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A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.

The interaction between different HO(z.rad;) radical scavengers in a three-component antioxidant system has been investigated by means of 19F-NMR spectroscopy. This system is composed of bovine serum albumin (BSA), trolox, and N-(4-hydroxyphenyl)-trifluoroacetamide (CF(3)PAF). The antioxidant capacity of BSA and trolox has been assessed by measuring the amount of trifluoroacetamide (TFAM) arising from the radical mediated decomposition of CF(3)PAF. When assayed separately, both trolox and BSA behaved as antioxidants, as they were effective to protect CF(3)PAF from HO* radical-mediated decomposition. By contrast, trolox enhanced the production of TFAM in the presence of BSA, thus behaving as a pro-oxidant. Urate, carnosine, glucose, and propylgallate showed antioxidant properties both with or without BSA. CF(3)PAF and trolox were found to bind to BSA with association constants in the order of 5 x 10(3)M(-1) and to compete for the same binding sites. These results have been discussed in terms of BSA-catalysed cross-reactions between trolox-derived secondary radicals and CF(3)PAF.

Zinc finger domains of the classical type represent the most abundant DNA binding domains in eukaryotic transcription factors. Plant proteins contain from one to four zinc finger domains, which are characterized by high conservation of the sequence QALGGH, shown to be critical for DNA-binding activity. The Arabidopsis thaliana SUPERMAN protein, which contains a single QALGGH zinc finger, is necessary for proper spatial development of reproductive floral tissues and has been shown to specifically bind to DNA. Here, we report the synthesis and UV and NMR spectroscopic structural characterization of a 37 amino acid SUPERMAN region complexed to a Zn(2+) ion (Zn-SUP37) and present the first high-resolution structure of a classical zinc finger domain from a plant protein. The NMR structure of the SUPERMAN zinc finger domain consists of a very well-defined betabetaalpha motif, typical of all other Cys(2)-His(2) zinc fingers structurally characterized. As a consequence, the highly conserved QALGGH sequence is located at the N terminus of the alpha helix. This region of the domain of animal zinc finger proteins consists of hypervariable residues that are responsible for recognizing the DNA bases. Therefore, we propose a peculiar DNA recognition code for the QALGGH zinc finger domain that includes all or some of the amino acid residues at positions -1, 2, and 3 (numbered relative to the N terminus of the helix) and possibly others at the C-terminal end of the recognition helix. This study further confirms that the zinc finger domain, though very simple, is an extremely versatile DNA binding motif.

Nitrazine Yellow is an indicator used in microscopy with variations of absorption visible spectrum in pH range 6.0–7.0. The stated structure, deriving from the attack of 2′,4′-dinitrobenzenediazonium salt to ortho hydroxy position of 1-naphthol-3,6-disulphonic acid, appears incoherent with the alochromic behaviour. Nmr analysis and, in particular, HMQC and HMBC two-dimensional techniques showed that the real structure comes from an attack of the diazonium salt to para hydroxy position.

New insights into the understanding of the changes induced in the iron domain of neuromelanin (NM) upon development of Parkinson’s disease (PD) have been gained by electron paramagnetic spectroscopy (EPR). The results of this study are compared with a previously reported variable temperature analysis of X-band EPR spectra of a NM specimen obtained from control brain tissues. The availability of high sensitivity instruments operating in the Q-band (34.4 GHz) allows us to deal with the low amounts of NM available from PD brains. The organization of iron in NM is in the form of polynuclear superparamagnetic/antiferromagnetic aggregates, but the lack of one or more signals in the EPR spectra of NM from PD suggests that the development of the pathology causes NM to decrease its ability to bind iron. Furthermore, the detection of the Mn(II) signal in the Q-band spectra is exploited as an additional internal probe to assess minor structural differences in iron domains of PD and control NM specimens.

Nuclear Magnetic Relaxation field-cycling relaxometry is a technique, able to report on water mobility in tissues. By means of this technique, post-mortem specimens from both controls and idiopathic Parkinson's disease patients have been investigated. Results show different relaxometric behavior between the groups, which is consistent with protein aggregation in Parkinson's disease specimens.

Neuromelanin is a dark brown pigment suspected of being involved in the pathogenesis of Parkinson's disease. This pigment can be isolated from normal human substantia nigra by a procedure that includes an extensive proteolytic treatment. In this study we used such a procedure to extract the neuromelanin pigment from a pool of substantia nigra from patients affected by Parkinson's disease. 13C Cross polarization magic angle spinning nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy were used to characterize the solid residue obtained from the extraction procedure. We found that the pigment extracted from the substantia nigra of parkinsonian patients was mainly composed of highly cross-linked, protease-resistant, lipo-proteic material, whereas the neuromelanin macromolecule appears to be only a minor component of this extract. A synthetic model of melanoprotein has been prepared by enzymatic oxidation of dopamine in the presence of albumin. Once it has undergone the same proteolytic treatment, this model system yields a 13C-NMR spectrum which is similar to that observed for the parkinsonian midbrain extract. These results are consistent with the view that oxidative stress has a relevant role in the pathogenesis of Parkinson's disease.

The relaxometric properties of two Gd(III) DTPA-like complexes (DTPA = diethylenetriamine-N,N',N,N"'-pentaacetic acid) bearing different substituents for binding to human serum albumin (HSA) are compared. In spite of the structural differences of the recognition synthon and of the residual electric charge, the two chelates display an analogous binding affinity for the serum protein. Upon formation of the adducts with HSA, the exchange rates of the coordinated water appear slowed down by an amount corresponding to ca. 50% of the rates found for the free complexes. The relaxivity of [Gd(BOM)3DTPA (H2O)]2- is significantly higher than that of MS-325 either in the free complex or in the macromolecular adduct. Finally, the effect of pH on the stability of the HSA adducts and on the values of their relaxivities has been investigated.

The interactions between iron and neuromelanin (NM) have been studied by means of EPR spectroscopy. The variable temperature EPR spectral features of a specimen of NM extracted from normal human midbrains clearly indicate that iron is present as polynuclear oxy-hydroxy ferric aggregates as well as isolated Fe(III) centres. Ferric oxy-hydroxy phases are typical of the iron storage proteins ferritin and hemosiderin, but the comparison of the variable temperature EPR spectra of ferritin and NM highlights significant differences between the two iron(III)oxy-hydroxy domains. Moreover, further investigations on melanin models synthesised in the presence of either ferritin or a ferric salt as iron sources suggest that the same pathway of formation and inclusion of the polynuclear iron oxide is operating in NM and in the model systems, whatever is the source of iron.

The cover picture shows a molecular model of the interaction between the new CCK8 analogue, Cycle29,34[Dpr29,Lys34]-CCK8 (shown as a CPK model) and the receptor fragment CCKA-R(1–47) (represented by a pink ribbon). The introduction of the cyclic constraint between the Dpr29 sidechain and the CCK8 C terminus (Lys34) decreases the flexibility of the molecule to stabilize the bioactive conformation of Cycle29,34[Dpr29,Lys34]-CCK8. The Trp30 and Met31 side chains are in favorable orientations for interaction with the CCKA receptor. Expansions of the aromatic/amide regions of the 1H NMR spectra of Cycle29,34[Dpr29,Lys34]-CCK8 in aqueous solution (top) and in presence of dodecylphosphocholine-d38 micelles (bottom) are shown in the inset. Further details can be found in the article by Morelli and co-workers on p. 1176 ff.

The cover picture shows the NMR structure of the SUPERMAN zinc finger domain, which is the first high-resolution structure of a classical zinc finger domain from a plant protein. The structure consists of a very well-defined ββα motif, typical of all the other Cys2–His2 zinc fingers so far structurally characterized. As a consequence, the QALGGH sequence, which is highly conserved in plant protein classical zinc finger domains, is located at the N terminus of the α helix. Interestingly, this domain region, in animal protein zinc fingers, is constituted of hypervariable residues deputed to the recognition of the DNA bases. Therefore, a peculiar DNA recognition code for the QALGGH zinc finger domain is proposed in the article by Fattorusso and co-workers on p. 171 ff.

A gadolinium(III) paramagnetic complex bearing salicylate functionalities can be used to evaluate the OH˙ radical production by measuring water proton relaxation times.