Proton-magnetic-resonance spectroscopic study of the histidine residues of bovine alpha-lactalbumin.

A study of the three histidine residues of bovine alpha-lactalbumin has been made using proton magnetic resonance (PMR) spectroscopy in order to obtain information on their environments in the protein and thereby to test in part the previously proposed structure. PMR titration curves are obtained for the H-4 resonances using difference spectroscopy and for the H-2 resonances and the 1-H-2-H exchange rates of the H-2 protons have been measured. The assignment of resonances to particular histidine residues is achieved by utilising their selective reaction with iodoacetate in conjunction with a PMR study of the carboxymethylation of alpha-N-acetyl-L-histidine. The H-2 and H-4 resonances labelled 1, 2 and 3 starting from the downfield end of the spectrum are assigned to histidine residues 107, 68 and 32 respectively. Their apparent pK values at low ionic strength and 20 degrees C are 5.78, 6.49 and 6.51 respectively. The experimental results on two histidine residues are consistent with the predictions of the proposed structure, which indicate that histidine-68 is an external residue and histidine-32 is partially buried and in the vicinity of aromatic residues. The experimental data on histidine 107 can also be rationalised with less certainty in terms of the proposed structure, which indicates a partially buried residue that may be involved in hydrogen bonding.     

Netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex. Antibiotic binding at adenine . thymine base pairs in the minor groove of the self-complementary octanucleotide duplex.

The structure of the netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex (one antibiotic molecule/self-complementary octanucleodide duplex) and its dynamics as a function of temperature have been monitored by the nuclear magnetic resonances of the Watson-Crick protons, the nonexchangeable base and sugar protons and the backbone phosphates. The antibiotic forms a complex with the nucleic acid duplex at the dA . dT-containing tetranucleotide segment dA-dA-dT-dT, with slow migration amongst potential binding sites at low temperature. The downfield shifts in the exchangeable protons of netropsin on complex formation demonstrate the contributions of hydrogen-bonding interactions between the antibiotic and the nucleic acid to the stability of the complex. Complex formation results in changes in the glycosidic torsion angles of both thymidine residues and one deoxyadenosine residue as monitored by chemical shift changes in the thymine C-6 and adenine C-8 protons. The close proximity of the pyrrole rings of the antibiotic and the base-pair edges in the minor groove is manifested in the downfield shifts (0.3--0.5 ppm) of the pyrrole C-3 protons of netropsin and one adenine C-2 proton and one thymine N-3 base-pair proton on complex formation. The internucleotide phosphates of the octanucleotide undergo 31P chemical shift changes on addition of netropsin and these may reflect, in part, contributions from electrostatic interactions between the charged ends of the antibiotic and the backbone phosphates of the nucleic acid.     

Epidermal growth factor from the mouse. Physical evidence for a tiered beta-sheet domain: two-dimensional NMR correlated spectroscopy and nuclear Overhauser experiments on backbone amide protons

When H2O-exchanged, lyophilized mouse epidermal growth factor (mEGF) is dissolved in deuterium oxide at low pH (i.e., below approximately 6.0), 13 well-resolved, amide proton resonances are observed in the downfield region of an NMR spectrum (500 MHz). Under the conditions of these experiments, the lifetimes of these amide protons in exchange for deuterons of the deuterium oxide solvent suggest that these amide protons are hydrogen-bonded, backbone amide protons. Several of these amide proton resonances show splittings (i.e., JNH alpha-CH) of approximately 8-10 Hz, indicating that their associated amide protons are in some type of beta-structure. Selective nuclear Overhauser effect (NOE) experiments performed on all amide proton resonances strongly suggest that all 13 of these backbone amide protons are part of a single-tiered beta-sheet structural domain in mEGF. Correlation of 2D NMR correlated spectroscopy data, identifying scaler coupled protons, with NOE data, identifying protons close to the irradiated amide protons, allows tentative assignment of some resonances in the NOE difference spectra to specific amino acid residues. These data allow a partial structural model of the tiered beta-sheet domain in mEGF to be postulated.     

Proton magnetic resonance studies of carbonic anhydrase. I. Identification of histidine resonances.

Nuclear magnetic resonance (nmr) spectra of human carbonic anhydrase B recorded in deuterium oxide reveal seven discrete single proton resonances between 7 and 9 ppm downfield from sodium 2,2-dimethyl-i-silapentane-5-sulfonate. Simplification of spectra by use of Fremy's salt, comparison of peak widths at intersections, and evaluation of the results of inhibition and modification experiments permit determination of the pH dependencies of these resonances. Five of these peaks change position with increasing pH; three move upfield by approximately 95 Hz and two move downfield by 10 and 23 Hz. The first three reflect residues with pK values of 7.23, 6.98, and 6 and can be assigned to the C-2 protons of histidines. The two remaining pH dependent resonances reflect groups with pK values of 8.2 and 8.24. Their line widths and T1 values are comparable to those of the first group, and they also appear to reflect C-H protons of histidines. Despite the structural and functional similarities of the B and C isozymes of human carbonic anhydrase, few of the low field resonances appear to be common to both. Six histidine C-2 protons are observed in the C enzyme and reflect groups with pK values of approximately 7.3, 6.5, 5.7, 6.6, 6.6, and 6.4. A seventh peak contains two protons and moves upfield with increasing pH without titrating. A final resonance to low field moves downfield with increasing pH and reflects a group with a pK between 6 and 7. Its behavior resembles that of peak 1 of the human B enzyme, and it also appears to be a histidine C-H proton. This peak may reflect a conserved residue in the two isozymes that plays an important role in enzymatic function, as discussed in the following paper.     

NMR study of the phosphate-binding loops of Thermus thermophilus elongation factor Tu.

The phosphoryl-binding loops in the guanosine diphosphate binding domain of elongation factor Tu were studied by 15N heteronuclear proton-observe NMR methods. Five proton resonances were found below 10.5 ppm. One of these was assigned to the amide group of Lys 24, which is a conserved residue in the phosphoryl-binding concensus loop of purine nucleotide binding proteins. The uncharacteristic downfield proton shift is attributed to a strong hydrogen bond with a phosphate oxygen. The amide protons from the homologous lysines in N-ras p21 [Redfield, A.G., & Papastavros, M.Z. (1990) Biochemistry 29, 3509-3514] and the catalytic domain of Escherichia coli elongation factor Tu [Lowry, D.F., Cool, R.H., Redfield, A.G., & Parmeggiani, A. (1991) Biochemistry 30, 10872-10877] also resonate downfield in similar positions. We propose that the downfield shift of this lysine amide proton is a spectral marker for this class of proteins. We also have studied the temperature dependence of the downfield resonances and find a possible conformation change at 40 degrees C.     

One- and two-dimensional 1H-NMR characterization of two series of sulfated disaccharides prepared from chondroitin sulfate and heparan sulfate/heparin by bacterial eliminase digestion.

The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the alpha and beta anomers of these disaccharides, except those containing N-sulfated GlcN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the alpha anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3 ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15 ppm, respectively, but that of C-1 was shifted downfield by 0.25 ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of delta GlcA (D-gluco-4-ene-pyranosyluronic acid), the signal of the H-3 proton of delta GlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15 ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive.     

A proton nuclear magnetic resonance investigation of proximal histidyl residues in human normal and abnormal hemoglobins. A probe for the heme pocket.

Proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate the conformations of proximal histidyl residues of human normal adult hemoglobin, hemoglobin Kempsey [beta 99(G1) Asp leads to Asn], hemoglobin Osler [beta 145(HC2) Tyr leads to Asp], and hemoglobin McKees Rocks [beta 145(HC2) Tyr leads to Term] around neutral pH in H2O at 27 degrees C, all in the deoxy form. Two resonances that occur between 58 and 76 ppm downfield from the water proton signal have been assigned to the hyperfine shifted proximal histidyl NH-exchangeable protons of the alpha- and beta-chains of deoxyhemoglobin. These two resonances are sensitive to the quaternary state of hemoglobin, amino acid substitutions in the alpha 1 beta 2-subunit interface and in the carboxy-terminal region of the beta-chain, and the addition of organic phosphates. The experimental results show that there are differences in the heme pockets among these four hemoglobins studied. The structural and dynamic information derived from the hyperfine shifted proximal histidyl NH-exchangeable proton resonances complement that obtained from the ferrous hyperfine shifted and exchangeable proton resonances of deoxyhemoglobin over the spectral region from 5 to 20 ppm downfield from H2O. The relationship between these findings and Perutz's stereochemical mechanism for the cooperative oxygenation of hemoglobin is discussed.     

An unusual heparan sulfate isolated from lobsters (Homarus americanus)

An unusual heparan sulfate was isolated from lobsters (Homarus americanus). The polysaccharide has a composition and properties intermediate to heparin and the more common heparan sulfates. The sulfate and D-glucuronic acid content is high, while anticoagulant activity is low. The major repeating unit appears to consist of D-glucuronic acid and D-glucosamine N-O-disulfate. Some N-acetyl groups are present, the content of L-iduronic acid O-sulfate is low, and monosulfated or nonsulfated disaccharide-repeating units (very common in heparan sulfates) appear to be very rare. The data obtained again emphasize the heterogeneity of heparan sulfates and the need for adequate characterization when dealing with unusual or unexplored sources.     

d-CpCpGpG and d-GpGpCpC self-complementary duplexes: Nmr studies of the helix-coil transition

We have monitored the helix-coil transition of the self-complementary d-CpCpGpG and d-GpGpCpC sequences (20mM strand concentration) at the base pairs, sugar rings, and backbone phosphates by 360-MHz proton and 145.7-MHz phosphorus nmr spectroscopy in 0.1M phosphate solution between 5 and 95°C. The guanine 1-imino Watson-Crick hydrogen-bonded protons, characteristic of the duplex state, are observed below 10°C, with solvent exchange occurring by transient opening of the tetranucleotide duplexes. The cytosine 4-amino Watson-Crick hydrogen-bonded protons resonate 1.5 ppm downfield from the exposed protons at the same position in the tetranucleotide duplexes, with slow exchange indicative of restricted rotation about the C-N bond below 15°C. The guanine 2-amino exchangeable protons in the tetranucleotide sequence exhibit very broad resonances at low temperatures and narrow average resonances above 20°C, corresponding to intermediate and fast rotation about the C-N bond, respectively. Solvent exchange is slower at the amino protons compared to the imino protons since the latter broaden out above 10°C. The well-resolved nonexchangeable base proton chemical shifts exhibit helix-coil transition midpoints between 37 and 42°C. The transition midpoints and the temperature dependence of the chemical shifts at low temperatures were utilized to differentiate between resonances located at the terminal and internal base pairs while the H-5 and H-6 doublets of individual cytosines were related by spin decoupling studies. For each tetranucleotide duplex, the cytosine H-5 resonances exhibit the largest chemical shift change associated with the helix-coil transition, a result predicted from calculations based on nearest-neighbor atomic diamagnetic anisotropy and ring current contributions for a B-DNA duplex. There is reasonable agreement between experimental and calculated chemical shift changes for the helix-coil transition at the internal base pairs but the experimental shifts exceed the calculated values at the terminal base pairs due to end-to-end aggregation at low temperatures. Since the guanine H-8 resonances of the CpCpGpG and d-CpCpGpG sequences exhibit upfield shifts of 0.6–0.8 and <0.1 ppm, respectively, on duplex formation, these RNA and DNA tetranucleotides with the same sequence must adopt different base-pair overlap geometries. The large chemical shift changes associated with duplex formation at the sugar H-1′ triplets are not detected at the other sugar protons and emphasize the contribution of the attached base at the 1′ position. The coupling sum between the H-1′ and the H-2′ and H-2″ protons equals 15–17 Hz at all four sugar rings for the d-CpCpGpG and d-GpGpCpC duplexes (25°C), consistent with a C-3′ exo sugar ring pucker for the deoxytetranucleotides in solution. The temperature dependent phosphate chemical shifts monitor changes in the ω,ω′ angles about the O-P backbone bonds, in contrast to the base-pair proton chemical shifts, which monitor stacking interactions.     

Non-covalent DNA groove-binding by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

The cooked meat mutagen 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) is metabolized in vivo to electrophilic intermediates that covalently bind to DNA guanines. Here we address the mechanism of PhIP's non-covalent interaction with DNA by using spectroscopic and computational methodologies. NMR methodologies indicated that upon addition of DNA, PhIP aromatic protons underwent a small, 0.11-0.12 p.p.m. upfield shift. DNA phosphorus resonances of non-covalent PhIP-DNA complexes broadened and slightly shifted upfield, while DNA base imino proton resonances shifted slightly downfield relative to DNA alone. UV and fluorescence spectra of PhIP titrated with DNA showed no detectable shifting and hypochromism of absorbance or fluorescence bands. In the presence of DNA, PhIP fluorescence was efficiently quenched by acrylamide, but not by silver ion. Further, the NMR spectra suggest that PhIP is in fast exchange with the DNA, and is slightly specific for adenine-thymine (A-T) sequences. Finally, structural arguments based on quantum chemistry calculations suggested that PhIP and its metabolites are unlikely to intercalate into DNA. These data collectively indicate that PhIP non-covalently binds in a groove of DNA.     

Formation of intermolecular and intramolecular hydrogen bonds in histidine-binding protein J of Salmonella typhimurium upon binding L-histidine. A proton nuclear magnetic resonance study

Histidine-binding protein J of Salmonella typhimurium has been chosen as a model system for a proton nuclear magnetic resonance spectroscopic investigation of binding protein-ligand interaction. This interaction is involved in the recognition step of the osmotic shock-sensitive active transport systems. When J protein binds L-histidine, four new, low-field, exchangeable proton resonances appear in the region +7 to +12 parts per million downfield from the water proton resonance (or +11.7 to +16.7 parts per million downfield from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate). Due to their chemical shift range and other properties, they indicate the formation of both intra- and intermolecular hydrogen bonds. Experiments with 15N-labeled compounds confirm this conclusion. The specificity of the hydrogen-bond formation is demonstrated by observing the effects of substrate analogs, temperature, pH, and mutations on the exchangeable proton resonances. Proton-proton nuclear Overhauser effect measurements suggest that two of these exchangeable proton resonances (at +7.2 and +10.6 parts per million from H2O) are most likely from intramolecular hydrogen-bonded protons, while the other two (at +7.1 and +9.5 parts per million from H2O) are intermolecular hydrogen bonds. Our finding of L-histidine-induced hydrogen-bond formation in histidine-binding protein J in the solution state is an excellent demonstration of the production of specific conformational changes in a periplasmic binding protein upon binding of ligand.     

Helix-coil transition of the self-complementary dG-dG-dA-dA-dT-dT-dC-dC duplex.

The helix-coil transition of the octanucleotide self-complementary duplex dG-dG-dA-dA-dT-dT-dC-dC has been monitored at the Watson-Crick protons, the base and sugar nonexchangeable protons and the backbone phosphates by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The melting transition of the octanucleotide monitored by ultraviolet absorbance spectroscopy is characterized by the thermodynamic parameters delta H degree = -216.7 kJ/mol and delta S degree (25 degrees C) = -0.632 KJ mol-1 K-1 in 0.1 M NaCl, 10 mM phosphate solution. Correlation of the transition midpoint values monitored by the ultraviolet absorbance studies at strand concentrations below 0.2 mM and by NMR studies at 5.3 mM suggest that both methods are monitoring the octanucleotide duplex-to-strand transition. The NMR spectra of the Watson-Crick ring NH protons of the octanucleotide duplex have been followed as a function of temperature. The resonance from the terminal dG.dC base pairs broadens out at room temperature while the resonances from the other base pairs broaden simultaneously with the onset of the melting transition. The nonexchangeable base and sugar H-1' protons are resolved in the duplex and strand states and shift as average peaks through the melting transition. The experimental shifts on duplex formation have been compared with calculated values based on ring-current and atomic diamagnetic anisotropy contributions for a B-DNA base-pair-overlap geometry in solution. Several nonexchangeable proton resonances broaden in the fast-exchange region during the duplex-to-strand transition and the excess widths yield a duplex dissociation rate constant for the octanucleotide of 1.9 x 10(3) s-1 at 32 degrees C (fraction of duplex = 0.86) in 0.1 M NaCl, 10 mM phosphate buffer. The 31P resonances of the seven internucleotide phosphates are distributed over 0.6 ppm in the duplex state, shift downfield during the duplex-to-strand transition and undergo additional downfield shifts during the stacked-to-unstacked strand transition with increasing temperature.     

A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult hemoglobin

High-resolution proton nuclear magnetic resonance spectroscopy and nuclear Overhauser effects for the low-field exchangeable proton resonances of human normal adult hemoglobin in aqueous solvents are being used to confirm and extend the assignments of these resonances to specific protons at the intersubunit interfaces of the molecule. Most of these exchangeable proton resonances of human normal adult hemoglobin have been found to be absent in the spectra of isolated alpha or beta subunits. This finding indicates that they are specific spectral markers for the quaternary structure of the hemoglobin tetramer. Based on the nuclear Overhauser effect results, we have assigned the exchangeable proton resonance at +7.4 ppm downfield from H2O to the hydrogen-bonded proton between alpha 103(G10)His and beta 108(G10)Asn at the alpha 1 beta 1 interface. The nuclear Overhauser effect results have also confirmed the assignments of the exchangeable proton resonances at +9.4 and +8.2 ppm downfield from H2O previously proposed by workers in this laboratory based on a comparison of human normal adult hemoglobin and appropriate mutant hemoglobins. This independent confirmation of previously proposed assignments is necessary in view of the possible long-range conformational effects of single amino-acid substitutions in mutant hemoglobin molecules.     

Complete structure of lipid A obtained from the lipopolysaccharides of the heptoseless mutant of Salmonella typhimurium

A highly purified monophosphoryl lipid A, TLC-3 fraction obtained from the lipopolysaccharides of the heptoseless mutant Salmonella typhimurium G30/C21 was converted to the dimethyl pentatrimethylsilyl derivative and analyzed by proton NMR spectroscopy at 400 MHz. Substantial downfield shifts of the resonances for protons at the 3- and 3'-carbons of the glucosamine disaccharide to 5.06 and 5.15 ppm, respectively, occurred from the normal range of 3.5-4.1 ppm, indicating that these two positions on the sugar rings were acylated. Significant downfield shift of the resonances for protons at the 4- and 6'-carbons did not occur, indicating the absence of acyl groups at these two positions. Since positive ion fast atom bombardment mass spectrometry previously established the presence of hydroxymyristoyl and myristoxymyristoyl esters at the reducing end and distal subunits, respectively, these acyl groups must be attached to the oxygen of the corresponding 3- and 3'-carbons of lipid A. With these results, we can now describe the complete structure of the monophosphoryl lipid A, TLC-3 from S. typhimurium.     

1H-nuclear magnetic resonance spectroscopy of reducing-residue anomeric protons of pertrifluoroacetylated carbohydrates

Eight monosaccharides (L-arabinoside, L-fucose, D-galactose, D-glucose, D-lyxose, D-mannose, L-rhamnose, and D-xylose), eight disaccharides (cellobiose, gentiobiose, isomaltose, lactose, maltose, nigerose, sophorose, and xylobiose), and three trisaccharides (isomaltotriose, maltotriose, and xylotriose) were derivatized with N-methylbis-(trifluoroacetamide) in pyridine solution to form trifluoroacetylated derivatives. These were analyzed by 1H-n.m.r. spectroscopy to determine the characteristics of the spectra and distributions of the reaction products. Peaks corresponding to reducing-residue anomeric protons were located significantly downfield of all others, and were in general 0.4 p.p.m. or more downfield of equivalent signals from the same carbohydrates when they were free or derivatized with other groups. Neither the location of anomeric proton peaks relative to each other nor the degree of spin-spin coupling between H-1 and H-2 varied greatly with type of derivatization. Spin-spin coupling, however, decreased for some beta-pyranose forms of xylobiose and the three trisaccharides. In all examples except some where H-2 was oriented equatorially to a pyranose ring, the proportion of the alpha-pyranose was either enhanced or not changed in concentration by trifluoroacetylation.     

Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of peptide-binding on the proton n.m.r. spectrum of bovine neurophysin-I

The effects of binding L-phenylalanyl-L-phenylalanine amide and related peptides on the 220 MHz and 300 MHz proton n.m.r. spectra of bovine neurophysin-I were studied. Throughout both the aliphatic and aromatic proton regions, marked binding-induced changes in the protein spectrum occur which are best explained by invoking conformational change within the neurophysin dimer, in addition to direct perturbation of individual protein protons by bound peptide. In the region downfield from 6 p.p.m., a new resonance, centered at 6.45 p.p.m. was resolved in 300 MHz spectra. This resonance is tentatively assigned to a non-exchangeable -NH and undergoes a reversible binding-induced broadening. Also in this region, the binding-induced chemical shift change in the ortho ring protons of Tyr-49 was used to explore additional aspects of the kinetics of peptide-binding. The results indicate that peptides with affinities greater than or equal to 10(4) M-1 exhibit slow to intermediate exchange rates on the time scale of the Tyr-49 chemical shift change, but that fast exchange can be achieved with peptides having affinities approximately equal to 10(2) M-1.     

Antithrombin III and its interaction with heparin. Comparison of the human, bovine, and porcine proteins by 1H NMR spectroscopy

1H NMR has been used to characterize and compare the structures of antithrombin III from human, bovine, and porcine plasma as well as to investigate the interactions of each of these proteins with heparin fragments of defined length. The amino acid compositions of the three proteins are very similar, which is reflected in the gross features of their 1H NMR spectra. In addition, aromatic and methyl proton resonances in upfield-shifted positions appear to be common to all three proteins and suggest similar tertiary structures. Human antithrombin III has five histidine residues, bovine has six, and porcine has five. The C(2) proton from each of these residues gives a narrow resonance and titrates with pH; the pKa's are in the range 5.15-7.25. It is concluded that all histidines in each protein are surface residues with considerable independent mobility. The carbohydrate chains in each protein also give sharp resonances consistent with a surface location and motional flexibility. The 1H spectra are sensitive to heparin binding. Although heparin resonances obscure protein resonances in the region 3.2-6.0 ppm, difference spectra between antithrombin III with and without heparin show clear perturbation of a small number of aromatic and aliphatic protein protons. These resonances include those of histidine C(2) and C(4) protons, of 10-20 other aromatic protons, of a methyl group, and also of protons with chemical shifts similar to those of lysine and/or arginine side chains. For human antithrombin III, it was shown that heparin fragments 8, 10, and 16 sugar residues in length result in almost identical perturbations to the protein. In contrast, tetrasaccharide results in fewer perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton magnetic resonance studies of ultraviolet-irradiated apurinic acid

In apurinic acid, a single-stranded polydeoxyribonucleotide easily obtained upon depurination of DNA, the proton resonances arising from thymine and cytosine are readily observable in aqueous solution of 25°C. Two methyl thymine resonances, centered at 1.88 ppm and separated by 0.045 ppm, are observed. We attribute the downfield methyl resonance to thymines with no pyrimidine nearest neighbors and the upfield methyl resonance to thymines having pyrimidine neighbors in the 3′ and/or 5′ positions. Upon ultraviolet irradiation, the upfield methyl and thymine H-6 resonances decrease in amplitude and two methyl resoances appear at 1.63 and 1.52 ppm, corresponding, respectively, to cytosine-thymine and thymine-thymine cyclobutane dimers. Photoreversal eliminates these two minor methyl resonances from the pmr spectrum. We conclude that apurinic acid provides a suitable model system for pmr studies of chemically modified pyrimidine bases in DNA.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.