First structural investigation of the restriction ribonuclease RegB: NMR spectroscopic conditions, 13C/15N double-isotopic labelling and two-dimensional heteronuclear spectra

The bacteriophage T4 genome-encoded ribonuclease RegB is the unique well-defined restriction endoribonuclease. This protein cleaves with an almost absolute specificity its RNA substrate in the middle of the GGAG tetranucleotide mainly found in the Shine-Dalgarno sequence (required for the prokaryotic initiation of the translation). This protein has no significant homology to any known ribonuclease and its structure has never been investigated. The extreme toxicity of this ribonuclease prevents the expression of large quantities for structural studies. Here, we show that the toxicity of RegB can be bypassed by using the RegB H48A point mutant and explain why resolving the structure of this mutant is relevant. For nuclear magnetic resonance (NMR) purposes, we report the preparation of highly pure (13)C/(15)N double-labelled 1.2mM samples of RegB H48A using a high yield expression procedure in minimal medium (30 mg/L). We also present a set of solution conditions that maintain the concentrated samples of this protein stable for long periods at the NMR-required temperature. Finally, we present the first (1)H/(15)N and (1)H/(13)C two-dimensional NMR spectra of RegB H48A. These spectra show that the protein is folded and that the full structural analysis of RegB by NMR is feasible.     

The determination of intracellular sodium concentration in human red blood cells: nuclear magnetic resonance measurements.

The intracellular sodium concentration and intracellular volume of human red blood cells were determined from 23Na and 1H NMR spectra. It is shown that sodium dissolved in the intracellular water has a concentration higher than that previously published. The intracellular sodium concentration measured was 11.4 +/- 3.1 mM. A comparison of different NMR methods used to determine sodium concentration is given.     

NMR study of the positions of His-12 and His-119 in the ribonuclease A-uridine vanadate complex.

The binding of uridine vanadate to ribonuclease A has been investigated by one- and two-dimensional 1H NMR. The homonuclear Nuclear Overhauser and exchange spectroscopy spectrum of the uridine vanadate/RNase A complex exhibits cross peaks between both the C5H and C6H protons of uridine vanadate and the H epsilon 1 proton of His-12 of ribonuclease A. These cross peaks suggest that the H epsilon 1 proton of His-12 is in the vicinity of the uracil base of uridine vanadate, as observed in the crystallographic structure of the uridine vanadate/RNase A complex. However, no cross peaks are observed between the C5H and C6H protons of uridine vanadate and the H epsilon 1 proton of His-119 of ribonuclease A, although they were predicted based upon the distances calculated from coordinates of the crystallographic structure of the complex. These results suggest that there is a significant difference between the positioning of the His-119 side chain in the solution and in the crystallographic structures.     

The study of diabetic cataractogenesis in the intact rabbit lens by deuterium NMR spectroscopy

The polyol pathway has been implicated in the process of diabetic cataractogenesis. We report the use of deuterium (2H) spectroscopy for dynamically monitoring the polyol and glycolytic pathways in the single intact rabbit lens. Using 2H labeled C-1 D-glucose, the formation of sorbitol from glucose and the metabolism of sorbitol to fructose was dynamically monitored at 5.5 mM and 35.5 mM glucose concentrations. The accumulation of sorbitol at 35.5 mM glucose concentration was prevented by the inhibition of aldose reductase using an inhibitor (Sorbinil). 2H spectra were obtained in short acquisition times because of the short T1's of deuterated metabolites. A further advantage of 2H spectroscopy is that the natural abundance resonance of water (HDO) can be used as an internal reference standard. These findings confirm previous studies and demonstrate for the first time by NMR spectroscopy activity in the polyol pathway at low glucose concentrations.     

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments.

The deuterium exchange kinetics of the C(2) protons of the four histidine residues of native bovine pancreatic ribonuclease A have been followed at pH 6.5 and 8.0 by proton magnetic resonance spectroscopy (1H NMR). Comparison of the order of exchange of the histidine peaks with tritium exchange rates into individual histidine residues [Ohe, M., Matsuo, H., Sakiyama, F., and Narita, K. (1974), J. Biochem. (Tokyo) 75, 1197] supports the previous assignment of histidine NMR peaks H(1) and H(4) to histidine-105 and histidine-48 but requires reassignment of peaks H(2) and H(3) to histidine-119 and histidine-12, respectively. Ribonuclease A samples having differentially deuterated histidines have been used to verify the existence of crossover points in the histidine proton magnetic resonance titration curves and to observe the discontinuous titration curve of histidine-48. Proton magnetic resonance peaks have been assigned to the C(4) protons of the four histidine residues of ribonuclease A on the basis of their unit proton areas and by matching their titration shifts with the more readily visible C(2)-H peaks of the histidines. The pK' values derived from the C(4)-H data agree, within experimental limits, with those derived from C(2)-H data. The C(4)-H peaks were assigned to histidine-12, -48, -105, and -119 of ribonuclease A on the basis of their pH dependence, pK' values, shifts of their pK' values in the presence of inhibitor cytidine 3'-phosphate, and by comparison with the assignments of the histidine C(2)-H peaks above.     

A ribonuclease with antimicrobial, antimitogenic and antiproliferative activities from the edible mushroom Pleurotus sajor-caju

A 12 kDa ribonuclease preferential for poly U and with much lower activity toward poly A, poly G and poly C was isolated from fresh fruiting bodies of the mushroom Pleurotus sajor-caju. A purification procedure involving ion exchange chromatography on CM-cellulose, affinity chromatography on Red-Sepharose and Heparin-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75 was used. The ribonuclease was adsorbed on all of the first three types of chromatographic media. It exhibited some activity toward herring sperm DNA and calf thymus DNA. The ribonuclease activity was unaffected in the presence of KCl (10 and 100 mM) and NaCl (100 mM and 1 M), but was strongly inhibited by CuSO4 (0.01 and 0.1 mM) and less potently inhibited by other divalent salts including MgCl2, CaCl2, ZnCl2, ZnSO4 and FeSO4. The optimal pH was 5.5 and the ribonuclease was stable up to 60 degrees C for 1 h. The ribonuclease inhibited mycelial growth in the fungi Fusarium oxysporum and Mycosphaerella arachidicola with an IC50 value of 95 and 72 microM, respectively. Out of the 12 species of bacteria tested, only Pseudomonas aeruginosa and Staphylococcus aureus were inhibited in growth by the ribonuclease. Viability of the tumor cells HepG2 (hepatoma) and L1210 (leukemia) was reduced with an IC50 of 0.22 and 0.1 microM, respectively in the presence of the ribonuclease. The ribonuclease inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 158 nM and 3H-methyl-thymidine uptake by murine splenocytes with an IC50 of 65 nM.     

DNA hairpin structures in solution: 500-MHz two-dimensional 1H NMR studies on d(CGCCGCAGC) and d(CGCCGTAGC)

A hairpin structure contains two conformationally distinct domains: a double-helical stem with Watson-Crick base pairs and a single-stranded loop that connects the two arms of the stem. By extensive 1D and 2D 500-MHz 1H NMR studies in H2O and D2O, it has been demonstrated that the DNA oligomers d(CGCCGCAGC) and d(CGCCGTAGC) form hairpin structures under conditions of low concentration, 0.5 mM in DNA strand, and low salt (20 mM NaCl, pH 7). From examination of the nuclear Overhauser effect (NOE) between base protons H8/H6 and sugar protons H1' and H2'/H2", it was concluded that in d(CGCCGCAGC) and d(CGCCGTAGC) all the nine nucleotides display average (C2'-endo,anti) geometry. The NMR data in conjunction with molecular model building and solvent accessibility studies were used to derive a working model for the hairpins.     

A Fourier transform infrared investigation of the structural differences between ribonuclease A and ribonuclease S

Fourier transform infrared spectroscopy was used to investigate the small conformational differences which exist between ribonuclease A and ribonuclease S in aqueous systems. Deconvolution and derivative methods were used to observe the overlapping components of the amide I and II bands. These proteins give identical spectra in H2O and after complete exchange in 2H2O. However structural differences are revealed by monitoring the rate of 1H-2H exchange by Fourier transform infrared spectroscopy. At equivalent times of exposure in 2H2O buffer ribonuclease S undergoes greater isotopic exchange than ribonuclease A. Thus complete exchange takes place for ribonuclease S but not ribonuclease A after incubation at room temperature for 8 days. Complete 1H-2H exchange of ribonuclease A was achieved by incubation at 62 degrees C for 30 min. The available X-ray data and comparison with the infrared spectra of other soluble proteins was used to assign the components of the amide I and II bands to various secondary structures. In particular, band shifts observed during the later stages of exchange are associated with slowly exchanging residues in beta-strand and alpha-helical regions. The higher rate of exchange for ribonuclease S is associated with a greater conformational flexibility and a more open structure. The results show that it is necessary to be cautious in making band assignments based on exchange methods unless the extent of exchange is known. Furthermore, it is seen that the combination of Fourier transform infrared spectroscopy and hydrogen-deuterium exchange is a powerful technique for revealing small differences in protein secondary structure.     

Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy

Summary The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). ¹H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pK_a values of the histidine residues obtained using ¹H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D ¹H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.     

Biochemical characterization of RNase HII from Aeropyrum pernix

Aeropyrum pernix contains one homolog of ribonuclease H (RNase H), A. pernix RNase HII (Ape-RNase HII). Activity characterization showed that Ape-RNase HII exhibited the highest activity in the presence of 5 mM Mn(2+), 1 mM Co(2+), or 10 mM Mg(2+), respectively; however, its cleavage efficiencies at different cleavage sites for Mn(2+) and Mg(2+) were different. Ape-RNase HII cleaved 12-bp RNA/DNA substrates at multiple sites and the optimum pH value was 11.0. Moreover, 16-bp DNA-r4-DNA/DNA and 13-bp DNA-r1-DNA/DNA chimeric substrates were cleaved at DNA-RNA junction. Ape-RNase HII was thermostable and the stabilization was enhanced with increased salt concentration. This work is believed to be the first in vitro functional study of Ape-RNase HII and the results should contribute to the analysis of RNase H of other archaeal species.     

Characterizing bathocuproine self-association and subsequent binding to Alzheimer's disease amyloid beta-peptide by NMR.

Aggregated amyloid beta-peptide (A beta) is the primary constituent of the extracellular plaques and perivascular amyloid deposits associated with Alzheimer's disease (AD). Deposition of the cerebral amyloid plaques is thought to be central to the disease progression. One such molecule that has previously been shown to 'dissolve' deposited amyloid in post-mortem brain tissue is bathocuproine (BC). In this paper 1H NMR chemical shift analysis and pulsed field gradient NMR diffusion measurements were used to study BC self-association and subsequent binding to A beta. The results show that BC undergoes self-association as its concentration increases. The association constant of BC dimerization, Ka, was estimated to be 0.64 mM(-1) at 25 degrees C from 1H chemical shift analysis. It was also found that dimerization of BC appeared to be essential for its binding to A beta. From the self-association constant of BC, Ka, the fraction of dimeric BC in the complex was obtained and the dissociation constant, Kd, of BC bound to A beta40 peptide was then determined to be approximately 1 mM.     

DNA interaction with RNase A alters protein conformation

DNA-RNase H adducts were used for site specific cleavage of RNA and DNA-RNA duplexes, whereas nonspecific DNA interaction with ribonuclease A (RNase A) has been observed. The aim of this study was to examine the complexation of calf-thymus DNA with RNase A at physiological condition, using constant DNA concentration (12.5 mM) and various protein contents (1 microM to 270 microM). FTIR, UV-visible, and CD spectroscopic methods were used to analyse protein binding mode, the binding constant and the effects of nucleic acid-enzyme interaction on both DNA and protein conformations. Our structural analysis showed a strong RNase-PO2 binding and minor interaction with G-C bases with overall binding constant of K = 6.1 x 10(4) M(-1). The RNase-DNA interaction alters the protein secondary structure with a major reduction of the alpha-helix and increase of the beta-sheet and random structure, while DNA remains in the B-family structure.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

NMR measurements of the diffusional permeability of water in cultured colonic epithelial cancer cells

The water residence time and diffusional water permeability in colonic epithelial T84 cancer cells was measured using (1)H NMR spectroscopy; the values estimated were 35.2+/-2.8 ms and (7.4+/-0.6)x10(-3)cms(-1), respectively. Water permeability was inhibited to approximately 10% of its original value by the mercurial diuretic, p-chloromercuribenzenesulfonate (PCMBS; 1mM), and fully restored by dithiothreitol (DTT; 1mM). The permeability was also inhibited reversibly to approximately 55%, by extracellular glibenclamide (1mM), an inhibitor of some ATP-binding cassette (ABC) transporters, including the cystic fibrosis transmembrane conductance regulator (CFTR). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IMBX; 0.1-1mM) and the adenylate cyclase activator, forskolin (0.1-1mM) did not alter water permeability. It is concluded that in T84 cells water diffuses through the membrane lipid bilayer and via channels that are inhibited by PCMBS, including the channels that are known to be inhibited by glibenclamide.     

Local secondary structure in ribonuclease A denatured by guanidine · HCl near 1 °C

Recent work has shown that a short α-helix can be stable in water near 1 °C when stabilized by specific interactions between side-chains, while earlier “host-guest” results with random copolymers have shown that a short α-helix is unstable in water at all temperatures in the absence of stabilizing side-chain interactions. As regards the mechanism of protein folding, it is now reasonable on energetic grounds to consider isolated α-helices and β-sheets as the first intermediates on the pathway of protein folding. Proton nuclear magnetic resonance is used here to detect isolated secondary structures in ribonuclease A denatured by guanidine · HCl (GuHCl). Temperatures near 1 °C are used because the low-temperature stability of the C-peptide helix may be a general property of isolated secondary structures in water.Our procedure is to titrate with GuHCl the C2H resonance lines of the four histidine residues of denatured ribonuclease A. Studies of model peptides (C-peptide (lactone) and C-peptide carboxylate, residues 1 to 13 of ribonuclease A; S-peptide, residues 1 to 20) show linear titration curves for the C2H resonance of His12 above 0.5 M-GuHCl, once helix unfolding is complete. Deviations from this line are used to monitor helix formation. The GuHCl titration curves of the other three histidine residues are also linear, once unfolding is complete. The results show that the helix found in C-peptide and S-peptide is also found in denatured ribonuclease A, where it behaves as an isolated helix not stabilized significantly by interactions with other chain segments. Studies of denatured S-protein show that the remaining three His residues, His48, His105 and His119, are involved in structure only below 1 m-GuHCl at 9 °C, pH 1.9. The nature of this structure is not known. The main conclusion from this work is that the His12 helix can be observed as a stable, isolated helix in denatured ribonuclease A near 1 °C, and that none of the other three His residues is involved in a comparably stable local structure. In native ribonuclease A, His12 is within an α-helix and the other three His residues are involved in a 3-stranded β-sheet structure.The helix-coil transition of C-peptide has also been studied for other side-chain resonances by GuHCl titration. Typically, but not always, the titration curves are linear after helix unfolding takes place and resonance lines from different residues of the same amino acid type can be resolved in GuHCl solutions. This is true of the four histidine residues of ribonuclease A although their pK values in 5 m-GuHCl are nearly the same. In C-peptide, the βCH₃ resonance of Ala6 is affected strongly by GuHCl while the lines of Ala4 and Ala5 are shifted only weakly by GuHCl. Evidently the interactions between GuHCl and side-chains in an unfolded peptide depend upon neighboring groups.     

Glycosylation in vitro of an asparagine sequon catalysed by preparations of yeast cell membranes.

Preparations of yeast cell membranes can catalyse in vitro the N-acetyl-beta-D-glucosaminylation of the asparagine sequon at residues 34--36 of bovine pancreatic ribonuclease A. The relevant glycopeptides were isolated from tryptic hydrolysates of the glycosylated ribonuclease and analysed. The donor used was UDP-N-acetyl-D-glucosamine, although the mechanism of the transfer is unknown. Mn2+ ions at concentrations of 25 mM double the activity of the enzymic transfer.     

4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization

The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM < c < mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9.     

1H NMR study on the interaction of cupric ion with ribonuclease A.

The reassignment of the 1H NMR C-2 histidine signals of the bovine pancreatic ribonuclease A has required a revision of the 1H NMR data on the role of the different histidines in their interaction with the Cu2+. The results of our measurements carried out at p2H 5.5 and 7.0 reduce the importance of His-12 as main site of interaction. At p2H 5.5 a very strong binding site involves His-119, while a weaker one contains certainly His-105. On the contrary, at p2H 7.0 the histidines 105 and 119 seem to possess binding constants of the same order of magnitude and in addition they provide stronger ligands for the Cu2+ than His-12. The comparison with X-ray data in the crystal shows numerous analogies. Finally, preliminary results on the competitive inhibition effect between the Cu2+ and 2',3'-cytidine monophosphoric acid are discussed.