Conformational transitions and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state 13C NMR.

Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high-resolution solid-state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local alpha-helical form is present around Gly10 whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a local beta-sheet form as viewed from Gly10 and Phe22, and both beta-sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from alpha-helix to beta-sheet, and from random coil to beta-sheet forms occurred in the central and C-terminus regions, respectively, during the fibril formation. The increased 13C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two-step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local beta-sheet conformation at the central region and exhibited a random coil at the C-terminus region. Not only a hydrophobic interaction among the amphiphilic alpha-helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel beta-sheets at pH 7.5 and a mixture of antiparallel and parallel beta-sheets at pH 3.3.

Conformational studies on the specific cleavage site of Type I collagen (alpha-1) fragment (157-192) by cathepsins K and L by proton NMR spectroscopy.

Cathepsins K and L are cysteine proteinases which are considered to play an important role in bone resorption. Type I collagen is the most abundant component of the extracellular matrix of bone and regarded as an endogenous substrate for the cysteine proteinases in osteoclastic bone resorption. We have synthesized a fragment of Type I collagen (alpha-1) (157-192) as a substrate for the cathepsins and found that cathepsins K and L cleave the fragment at different specific sites. The major cleavage sites for cathepsin K were Met159-Gly160, Ser162-Gly163 and Arg165-Gly166, while those for cathepsin L were Gly166-Leu167 and Gln180-Gly181. The structure of the fragment was analyzed in aqueous solution by circular dichroism and proton NMR spectroscopy and the difference in the molecular recognition of collagen by cathepsins K and L was discussed from the structural aspect.

Phosphorus-31 nuclear magnetic resonance study of healthy human saliva.

Phosphorus nuclear magnetic resonance spectra of whole, mandibular and parotid salivas from healthy humans all presented single broad inhomogeneous lines, the widths arising from the chemical-shift distribution of the resonances of the constituent inorganic phosphate compounds. Spin lattice relaxation of phosphorus in the stimulated parotid saliva was characterized by a single time constant of T1, whereas the spin spin relaxation was found to be determined by the chemical exchange rates. The results indicate that calcium phosphates found in parotid saliva are involved in at least two chemical exchange processes. The dissociation rates for the fast and slow exchange were estimated to be 1.5/sec and 0.2/sec, respectively.

Structural study on silkworm eclosion hormone fragment (1-34) in solution by proton nuclear magnetic resonance spectroscopy.

Eclosion hormone (EH) is a neuropeptide hormone which controls the ecdysis behavior in insect. The three dimensional structure of the N-terminal fragment (1-34) of the eclosion hormone which was predicted to contain a compact region crucial for the EH activity was studied in 50% d3-trifluoroethanol(TFE)/50% H2O at pH 3 and 298 K by 1H NMR spectroscopy with the combined use of distance geometry and molecular dynamics calculations. NMR results indicated that the fragment actually assumes an alpha-helix between Ala10 and Gln20, but no rigid structure is present from Cys21 through the C-terminus and for the N-terminal region (Ser1-Asp9). The elucidated structure was compared with the predicted structure of the native EH for the further development of the design of the insecticide.

Structural studies on endothelin receptor subtype B specific agonist IRL 1620 [suc-[Glu9, Ala11,15]ET-1(8-21)] and its analogs with dipalmitoyl phosphatidylcholine vesicles by NMR spectroscopy.

IRL 1620 ¿suc-[Glu9,Ala11,15]ET-1(8-21)¿ is a potent and specific agonist for the ET(B) receptor. Five analogs of IRL 1620 were synthesized in this study. These were all C-terminal linear peptides of endothelin 1 (ET-1) comprising 14 amino acid residues and exhibiting highly potent ET(B) receptor binding affinities. The peptides consisted of three pairs and each component of the pairs differed from its partner in only the 18th residue, i.e. Asp was replaced by Gly. The replacements resulted in more than a 10-fold increase in affinity to the ET(A) receptor. The structures of these peptides were investigated in the presence of phospholipid vesicles (dipalmitoyl phosphatidylcholine) by NMR spectroscopy. By the replacement of Asp by a less bulky Gly, the C-terminal tripeptide region folded back toward the helical region, making it shorter than the Asp-substituted peptide helical region. Such a folded conformational feature may explain the increased binding affinity to ET(A) receptor.

Comparative study of chicken and human parathyroid hormone-(1-34)-peptides in solution with SDS.

Molecular conformations of chicken [cPTH-(1-34)] and human [hPTH-(1-34)] parathyroid hormone fragments in aqueous solutions with various concentrations of SDS were investigated by CD, fluorescence and NMR spectroscopy techniques. In the presence of SDS, chicken and human PTH-(1-34) adopt an a-helical structure making up 32-38% of all the peptide amino acids. The process of the a-helical formation of these two fragments is considerably different. The CD spectral change of hPTH-(1-34) was characteristic of a monotonous increase in the negative peak at 222 nm with increasing SDS concentrations. However, for cPTH-(1-34) a beta-turn is formed first, followed by alpha-helix formation upon an increase in SDS concentrations. The change of the tryptophan fluorescence spectra of cPTH-(1-34) is well correlated with the changes in CD spectra, suggesting that the side chain of Trp23 is involved in the conformational change from random coil to alpha-helix via beta-turn. The three-dimensional structure of cPTH-(1-34) with SDS micelle was elucidated by 1H-NMR at pH 3.8 and 300 K, with the combined use of distance geometry and restrained molecular dynamics calculations. NMR results indicated that it contains two helices encompassing residues 7-12 and 24-30, respectively. The C-terminal helix in the residue range of 24-30 is amphiphilic, which is stabilized by the hydrophobic interactions among Trp23, Leu24 and Lys27.

Effect of excess cadmium ion on the metal binding site of cabbage histidinol dehydrogenase studied by 113Cd-NMR spectroscopy.

The enzymatic reaction of histidinol dehydrogenase (HDH) was stimulated by about maximally 75% on the addition of Cd2+ ion to the reaction mixture. 113Cd-substituted HDH in the presence of excess Cd2+ has been studied by 113Cd-NMR. 113Cd2+ less than 1 equiv. per subunit preferentially binds to the catalytic metal binding site of the apoenzyme. Further addition of the metal ions causes the structural change of the enzyme including the catalytic metal binding site. HDH takes at least three discernible states, which may correspond to the more or less active forms of the enzyme induced by metal ions.

Characterization of human calcitonin fibrillation in aqueous urea solution by 1H NMR spectroscopy.

The inhibitory effects of urea on the normally rapid fibrillation of human calcitonin (hCT) were investigated by 1H NMR. From subtle differences in the chemical shift of hCT in the presence and absence of urea, the occurrence of weak interactions between urea and hCT was confirmed. The chemical shifts on the NH and C alpha protons of residues in the C-terminal (Gln24-Pro32) region were unaffected by the urea interactions, while the chemical shifts of the N-terminal (Cys1-Cys7) and central (Met8-Pro23) residues were observed to move significantly downfield with increasing urea concentrations. These findings suggest that urea serves to stabilize the monomeric form of hCT and to promote the concentration of the extended hCT conformer. However, it was also found that even by storing hCT in urea the fibrillation process cannot be circumvented, as the gradual carbamylation of the N-terminus takes place. The time course of 1D and 2D spectra of carbamylated hCT showed that cross peaks of residues in the N-terminal and central regions disappear faster than those in the C-terminal regions, indicating that the fibrillation of carbamylated hCT is initiated in the N-terminal and central regions. It is postulated that carbamylation increases the hydrophobicity of the N-terminal region and hence fosters fibrillation, even in urea solution.

113Cd nuclear magnetic resonance studies of cabbage histidinol dehydrogenase.

Histidinol dehydrogenase (HDH), a dimeric protein, catalyzes two sequential oxidation reactions to yield L-histidine from L-histidinol via L-histidinal. HDH contains 1 mol of Zn(II) per mol of subunit, and removal of this metal abolishes the enzymatic activity. On substitution of Zn(II) with 113Cd(II), the enzyme ([113Cd]HDH) showed similar catalytic activity. The 113Cd NMR spectra of [113Cd]HDH were measured under various conditions. The 113Cd NMR spectrum of [113Cd]HDH showed a resonance at 110 ppm, which indicates that the metal ion is bound to the protein by a combination of nitrogen and oxygen ligands. 113Cd NMR spectra of [113Cd]HDH were measured as complexes with two substrates (L-histidinol and DL-histidinal) and four inhibitors (imidazole, histamine, L-histidine, and DL-4-(4-imidazolyl)-3-amino-2-butanone) in the absence and presence of NAD+. Significant shifts of [113Cd]-HDH resonance in the presence of the ligand indicate that the metal ion is located in the catalytic site of HDH and that substrates and inhibitors interact with the metal ion. The role of the metal ion in the HDH reaction is discussed.

Solution structure of the phosphorylated sites of ribosomal protein S6 by 1H NMR spectroscopy.

An increase in the rate of protein synthesis is found to be accompanied by phosphorylation of the 40S ribosomal protein S6. Treatment of S6 by cyanogen bromide produced three fragments, and one of the fragments of S6, which is a C-terminal portion of S6 (M(r) approximately 4,000), contains all phosphorylation sites of S6. The C-terminal fragment of S6 contains seven serines. S6 kinase phosphorylates S6 specifically, i.e. five serines in the C-terminal of S6 are phosphorylated. The three-dimensional structure of S6 peptide was studied in 50% trifluoroethanol/50% H2O solution by 1H NMR with combined use of distance geometry and restrained molecular dynamics calculations. NMR results indicated that it takes an alpha-helix between Glu5 and Arg21 and a distorted helical structure for the following three residues, but no rigid structure was present from Ser25 through the C-terminus and for the N-terminal region (Lys1-Lys4). The specificity of the phosphorylation of the peptide is discussed from a structural aspect.

Study of human calcitonin fibrillation by proton nuclear magnetic resonance spectroscopy.

The fibrillation of human calcitonin (hCT) has been investigated by NMR in aqueous solution. The time course of proton one- and two-dimensional NMR spectra of hCT (80 mg/mL at pH 2.9) was measured during the fibrillation. It showed a gradual broadening of the peptide peaks, followed by a rapid broadening and subsequent disappearance of the peaks. The gradual broadening can be attributed to equilibrium between monomer and associated hCT, whereas the rapid broadening can be attributed to formation of aggregates and to gelation of the peptide solution. All the peptide peaks did not broaden and disappear simultaneously. Peaks of residues in the N-terminal (Cys1-Cys7) and central (Met 8-Pro23) regions broadened and disappeared faster during the gradual broadening than those in the C-terminal region (Gln24-Pro32). Moreover, in the N-terminal and central residues, peaks of Cys1, Leu4,9, Met 8, Tyr12, Asp15, and Phe16,19,22 disappeared faster than those of Asn3,17, Ser5, Cys7, Gln14, Lys18, and His20. Hydrogen-deuterium exchange of amide protons indicated the formation of hydrogen bonds caused by association of hCT molecules. The amphiphilicity of the peptide appears to be important for the hCT association.