1H-NMR investigation of the influence of the heme orientation on functional properties of myoglobin.

There are two interconverting forms of myoglobin, which differ in the orientation of the heme by a 180 degrees rotation around the alpha,gamma-meso axis; the proteins possessing the same heme orientation, as found in the single crystal, and the reversed heme orientation are called the major and minor forms, respectively. Structures and functional properties of these two forms have been investigated by NMR. Heme peripheral side-chain and non-coordinated amino acid proton resonances of the minor form in its met-cyano form have been assigned and the comparison of the shift between the corresponding resonances of the two forms revealed that the heme electronic structure is not largely influenced by the heme rotation. On the other hand, the exchange rate of His E7 NepsilonH proton of the minor form is larger by a factor of 3-5 than that of the major one, indicating that the stability of the hydrogen bond between Fe-bound ligand and His E7 is slightly weaker in the minor form that in the major one. The rate of autoxidation of oxy-myoglobin and azide affinity of met-aquo myoglobin were also found to depend on the orientation of the heme.

Molecular mechanism for ligand stabilization in the mollusc myoglobin possessing the distal Val residue.

Myoglobin extracted from the triturative stomach of Dolabella auricularia, a common mollusc found on the Japanese coast, possesses naturally occurring substitution at the distal E7 position (Val-E7) and its oxygen affinity is only slightly lower than those of the common mammalian myoglobins possessing the usual His-E7. 1H nuclear magnetic resonance studies of Dolabella met-cyano myoglobin have revealed that a guanidino NH proton of Arg-E10 is hydrogen-bonded to the Fe-bound CN-. The role of Arg-E10 as a hydrogen-bond donor for Fe-bound ligand in the present myoglobin appears to be responsible for its relatively high ligand affinity.

N.m.r. study of interaction between sugar and peptide moieties in mucin-type model glycopeptides.

In order to investigate the structural properties of the sugar and peptide linkage region in glycoprotein, some glycopeptides were synthesized as a model for AFGP (antifreeze glycoprotein), which is one of the mucin-type glycoproteins. The results from n.m.r. measurements in DMSO and aqueous conditions revealed that the glycopeptides form an intramolecular hydrogen bond between the amide proton of N-acetylgalactosamine (GalNAc) and the carbonyl oxygen of threonine (Thr) to which the GalNAc is covalently linked. This intramolecular hydrogen bond may play an important role in determining the orientation of the sugar moiety relative to the protein backbone. The roles for the activity of the proline (Pro) residue in AFGP were also discussed.

Hydration of oligosaccharides: anomalous hydration ability of trehalose.

The disaccharide trehalose extensively exists in anhydrobiotic organism and is considered to play an important role in preserving the integrity of biomembrane. However, the preserving mechanism remains unclear. In this report, we examine the hydration abilities of trehalose and several oligosaccharides composed of alpha-D-glucopyranosyl residues. The unfrozen water fraction per molecule was determined from differential scanning calorimetry measurements of their aqueous solutions. Further, the NMR relaxation time of the natural abundance 17O of water is measured for several saccharide solutions. These results indicate that trehalose has the highest hydration ability among the saccharides studied. In other words, trehalose can effectively lower the mobility of water molecules hydrogen-bonded with saccharides. It is thus reasonable that, among the disaccharides studied, trehalose exhibits the maximum stabilizing effect on the bilayer structure of lipid whose acyl chains are bonded with each other by the apolar interaction, because the apolar interaction is strengthened with the stabilization of the surrounding water structure.

1H-NMR comparative study of the active site in shark (Galeorhinus japonicus), horse, and sperm whale deoxy myoglobins.

1H-NMR spectra of deoxy myoglobins (Mbs) from shark (Galeorhinus japonicus), horse, and sperm whale have been studied to gain insights into their active site structure. It has been demonstrated for the first time that nuclear Overhauser effect (NOE) can be observed between heme peripheral side-chain proton resonances of these paramagnetic complexes. Val-E11 methyl and His-F8 C delta H proton resonances of these Mbs were also assigned from the characteristic shift and line width. The hyperfine shift of the former resonance was used to calculate the magnetic anisotropy of the protein. The shift analysis of the latter resonance, together with the previously assigned His-F8 N delta H proton resonance, revealed that the strain on the Fe-N epsilon bond is in the order horse Mb approximately whale Mb < shark Mb and that the hydrogen bond strength of the His-F8 N delta H proton to the main-chain carbonyl oxygen in the preceding turn of the F helix is in the order shark Mb < horse Mb < whale Mb. Weaker Feporphyrin interaction in shark Mb was manifested in a smaller shift of the heme methyl proton resonance and appears to result from distortion of the coordination geometry in this Mb. Larger strain on the Fe-N epsilon bond in shark Mb should be to some extent attributed to its lowered O2 affinity (P50 = 1.1 mmHg at 20 degrees C), compared to whale and horse Mbs.

Kinetic characterization of the acid-alkaline transition in Dolabella auricularia ferric myoglobin using 1H-NMR saturation transfer experiments.

The acid-alkaline transition in ferric myoglobin of the mollusc, Dolabella auricularia, exerts the changes in both the coordination and spin states of the heme iron. Slower transition rate, compared to the NMR time scale, in this myoglobin allowed the observation of separate signals arising from the two forms, and pH titration yielded a pK value of 7.8. 1H-NMR saturation transfer experiments have been successfully used not only to provide the first signal assignments for the heme methyl proton resonances of the Met-hydroxyl form of the myoglobin, but also to determine the kinetics of the transition.

Biosynthesis of poly(3-hydroxyalkanoates) from threonine.

A series of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) was biosynthesized by Alcaligenes eutrophus from an amino acid, threonine. The 3HV content of these polyesters ranged from less than 0.1% to 30%.

Biosynthesis and n.m.r. studies of deuterated poly(3-hydroxybutyrate) produced by Alcaligenes eutrophus H16.

Alcaligenes eutrophus H16 was grown on mixtures of 1H- and 2H-acetate as carbon sources. The accumulation of deuterated poly(3-hydroxybutyrate) (P(3HB)) was observed. The deuterium distributions in the isolated P(3HB)s were determined from 1H and 2H-n.m.r. spectra and confirmed by 13C-n.m.r. spectra. Although one would expect to synthesize P([2,2,4,4,4-2H5]3HB) when the cells were grown on 2H-acetate as the sole carbon source, the methyl, methylene and methine groups of the P(3HB) contained both deuterium and proton. This observation indicates some substitution from 2H to 1H during the P(3HB) synthesis. The 2H content in the methyl groups was larger than that in the methylene groups, which suggests a kinetic isotope effect in the P(3HB) synthesizing process. The deuterium distributions in the two magnetically non-equivalent methylene protons were determined to be different, which indicates stereoselectivity at the C2 site.

Orientation and mobility of the heme vinyl groups in myoglobins with the aid of NOE and MATDUHM NMR.

The heme vinyl substituents in a shark (Galeorhinus japonicus) myoglobin in its met-cyano form (MbCN) have been characterized by NMR and the results were compared with those of the well-studied sperm whale (Physter catodon) myoglobin. Their orientation has been inferred from NOE connectivities and the analysis of the hyperfine shifts based on the principal magnetic tensor determined by MATDUHM (Magnetic Anisotropy Tensor Orientation Determination Utilizing the Heme Methyls) [Yamamoto, Y., Nanai, N. and Chujo, R.(1990) J. Chem. Soc., Chem. Commun. 1556-1557]. It has been shown that the C3-vinyl group is oriented roughly orthogonal to the heme plane in both G. japonicus and P. catodon MbCNs at 35 degrees C and their C8-vinyl groups, on the other hand, are close to in-plane orientation. Although CO form of myoglobin (MbCO) and MbCN have been thought to be isostructural to each other, the C8-vinyl orientation for P. catodon MbCN is found to be different from the orthogonal orientation indicated in the crystal structure analysis of MbCO [Hanson, J.C. and Schoenborn, B.P. (1981) J. Mol. Biol. 153, 117-146]. Their mobility has been characterized quantitatively from the study of time-dependent NOE build-up between the selected pair of the vinyl proton resonances. It has been revealed that the heme C3- and C8-vinyl groups of approximately 1 mM G. japonicus MbCN at 45 degrees C undergo internal motion with the correlation time of 1.9 and 2.4 ns, respectively. Therefore, their oscillatory motion is faster by a factor of 4-5 compared with the protein overall tumbling. Difference in the internal mobility between the two vinyl groups in the active site of this Mb is attributed to their differential contact with the apo-protein.

NMR study of Galeorhinus japonicus myoglobin. 1H-NMR evidence for a structural alteration on the active site of G. japonicus myoglobin upon azide ion binding.

The heme molecular structure of the met-azido form of the myoglobin from the shark Galeorhinus japonicus has been investigated by 1H NMR. A nuclear Overhauser effect (NOE) was clearly observed among the heme peripheral side-chain proton signals of this complex, which undergoes thermal spin equilibrium between high-spin (S = 5/2) and low-spin (S = 1/2) states, and the NOE connectivities provided the assignment of the resonances from the heme C13(1)H2 and C17(1)H2 protons. Chemical shift inequivalence of these proton resonances not only provided information about the orientation of these methylene protons with respect to the heme plane, but also allowed characterization of the time-dependent build-up of the NOE between them, which yields the correlation time for the internal motion of the inter-proton vector. The relatively large mobility found for the C17(1)H2 group suggests that the carboxyl oxygen of the heme C17 propionate is not anchored to the apo-protein by a salt bridge. It has been shown that the ferric high-spin form of G. japonicus Mb possesses a penta-coordinated heme [Suzuki, T. (1987) Biochim. Biophys. Acta 914, 170-176; Yamamoto, Y., Osawa, A., Inoue, Y., Chûjô, R. & Suzuki, T. (1990) Eur. J. Biochem. 192, 225-229] and that the conformation of both heme propionate groups is fixed with respect to the heme, as well as the apo-protein, by a salt bridge [Yamamoto, Y., Inoue, Y., Chûjô, R. & Suzuki, T. (1990) Eur. J. Biochem. 189, 567-573]. Therefore the weakening or interruption of the interaction between the C17 propionate and His FG3 upon the changes of the coordination and spin state of the heme iron, during azide ion binding to ferric high-spin G. japonicus Mb, is attributed to the displacement of the FG corner of the apoprotein away from the heme C17 propionate group. A similar structural alteration has been revealed by X-ray structural analyses of unliganded and liganded forms of ferrous hemoproteins [Baldwin, J. & Chothia, C. (1979) J. Mol. Biol. 129, 175-220; Phillips, S.E.V. (1980) J. Mol. Biol. 142, 531-554].

NMR study of Galeorhinus japonicus myoglobin. 1H-NMR study of molecular structure of the heme cavity.

The molecular structure of the active site of myoglobin from the shark, Galeorhinus japonicus, has been studied by 1H-NMR. Some hyperfine-shifted amino acid proton resonances in the met-cyano form of G. japonicus myoglobin have been unambiguously assigned by the combined use of various two-dimensional NMR techniques; they were compared with the corresponding resonances in Physter catodon myoglobin. The orientations of ThrE10 and IleFG5 residues relative to the heme in G. japonicus met-cyano myoglobin were semiquantitatively estimated from the analysis of their shifts using the magnetic susceptibility tensor determined by a method called MATDUHM (magnetic anisotropy tensor determination utilizing heme methyls) [Yamamoto, Y., Nanai, N. & Chûjô, R. (1990) J. Chem. Soc., Chem. Commun., 1556-1557] and the results were compared with the crystal structure of P. catodon carbonmonoxy myoglobin [Hanson, J. C. & Schoenborn, B. P. (1981) J. Mol. Biol. 153, 117-124]. In spite of a substantial difference in shift between the corresponding amino acid proton resonances for the two proteins, the orientations of these amino acid residues relative to the heme in the active site of both myoglobins were found to be highly alike.

A 1H-NMR study of electronic structure of the active site of Galeorhinus japonicus metmyoglobin.

The ferric high-spin form of the myoglobin from the shark Galeorhinus japonicus, which possesses a Gln residue at the distal site instead of the usual His residue, has been studied by 1H-NMR spectroscopy. Using the heme meso-proton (C5H, C10H, C15H and C20H) resonance shift as a diagnostic probe for identifying the coordination system of the iron center in ferric high-spin form of hemoprotein, it has been shown that G. japonicus metmyoglobin (metMb) possesses the pentacoordinated active site. The pH-dependence study of NMR spectra of G. japonicus metMb revealed the appearance of the hydroxyl form of metMb at high pH, indicating that the protein undergoes the transition between the acidic and alkaline forms. The pK value and the rate for this acid-alkaline transition in G. japonicus metMb were found to be approximately 10 and much less than 4 x 10(2) s-1, respectively. Since the pK value of the acid-alkaline transition for the pentacoordinated heme in Aplysia limacina metMb is 7.8 [Giacometti, G.M., Das Ros, A., Antonini, E. & Brunori, M. (1975) Biochemistry 14, 1584-1588] and that of the hexacoordinated heme in sperm whale metMb is 9.1 [Brunori, M., Antonini, E., Fasella, P., Wyman, J. & Rossi-Fanelli, A. (1968) J. Mol. Biol. 34, 497-504], the OH- affinity of the ferric heme iron does not appear to depend on its coordination system. The acid-alkaline transition rate in A. limacina metMb was reported to be much less than 1.5 x 10(2) s-1 [Pande, U., La Mar, G.N., Lecomte, J.T.J., Ascoli, F., Brunori, M., Smith, K.M., Pandey, R.K., Parish, D.W. & Thanabal, V. (1986) Biochemistry 25, 5638-5646] and therefore a slow transition rate may be unique to the pentacoordinated active site of Mb.

1H-NMR study of heme propanoate mobility in the active site of myoglobin from Galeorhinus japonicus.

Time-dependent NOE studies of the C13(1) and C17(1) methylene proton resonances of the heme peripheral propanoate groups have elucidated their mobility in the active site of the ferric high-spin form of Galeorhinus japonicus myoglobin. A large difference in the chemical shift due to the non-equivalence of the heme C13(1) and C17(1) methylene proton resonances allows selective irradiation of a given proton resonance by a high-power selective decoupler pulse in spite of their fast relaxation rates. NOE accumulation of the resonance of one methylene proton after saturation of the resonance of the other proton essentially follows the theoretical prediction derived using the two-spin approximation, and the cross-relaxation rates for the heme C13(1) and C17(1) methylene proton spin systems were quantitatively determined. The correlation time for the mobility of the internuclear vector connecting the heme C13(1) or C17(1) methylene protons was then calculated from the cross-relaxation rate and values of approximately 11 ns were obtained for both C13(1) and C17(1) methylene groups in 2 mM Galeorhinus japonicus myoglobin at 35 degrees C. The immobile C13(1) and C17(1) methylenes of the heme propanoate groups, together with a large difference in chemical shift between the methylene proton resonances, dictate their fixed orientation with respect to the protein moiety as well as the heme plane, and are therefore consistent with the immobile heme in the active site of myoglobin.

Heme methyl hyperfine shift pattern as a probe for determining the orientation of the functionally relevant proximal histidyl imidazole with respect to the heme in hemoproteins.

Heme methyl 1H and 13C resonances of met-cyano form of myoglobin from the shark, Geleorhinus japonicus (GJMbCN), have been assigned via 1H-13C heteronuclear shift correlated spectroscopy (COSY) connectivities and their hyperfine shifts were compared with those of the corresponding resonances of some hemoproteins. Variation of the heme methyl 1H hyperfine shift pattern correlates well with the angle (phi) between the projection of the proximal histidyl imidazole plane onto the heme plane and the NII-Fe-NIV vector. The alteration of the interaction of the heme peripheral side-chains and/or the iron-bound ligand with the surrounding amino acid residues cannot account for large differences in the shifts of the corresponding heme methyl resonances between GJMbCN and sperm whale MbCN. Since the heme methyl 1H shifts for GJMbCN fall in between those of the corresponding resonances for sperm whale Mb and Aplysia limacina Mb in which the phi values have been reported to be 19 degrees and 29 degrees, respectively, the phi value in GJMb is estimated to be slightly larger than 19 degrees.

Synthesis and conformational analysis of Aib-containing peptide modelling for N-glycosylation site in N-glycoprotein.

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a beta-turn structure with a hydrogen bond involving Thr and Aib4 NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d6 solution.

N.m.r. study on conformation of Ac-Thr(alpha-GalNAc)-Ala-Ala-OMe as a model for mucin type glycoprotein.

This study report on the results of high resolution 1H n.m.r. investigations on Ac-Thr(alpha-GalNAc)-Ala-Ala-OMe 1 as a mucin type model glycopeptide of antifreeze glycoprotein (AFGP) in both dimethyl sulfoxide (DMSO) and H2O. The temperature dependence of amide proton chemical shifts strongly suggested the presence of the intramolecular hydrogen bond between the amide proton of GalNAc and the carbonyl oxygen of the Thr residues. Due to this bond, the orientation of the sugar residue of 1 appears to be fairly restricted relative to its peptide backbone. Despite the lack of the clear evidence for such intramolecular hydrogen bond in H2O, 1H coupling constant data suggested the structural similarity of 1 in DMSO and H2O, indicating the presence of the intramolecular hydrogen bond even in H2O, which may play an important role in determining the orientation of the sugar moiety with respect to the peptide backbone in glycoprotein.

Determination of the functionally important heme peripheral vinyl group orientation in paramagnetic hemoprotein by 2D NMR.

2D NMR spectroscopies have been successfully used to characterize the heme peripheral vinyl groups in paramagnetic hemoprotein in spite of the difficulties from the rapid paramagnetic relaxation and the low digital resolution of the 2D NMR map. The scalar coupling network system among the vinyl protons is clearly identified in the COSY spectra from its characteristic cross-peak pattern and the dipolar coupling connectivities of the vinyl proton resonances with other heme side-chain proton resonances not only provide the specific assignment of vinyl beta-proton resonances but also allow the determination of the vinyl group orientation with respect to the heme plane.

[Clinical prospective study of surgical treatment of pterygium].

Pterygium is a kind of degenerative lesion of the ocular conjunctiva and is thought to be hyperaemia of fibrovascular tissue onto the cornea caused by various factors. The most frequently used treatment for pterygium at present is surgery. Various surgical procedures and postoperative treatments for pterygium have been reported, however, the postoperative recurrence rates are somewhat high. Therefore, until now we have had no satisfactory clinical management of pterygium. The results of our method for surgical treatment of pterygium are given and compared with those described in many other reports. From this comparison, our clinical surgical treatment of pterygium, which is the so-called "bare sclera" technique, is evaluated to give the best management without serious complications and recurrences.

Natural abundance 13C-NMR study of paramagnetic horse heart ferricytochrome c cyanide complex: assignment of hyperfine shifted heme methyl carbon resonances.

Hyperfine shifted heme methyl carbon resonances of paramagnetic horse heart ferricytochrome c cyanide complex (Cyt-c(CN)) have been observed for the first time in the natural abundance 13C-NMR spectrum and assigned using 1H-13C heteronuclear chemical shift correlated spectroscopy (1H-13C COSY). Individual heme methyl carbon NMR signal assignment permits a direct comparison between the hyperfine shifts of heme methyl carbon and attached methyl proton resonances which provides a useful information on the delocalization mechanism of the unpaired spin from the pi-conjugated system of porphyrin ring into the peripheral methyl side chains.