The interactions between nucleic acids and polyamines: I. High resolution carbon-13 and hydrogen-1 nuclear magnetic resonance studies of spermidine and 5'-AMP

In 0.5 M solution at pH 7.6, interaction of spermidine and 5'-AMP is demonstrated by downfield proton NMR shifts. Shifts of ribose and adenine protons support a model in which triprotonated spermidine engages the phosphate, anion with the C-3 diamine segment in a conformation to maximize interaction and the C-4 ammo segment extended to interact with adenine N-7 (base anti, 2'endo, g'g' and gg nucleoside conformation). Changes in carbon-13 chemical shifts for ribose C-5' (downfield), C-2' C-3', and C-4' (upfield) and for adenine C-6 and C-8 (upfield) support this model. In 0.006 M solution no significant changes in proton shifts and therefore no evidence for interaction was found. Spermidine and 5'-UMP (0.006 M) showed interaction at pH 10.5 (small upfield shifts in the proton nmr) interpreted as changing conformation by solvent interaction. In 0.006 M 3'-UMP at pH 10.5 small downfield proton shifts induced by spennidine are attributed to interactions with phosphate anion.     

Pentacopper(II) complexes of alpha-aminohydroxamic acids: uranyl-induced conversion of a 12-metallacrown-4 to a 15-metallacrown-5

Effects of uranyl on the pentacopper(II) complexes of alpha-leucinehydroxamic acid and alpha-tyrosinehydroxamic acid were studied in water and methanol by means of electrospray ionisation mass spectrometry (ES-MS), absorption spectrophotometry, circular dichroism spectroscopy and proton NMR spectroscopy. All the measurements were consistent with the complete conversion of a 12-metallacrown-4 to a 15-metallacrown-5 upon addition of one equivalent of the uranyl ion. The uranyl ion is accommodated in the cavity formed by five copper(II) ions and five alpha-aminohydroxamate ligands. The 15-metallacrown-5 inclusion complexes have a high affinity for the uranyl ion. Competition studies showed that even in the presence of a large excess of calcium(II), the 15-metallacrown-5 remained stable, and no exchange reactions between calcium(II) and uranyl were observed. Extraction of uranyl from the 15-metallacrown-5 was also not detected in the presence of a large excess of 18-crown-6. Trivalent lanthanide ions can be partially sequestered by the 15-metallacrown-5, however, even these trivalent ions are displaced by uranyl.     

Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo-halide ion binding to chloroperoxidase

The proton nuclear magnetic resonance spectra of several chloroperoxidase-inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide-chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin.     

A nuclear magnetic resonance study of axial ligation for the reduced states of chloroperoxidase,cytochrome P-450cam,and porphinatoiron(II) thiolate complexes

The reduced forms of cytochrome P-450_cam and chloroperoxidase were examined by proton NMR spectroscopy. The pH and temperature dependences of the proton NMR spectra of both ferrous enzymes are reported. A series of alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins was also examined. The proton NMR spectra of these complexes facilitated the assignment of resonances due to the axial ligand in the model compounds on the basis of their isotropic shifts and multiplicities. Comparison of model compound data with that for the reduced enzymes supports assignment of the methylene protons for the axial cysteinate of ferrous cytochrome P-450_cam and ferrous chloroperoxidase to proton NMR resonances at 279 and 200 ppm (pH 7.0, 298K), respectively. Differences in the active site structure of the two enzymes are further demonstrated by ¹⁵N-NMR spectroscopy of the cyanide complexes of the ferric forms.     

Nuclear magnetic resonance study of hydrogen-bonded ring protons in oligonucleotide helices involving classical and nonclassical base pairs.

A study of the exchangeable ring nitrogen protons in aqueous solutions of oligonucleotide complexes involving Watson-Crick base pairs as well as Hoogsteen pairs and other nonclassical hydrogen bonding schemes shows that resolvable resonances in the low-field (-10 to -16 ppm from sodium 4,4-dimethyl-4-silapentanesulfonate) region can be detected in a variety of structures other than double stranded helices. Ring nitrogen proton resonances arising from the following hydrogen-bonding situations are reported: (1) AT and GC Watson-Crick base pairs in a self-complementary octanucleotide, dApApApGpCpTpTpT; (2) U-A-U base triples in complexes between oligo-U15 and AMP; (3) C-G-C+ base triples in complexes between oligo-C17 and GMP at acid pH; (4) s4U-A-s4U base triples in complexes between oligo-s4U15 and AMP, all of which involve both Watson-Crick and Hoogsteen base pairing to form triplexes; (5) C-C+ base pairing between protonated and unprotonated C residues in oligo-C17 at acid pH; and (6) I4 base quadruples in the four strand association among oligo-I at high salt. The behavior of the dA3G-CT3 helix is consistent with both fraying of the terminal base pairs and presence of intermediate states as the helix opens. In the monomer-oligomer complexes, under the conditions used here, the exchange appears to be governed by the dissociation rate of monomer from the complex. These findings suggest that those tertiary structure hydrogen bonds in tRNA involving ring nitrogen protons should have representative resonances in the low-field (11-16 ppm) proton NMR region in H2O.     

The pentaammineruthenium(III)histidine complex in ribonuclease A: application to the assignment of histidine proton resonances

The assignment of two histidine proton resonances in the proton NMR spectrum of ribonuclease A has been made by forming a paramagnetic complex between pentaammineruthenium(III) and the N-3 nitrogen of a single histidine residue. Reaction of chloropentaammineruthenium(III)dichloride with ribonuclease A in 0.1 m Tris-HCl, pH 7.0, 25°C yields a variety of products in which various histidine residues have been labeled. Cation-exchange chromatography affords the isolation of a specific derivative, labeled at a single histidine residue, that retains 66% of the activity toward the hydrolysis of 2′,3′-cyclic CMP. The site of labeling was determined by peptide mapping to be histidine 105. The binding of ruthenium results in the disappearance of both a histidine C-2 and a C-4 proton resonance from the downfield region of the proton NMR spectrum, as expected from model compound studies. The assignment of these two resonances to histidine 105 is in agreement with a previous assignment (J. L. Markley, 1975, Biochemistry, 14, 3546–3554), thereby demonstrating the potential utility of this ruthenium reagent in the assignment of histidine resonances in the proton NMR spectra of other proteins.     

Proton magnetic resonance studies of ribonuclease T1. Assignment of histidine-40 peak and analysis of the active site.

Nuclear magnetic resonances of the C-2 protons of the three histidine residues in ribonuclease T₁ have been studied at 360 MHz as a function of pH to discuss the structure of the active site. Comparison of the order of deuterium exchange of the histidine peaks with tritium incorporation rates into individual histidines of the enzyme leads to the unambigous assignment of one of the C-2 proton peaks to histidne-40. It has been concluded that histidine-40 is in the active site, interacting with a charged group of pK 4.1, which is replaced by the phosphate group of guanosine-3′-monophosphate in the enzyme-inhibitor complex. Histidine-92 is most likely a binding site for the complex, where the existence of a hydrogen bond between N-7 of the inhibitor and the ring NH proton of the histidine is suggested on the basis of NMR data.     

A comparison by 220-MHz NMR of histidine hydronium ion titrations in porcine pancreatic ribonuclease and an extensively deglycosylated derivative.

220-MHz NMR was used to observe the titration behavior of the 5 histidine residues in porcine pancreatic ribonuclease (ribonucleate pyrimidine-nucleotido-2'-transferase (cyclizing), EC 3.1.4.22) and a derivative prepared by removal of 80% of the attached carbohydrate from this glycoprotein. Resonances due to histidine C-2 protons were observed over the full pH range for 3 of the residues; such resonances for the remaining 2 histidine residues broadened out as the pH was increased. Resonances due to histidine C-4 protons were also observed for 2 of the residues. The titration curves for both proteins were identical within experimental error. Resonances were assigned by comparison with histidine NMR titrations in ribonucleases from other species. Histidine 105, immediately adjacent to the site of attachment of a heterosaccharide side chain, has a C-2 proton chemical shift and pK that are insensitive to the large alteration in the bulk of the carbohydrate side chain. The chemical shifts of the C-2 proton of histidine 48 and of the C-4 proton of histidine 80, histidine residues that are close to one another and to another heterosaccharide side chain, show a similar insensitivity. The observations are direct evidence in support of the thesis that the heterosaccharides in porcine ribonuclease project away from the surface of the protein into the solution environment.     

Exchange mechanisms for hydrogen bonding protons of cytidylic and guanylic acids.

The pH dependence of buffer catalysis of exchange of the C-4 amino protons of cyclic cytosine 2',3'-monophosphate (cCMP) and the N-1 proton of cyclic guanosine 2',3'-monophosphate (cGMP) conforms to an exchange mechanism, in which protonation of the nucleobases at C(N-3) AND G(N-7) establishes the important intermediates at neutral to acidic pH. Rate constants for transfer of the G(N-1) proton to H2O, OH-, phosphate, acetate, chloracetate, lactate, and cytosine (N-3) were obtained from 1H nuclear magnetic resonance line width measurements at 360 MHz and were used to estimate the pK or acidity of the exchange site in both the protonated and unprotonated nucleobase. These estimates reveal an increase in acidity of the G(N-1) site corresponding to 2 to 3 pK units as the G(N-7) site is protonated: At neutral pH the G(N-1) site of the protonated purine would be ionized (pK = 6.3). Determinations of phosphate, imidazole, and methylimidazole rate constants for transfer of the amino protons of cCMP provide a more approximate estimate of pK = 7 to 9 for the amino of the protonated pyrimidine. A comparison of the intrinsic amino acidity in the neutral and protonated cytosine is vitiated by the observation that OH- catalyzed exchange in the neutral base is not diffusion limited. This leads to the conclusion that protonation of the nucleobase effects a qualitative increase in the ability of the amino protons to form hydrogen bonds: from very poor in the neutral base to "normal" in the conjugate acid.     

Origin of the ammonia used for mitochondrial citrulline synthesis as revealed by 13C-15N spin coupling patterns observed by 13C NMR.

The sources of ammonia used by isolated, intact rat liver mitochondria in the production of citrulline have been investigated in situ using a novel methodology based on the analysis of 13C-15N heteronuclear couplings observed by 13C NMR. Isolated mitochondria from rat liver were incubated with ornithine, 13CO3H- and 15NH4Cl, using unlabeled glutamate or glutamine as alternative, intramitochondrial nitrogen donors. The production of (7-13C, 8-15N) or (7-13C, 8-14N) citrulline was determined in situ by 13C NMR and the relative proportions of 15N- and 14N-citrullines confirmed by high resolution 13C NMR analysis of the C-7 citrulline resonance observed in perchloric acid extracts prepared at the end of the incubations. The 15N fractional enrichment of the intramitochondrial NH3 pool was manipulated either by modifying the 15N enrichment of added 15NH4Cl, or by altering the concentration of the unlabeled nitrogen donors in the incubation medium. Fractional 15N enrichments measured in the N-8 nitrogen of the resulting (7-13C) citrulline closely paralleled those of the external 15NH4Cl with minor dilutions derived from the unlabeled nitrogen contribution from the alternative substrates. In the presence of 10 mM 15NH4Cl, 10 mM glutamate contributed 4% of the citrulline N-8 nitrogen. Under similar conditions, the contribution of nitrogen from 10 mM glutamine to N-8 citrulline was 6%. These results indicate that the primary source of ammonia used for citrulline synthesis by isolated, intact rat liver mitochondria is extramitochondrial, providing also an illustration of the use of 13C-15N spin coupling patterns observed by 13C NMR, as a new tool in the study of ammonia metabolism.     

NMR studies of chromomycin A3 interaction with DNA

The binding of chromomycin A3 to calf thymus DNA and poly(dG-dC) has been studied by 13C and 1H NMR with emphasis on the mode of binding, the role of Mg2+, and pH effects. The most prominent changes in the DNA base pair 13C NMR resonances upon complexation with chromomycin were observed for G and C bases, consistent with the G-C preference exhibited by this compound. Comparison of the 13C spectrum of DNA-bound chromomycin A3 with that of DNA-bound actinomycin D, a known intercalator, showed many similarities in the base pair resonances. This suggested the possibility that chromomycin A3 binds via an intercalative mechanism. 1H NMR studies in the imino proton, low-field region of the spectrum provided additional evidence in support of this binding mode. In the low-field spectrum of chromomycin A3 bound to calf thymus DNA, a small shoulder was observed on the upfield side of the G-C imino proton peak. Similarly, in the chromomycin A3 complex with poly(dG-dC), a well-resolved peak was found upfield from the G-C imino proton peak. These results are expected for ligands that bind by intercalation. Furthermore, in both the calf thymus and poly(dG-dC) drug complexes (in the presence of Mg2+) a broad peak was also present downfield (approximately 16 ppm from TSP) from the DNA imino protons. This was attributed to the C-9 phenolic hydroxyl proton on the chromomycin chromophore. Visible absorbance spectra at different pH values showed that the role of Mg2+ in the binding of chromomycin A3 to DNA is more than simple neutralization of the drug's anionic change.     

Demonstration of different complexation modes between cobalt and 5' AMP, by direct NMR observation of the low-temperature complex

We report the first direct NMR observation of transition metal-nucleotide complexes. The phosphorus and proton spectra of a cobalt-5' AMP complex were observed in water, pH 7, -- 10 degrees C. This complex is different from the high temperature species : for instance its 31P chemical shift is -- 50 ppm, whereas the published value obtained indirectly for the high temperature form corresponds to ca. -- 1200 ppm. The -- 50 ppm complex is present in significant proportion at 20 degrees C and possibly at higher temperatures. Multiple complexation modes are also observed for Co-ATP.     

18,19-Dihydroxydeoxycorticosterone, a new metabolite produced from 18-hydroxydeoxycorticosterone by cytochrome P-450(11) beta. Chemical synthesis and structural analysis by 1H NMR

A new metabolite was produced from 18-hydroxydeoxycorticosterone by the cytochrome P-450(11) beta linked hydroxylase system purified from bovine adrenocortical mitochondria. It was identified as 18,19-dihydroxydeoxycorticosterone by chemical synthesis on the basis of high-performance liquid chromatography, gas chromatography-mass spectrometry, and proton nuclear magnetic resonance (1H NMR) spectroscopy, and detailed structural analysis of it was performed by 1H NMR spectroscopy. The methylene protons at the C-19 position of the steroid were nonequivalent and coupled with each other, having a coupling constant of 10.6 Hz. These protons had different coupling constants, 6.7 and 3.4 Hz, for the hydroxy proton at the C-19 position. Due to these couplings, the signals of the methylene protons were observed around 3.9 ppm as two double doublets. The methylene protons at the C-21 position were also nonequivalent, having a coupling constant of 11.1 Hz. Coupling constants between these methylene protons and the hydroxy proton at the C-21 position were 8.2 and 4.2 Hz, respectively. These results indicate that both hydroxymethyl groups at the C-19 and C-21 positions do not freely rotate in chloroform solution. The signals of hydroxy protons at the C-19 and C-21 positions were found at 1.25 and 1.87 ppm, respectively, by means of decoupling of the corresponding methylene protons. The hydroxy proton at the C-18 position was found to scarcely couple with any proton. This fact suggests that this hydroxy group is linked to the C-20 position, making a hemiketal bridge between the C-18 and the C-20.     

An isocratic separation of underivatized gentamicin components, 1H NMR assignment and protonation pattern

A simple method for the separation of the major components of commercial gentamicin sulfate (C-1, C-1a, C-2, C-2a) by high-performance liquid chromatography (HPLC) on an analytical and a semipreparative scale was developed. The method utilized ion-pair reversed-phase chromatography, isocratic elution with an aqueous solution containing 9% trifluoroacetic acid and 2.5% acetonitrile as the mobile phase at a flow rate of 2 and 9 mL/min for analytical and semipreparative columns, respectively. Detection was carried out at 213 nm without derivatization. The protonation pattern of the separated gentamicins was determined by potentiometry and 15N and 1H NMR. The full proton NMR assignment for gentamicin C-1 was obtained through the use of 1H 1D and 2D 1H-1H COSY measurements.     

Binding of thiocyanate to lactoperoxidase: 1H and 15N nuclear magnetic resonance studies

The binding of thiocyanate to lactoperoxidase (LPO) has been investigated by 1H and 15N NMR spectroscopy. 1H NMR of LPO shows that the major broad heme methyl proton resonance at about 61 ppm is shifted upfield by addition of the thiocyanate, indicating binding of the thiocyanate to the enzyme. The pH dependence of line width of 15N resonance of SC15N- in the presence of the enzyme has revealed that the binding of the thiocyanate to the enzyme is facilitated by protonation of an ionizable group (with pKa of 6.4), which is presumably distal histidine. Dissociation constants (KD) of SC15N-/LPO, SC15N-/LPO/I-, and SC15N-/LPO/CN- equilibria have been determined by 15N T1 measurements and found to be 90 +/- 5, 173 +/- 20, and 83 +/- 6 mM, respectively. On the basis of these values of KD, it is suggested that the iodide ion inhibits the binding of the thiocyanate but cyanide ion does not. The thiocyanate is shown to bind at the same site of LPO as iodide does, but the binding is considerably weaker and is away from the ferric ion. The distance of 15N of the bound thiocyanate ion from the iron is determined to be 7.2 +/- 0.2 A from the 15N T1 measurements.     

Neutral imidazole is the electrophile in the reaction catalyzed by triosephosphate isomerase: structural origins and catalytic implications

To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, 13C and 15N NMR titration studies have been carried out both on the wild-type enzyme and on a mutant isomerase in which the single remaining histidine (that at the active site) has been isotopically enriched in the imidazole ring. 15N NMR has proved especially useful in the unambiguous demonstration that the imidazole ring of histidine-95 is uncharged over the entire pH range of isomerase activity, between pH 5 and pH 9.9. The results require that the first pKa of histidine-95 is below 4.5. This abnormally low pKa rules out the traditional view that the positively charged imidazolium cation of histidine-95 donates a proton to the developing charge on the substrate's carbonyl oxygen. 15N NMR experiments on the enzyme in the presence of the reaction intermediate analogue phosphoglycolohydroxamate show the presence of a strong hydrogen bond between N epsilon 2 of histidine-95 and the bound inhibitor. These findings indicate that, in the catalyzed reaction, proton abstraction from C-1 of dihydroxyacetone phosphate first yields an enediolate intermediate that is strongly hydrogen bonded to the neutral imidazole side chain of histidine-95. The imidazole proton involved in this hydrogen bond then protonates the enediolate, with the transient formation of the enediol-imidazolate ion pair. Abstraction of the hydroxyl proton on O-1 now produces the other enediolate intermediate, which collapses to give the product glyceraldehyde 3-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tautomerism,acid-base equilibria,and H-bonding of the six histidines in subtilisin BPN' by NMR

We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every (15)N, (1)H, C(epsilon 1), and C(delta2) resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25 degrees C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/- 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C(epsilon 1)-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C(epsilon 1)-H(.)O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N(delta1)-H tautomer, exhibiting (13)C(delta1) chemical shifts approximately 9 ppm higher than those for N(epsilon 2)-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by (15)N-(1)H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02.     

Evaluation of the metal-ion-coordinating differences between the 2'-, 3'- and 5'-monophosphates of adenosine

The stability constants of the 1:1 complexes formed between Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+ and 2'AMP2-, 3'AMP2- or 5'AMP2- were determined by potentiometric pH titration in aqueous solution (I = 0.1 M, NaNO3; 25 degrees C). The experimental conditions were carefully selected such that self-association of the nucleotides and their complexes is negligibly small; i.e. it was made certain that the properties of the monomeric divalent-metal-ion--AMP [M(AMP)] complexes were studied. Based on recent measurements with simple phosphate monoesters, R-MP2- where R is a non-coordinating residue [Massoud, S. S. & Sigel, H. (1988) Inorg. Chem. 27, 1447-1453], it is shown that all the M(AMP) complexes of the alkaline earth ions, with the possible exception of Mg(5'AMP), have exactly the stability expected for a sole-phosphate coordination of the metal ion. The same property is revealed for the complexes with Mn2+, Co2+, Zn2+ or Cd2+ and 3'AMP2-; in case of Ni(3'AMP) and Cu(3'AMP) a slight stability increase just at the edge of the experimental-error limits is indicated. This slight stability increase is attributed to the formation of a macrochelate (possibly with N-3); in fact, additional information confirms macrochelation for Cu(3'AMP). About 45% of Cu(2'AMP) exists in aqueous solution as a macrochelate (probably involving N-3); the other M(2'AMP) complexes (M2+ = Mn2+, Co2+, Ni2+, Zn2+, Cd2+) form (if at all) only traces of a base-backbound species. Most pronounced is macrochelate formation with 5'AMP2-: all mentioned 3d ions and Zn2+ or Cd2+ form to some extent macrochelates via N-7 (the structures of these closed species are indicated). In case of M(5'AMP) the base-binding site is certain: replacement of N-7 by a CH unit (tubercidin 5'-monophosphate) eliminates any increased complex stability, whereas formation of the 1,N6-etheno bridge to form 1,N6-ethenoadenosine 5'-monophosphate results in the phenanthroline-like N-6,N-7 site which facilitates macrochelation significantly.     

TEDOR with adiabatic inversion pulses: Resonance assignments of <Superscript>13</Superscript>C/<Superscript>15</Superscript>N labelled RNAs

We have examined via numerical simulations the performance characteristics of different 15N RF pulse schemes employed in the transferred echo double resonance (TEDOR) experimental protocol for generating 13C-15N dipolar chemical shift correlation spectra of isotopically labelled biological systems at moderate MAS frequencies (omega(r) approximately 10 kHz). With an 15N field strength of approximately 30-35 kHz that is typically available in 5 mm triple resonance MAS NMR probes, it is shown that a robust TEDOR sequence with significant tolerance to experimental imperfections sa as H1 inhomogeneity and resonance offsets can be effectively implemented using adiabatic heteronuclear dipolar recoupling pulse schemes. TEDOR-based 15N-13C and 15N-13C-13C chemical shift correlation experiments were carried out for obtaining 13C and 15N resonance assignments of an RNA composed of 97 (CUG) repeats which has been implicated in the neuromuscular disease myotonic dystrophy.     

NMR study and comparison of the antigenic properties of a peptide recognized by two HIV-1 neutralizing antibodies

Fab–peptide complexes formed between a 15 residue peptide derived from the HIV-1 gp120 V3 loop and two of its cognate monoclonal antibodies, 5023A and 5025A, were studied using isotope-edited solution nuclear magnetic resonance (NMR) techniques. Since these antibodies neutralize HIV-1 virus with different strain specificities, this study was conducted to better understand the nature of these differences. The amide proton and nitrogen NMR resonances of specific residues were used to monitor the backbone of this peptide in these complexes. Three central residues of this peptide (‘RAF’) were found to be strongly affected by binding to both antibodies. Several other peptide residues were affected by binding to antibody 5023A but not 5025A. The antibody epitopes mapped by NMR are similar to those obtained previously via PEPSCAN at higher pH. One main difference between the PEPSCAN and NMR determined epitopes for 5023A involved two glycine residues of the peptide. By NMR, one of these glycines was more dramatically affected by antibody binding than predicted by PEPSCAN, while the other was much less so. © 1998 John Wiley & Sons, Ltd.