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.

The O 2 equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O 2 affinity. The O 2 affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O 2 carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O 2 -linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model. However, the interactions with allosteric effectors provide such "inert" hemoglobin unprecedented magnitudes of functional diversities not only of physiological relevance but also of extreme nature, by which hemoglobin can behave energetically beyond what can be explained by the Monod-Wyman-Changeux/Perutz model. Thus, the heterotropic effector-linked tertiary structural changes rather than the homotropic ligation-linked T/R quaternary structural transition are energetically more significant and primarily responsible for modulation of functions of hemoglobin. Hemoglobin (Hb)1 has played a pivotal role in the understanding of the mechanisms of allosteric enzymes. Monod et al.(1) designated Hb an honorary enzyme, since it used the same molecule (O 2 ) for signaling as well as regulation. With the advent of detailed molecular structure at atomic levels, the question of enzyme activity became one of molecular mechanism. In the case of an allosteric enzyme, there is a need to assume at least two possible structures, customarily labeled T and R (1), and to regulate ligand affinity in each structure. The first question of signaling is straightforward, for it involves alternate packing of interfaces, for example. Monod's original proposal (1) to assign deoxy-and oxy-Hbs to the T and R states acquired a structural foundation, since crystallographic studies (2) revealed that the three-dimensional molecular structures of deoxy-Hbs and ligated Hbs. The hemoglobin molecule, a heterotetramer, consists of two ␣-and two ␤-subunits, each of which contains one O 2 -binding heme group. These four subunits are paired as two dimers, ␣ 1 ␤ 1 and ␣ 2 ␤ 2 . The structural studies showed that deoxy-Hbs and ligated Hbs have two different modes of packing of the two dimers (the quaternary structures) with no major changes in the gross conformation of each of the subunits (the tertiary structures). Thus, Perutz (3) assigned "deoxy" and "oxy" Hbs to T and R quaternary states, which exhibited low and high O 2 affinity, respectively.The second question concerning regulation is the deeper one, and for Hb it has proved remarkably elusive. The essence of the question is to find a way in the low affinity T (deoxy) state to store free energy that is made available to bind ligands in the high affinity R (oxy) state. The MWC/Perutz stereochemical model...

We previously reported the de novo design of an amphiphilic peptide @YGG~IEKKIEA! 4 # that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded a-helix structure. The addition of Cd~II!, however, resulted in the reconstitution of the triple-stranded a-helix bundle, as revealed by circular dichroism~CD! spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant K d was determined to be 1.5 6 0.8 mM for Cd~II!. The triple-stranded complex formed by the 113 Cd~II! ion showed a single 113 Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl Ϫ ions. The 113 Cd NMR resonance was connected with the bH protons of the cysteine residue by 1 H-113 Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg~II! also induced the assembly of the peptide into a triple-stranded a-helical bundle below the Hg~II!0peptide ratio of 103. With excess Hg~II!, however, the a-helicity of the peptide was decreased, with the change of the Hg~II! coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.Keywords: coiled coil; de novo design; folding; helical structures; metalloproteins Studies on de novo designed proteins involve the construction of proteins with unique tertiary structures and the creation of new functional proteins. The coiled coil structure is often observed in natural proteins for the intermolecular assemblies of the functional domains. This motif, due to its structural simplicity and biomolecular significance, has been the subject of extensive analyses to understand the principles of de novo design, as well as protein folding and stability~Lau et al., 1984;O'Neil & DeGrado, 1990;Harbury et al., 1993!. Furthermore, the designed coiled coils can be fused to various functional peptides or domains of natural proteins for biological and medical applications~Pack & Plückthun, 1992; Hodges, 1996; Terskikh et al., 1997!.The designed coiled coil, which drastically changes its conformation depending on external stimuli, should be useful to control the associations and the functions of domains attached to the peptide. Among the various external stimuli, metal binding has been studied extensively, and the factors required for metal binding are well understood. A variety of metal binding sites have been designed, but they are on the surfaces of preformed artificial supramolecules, such as an a-helical bundle protein~Handel & Regan & Clarke, 1990;Regan, 1995;Dieckmann et...

We have demonstrated recently that nitrous acid or nitric oxide converts 2'-deoxyguanosine (dGuo) into 2'-deoxyoxanosine (dOxo) [Suzuki, T., Yamaoka, R., Nishi, M., Ide, H., & Makino, K. (1996) J. Am. Chem. Soc. 118, 2515-2516]. In the present study, we have measured susceptibility of the N-glycosidic bond of dOxo to spontaneous hydrolysis and its base-pairing stability to evaluate the biological significance of dOxo as a new lesion in DNA. When oligodeoxynucleotide d(T5OT6) (O = dOxo), isolated from nitrous acid-treated d(T5GT6), was incubated at pH 4.0 and 70 degrees C, hydrolysis of the N-glycosidic bond of dOxo occurred with a first-order rate constant. Comparison of the rate constants with those of dGuo and dXao indicates that the N-glycosidic bond of dOxo was as stable as that of dGuo in d(T5GT6) and hydrolyzed 44-fold more slowly than that of 2'-deoxyxanthosine (dXao), a simultaneously generated damage by nitrous acid and nitric oxide. For the estimation of the base-pairing stability, UV melting curves were measured for the duplexes of d(T5OT6).d(A6NA5) (N = A, G, C, and T) at neutral pH. The Tm values obtained were 15.3, 14.1, 19.3, and 16. 3 degrees C for N = A, G, C, and T, respectively, which are much lower than that of the intact duplex containing a G.C pair at the same position [d(T5GT6).d(A6CA5), Tm = 32.8 degrees C] but comparable with those of d(T5XT6).d(A6NA5) (X = dXao, Tm = 14.8-22.3 degrees C). CD spectra of the four duplexes containing dOxo showed preservation of the structure of the intact duplex at low temperature. UV and NMR pH-titration studies indicated the pKa for the ring-opening and -closing equilibrium to be 9.4, implying that dOxo is in the ring-closed form at physiological pH. This structure appears to be not suitable geometrically for the hydrogen bond formation with a specific counter base, thus causing equally low Tm values for all the counter bases. Consequently, these results imply that dOxo, a novel DNA lesion, may have an important and unique role in mutagenic events in cells.