 with a 24 kDa calcineurin fragment.

Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP-12/ FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP-12/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA). Analysis of 1H-13C HSQC data acquired for the FKBP-12/ 13C-FK506 and FKBP-12/13C-FK506/CnB/BBD complexes indicates that FKBP-12/FK506 and CnB/BBD are in fast exchange in the quaternary complex. Comparison of proton line widths shows significant broadening of resonances along the macrocycle backbone at 13-CH, 13-OMe, 15-OMe, 18-CH2, 20-CH, 21-CH, and 25-Me, as well as moderate broadening on the macrocycle backbone at 17-Me, 24-CH, and the pyranose 12-CH2 protons. The tri-substituted olefin and cyclohexyl groups also show moderate broadening at the 27-Me, 28-CH, and 30-CH2 positions, respectively. Unexpectedly, little line broadening was observed for the allyl resonances of FK506 in the quaternary complex, although 13C longitudinal relaxation measurements suggest this group also makes contacts with calcineurin. In addition, intermolecular transfer NOE peaks were observed for the allyl 37-CH2, 21-CH, 30-CH2, 13-OMe, 15-OMe, 17-Me, 25-Me, and 27-Me groups, indicating that these are potential sites on the FK506 molecule that interact with calcineurin.     

Studies of Pargyline-Monoamine Oxidase Binding Using a Spin Label Probe Analog

Abstract: Analogs of the monoamine oxidase (MAO) inhibitor pargyline with a nitroxide free radical moiety attached through an ether linkage to the para position on the benzene ring have been prepared and reacted with solubilized MAO preparations from rat and beef brain and pig liver. These compounds behave as normal irreversible inhibitors of catalytic activity, with some preference for B-type enzyme. When the reaction was monitored by electron spin resonance (ESR), line broadening effects indicative of binding and with an apparent relation to substrate specificity of the preparation were observed. In addition, there was a slow decrease in intensity of the ESR spectra, which could be retarded by the addition of other MAO inhibitors or increased O₂ and enhanced by flavin reduction. It appears to be related to development of the irreversible phase of MAO inhibition. Signal recovery with added O₂ and studies of a model reaction with free flavin, suggest the signal loss to be a line broadening effect due to interaction with an enzyme-generated paramagnetic species rather than to direct reduction of the nitroxide radical.     

31P nuclear magnetic resonance studies on relaxation parameters and line broadening of intracellular metabolites of HeLa cells.

The 40-MHz ³¹P nuclear magnetic resonance (nmr) spectrum of intact HeLa cells contains seven broad peaks with some detectable splittings. The linewidths were significantly broader than for those of cell-free systems such as cell extracts, indicating that the cellular environment is responsible for the unusual line broadening. Resolution of these peaks at 40 MHz is sufficient to make certain assignments and the relaxation parameters of some of the intracellular metabolites have been measured. The spin-lattice relaxation times (T₁) ranged from 0.3 s for adenosine triphosphate (ATP) to about 3 s for inorganic phosphate (P_i) and monophosphate compounds. Nuclear Overhauser enhancements (NOE) were induced by proton irradiation with the possible exception of ATP. The relaxation parameters were compared to those of cell-free compounds and in all cases T₁ and NOE were smaller for the intracellular metabolites. The relaxation parameters for ATP were affected the most. This behavior was mimicked with mixtures of cell-free metabolites containing paramagnetic ions. The larger change in both T₁ and NOE of intracellular ATP could be accounted for by selective binding of paramagnetic ions. This phenomenon also explains some of the line broadening in the cell spectrum especially that of ATP. The spin-spin relaxation times (T₂) of P₁ and monophosphate compounds as measured by a pulse technique did not account for the observed linewidths. This is due to the presence of chemical shift envelopes arising from pH heterogeneity. All resonances were broader at 146 MHz because of the line broadening by paramagnetic ions and the presence of chemical shift envelopes. Other mechanisms of line broadening may also be significant. There was little difference in resolution of spectra at 40 and 146 MHz. Water proton linewidths and T₂ values were measured for HeLa cells and for some minced tissue preparations. The water linewidth in tissue samples was broader than that in the cell suspension. The large linewidths in tissues arise mainly from chemical shift envelopes caused by magnetic field nonuniformity in the tissue samples. There appears to be a small chemical shift envelope from magnetic nonuniformity in HeLa cells as well. The ¹H results on envelopes were extrapolated to ³¹P studies on cells and tissues. Possible methods for reducing linewidths arising from the various proposed broadening mechanisms were discussed.