Search

We report a case of temporomandibular disorder patient with disc displacement without reduction, myofascial pain, limited opening and a novel, never described, nonsynonimous mutation of catechol-O-methyl-transferase (COMT) gene. COMT is one of the enzymes that metabolizes catecholamines, thereby acting as a key modulator of dopaminergic and adrenergic/noradrenergic neurotransmissions, which play a key role in pain modulation. This novel mutation, p.R58S, changed a codon (58 from arginine to serine) in the COMT protein. The introduction of a serine residue in a highly organised secondary structure, in critical regions of the protein, results in a structural alteration. Therefore, we speculate an influence of the mutation on the high pain sensitivity of the patient.

Raman microscopy is having increasing applications into molecular biology, ranging from studies on ligand binding [1] and secondary structure analysis [2]. Here we present results of a combined Raman microscopy and X-ray crystallography approach to study protein single crystals. The combined approach reinforces the interpretation of biophysical data. Both tetrameric hemoglobins and collagen-like polypeptides have been ivestigated. Tetrameric hemoglobins from Antarctic fish (AFHb) exibit a peculiar autoxidation pathway [3-5], that has been investigated via crystallography, and via Raman spectro/microscopy (both in solution and in crystal phase). The appearance of transient Raman bands both in solution and in crystals of AFHb at short time of air-exposure suggests that a bis-histidyl ferric adduct forms after the ferrous iron oxidizes to a penta-coordinated ferric state. A collagen-like polypeptide, (Pro-Pro-Gly)10, whose triple helix crystal structure has been solved at high resolution [6], has been also investigated. The comparison of amide I and amide III bands of the single crystal with the x-ray crystal structure of the triple helix provides an accurate reference to predict relationship between amide band frequencies and Ramachandran angles [7]. Acknowledgement We acknowledge Regione Campania and PNRA for financial support. References [1] P. R.Carey, J. Dong, Biochemistry 2004, 43, 8885-8893. [2] P. R. Carey, Ann. Rev. Phys. Chem. 2006, 57, 527-554. [3] A. Riccio, L. Vitagliano, G. di Prisco, A. Zagari, L. Mazzarella., Proc. Natl. Acad. Sci. USA 2002, 99, 9801-9806. [4] L. Vitagliano, G. Bonomi, A. Riccio, G. di Prisco, G. Smulevich, L. Mazzarella.. Eur. J. Biochem. 2004, 271, 1651-1659. [5] A. Vergara, M. Franzese, A. Merlino, L. Vitagliano, C. Verde, G. di Prisco, H. C. Lee, J. Peisach, L. Mazzarella, , Biophys. J. 2007, in press. [6] R. Berisio, L. Vitagliano, L. Mazzarella, A. Zagari,. Prot. Sci. 2002, 11, 262-270. [7] S.A. Asher, A. Ianoul, G. Mix, M. N. Boyden, A. Karnoup, M. Diem, R. Schweitzer-Stenner, J. Am. Chem. Soc. 2001, 123, 11775-11781.

Prion proteins are regarded as the main agents of transmissible spongiform encephalopathies. Understanding their fate in soil may be crucial to elucidate the dissemination of the prion in the environment, associated with a possible transmission of infectivity. Studies were performed with simplified model systems, derived by the birnessite-mediated oxidative polymerization of catechol, which simulate processes naturally occurring in soil. A benign full-length recombinant purified ovine protein (PrP) (residues 23–234), as well as a truncated-form ovPrP (tPrP) (103–234) were utilized. Catechol and prion protein interacted under experimental conditions that reproduced the interaction of PrP with soluble organic matter or with insoluble organic matter during or after formation of catechol polymers. PrP stability in all buffers and chemicals was preliminarily monitored by circular dicroism (CD) measurements. The disappearance of protein molecules from the solution, the decrease of UV–Visible absorbance of supernatants, and the FT-IR spectra and the elemental analyses of solid-phase residues indicated that both PrP and tPrP were involved in catechol polymerization by birnessite. Furthermore, a clear flocculation of soluble catechol–protein polymeric products in solid aggregates was observed when PrP was added to the supernatants. Different kinds of extracting agents were not able to desorb/extract PrP as well as tPrP from the formed solid aggregates, thereby highlighting the high stability of protein–organic and –organo-mineral complexes.

Recent studies have shown that elongation factors extd. from archaea/eukarya and from eubacteria exhibit different structural and functional properties. Along this line, it has been demonstrated that, in contrast to EF-Tu, Sulfolobus solfataricus EF-1a in complex with GDP (SsEF-1a×GDP) does not bind Mg2+, when the ion is present in the crystn. medium at moderate concn. (5 mM). To further investigate the role that magnesium plays in the exchange process of EF-1a and to check the ability of SsEF-1a×GDP to bind the ion, we have detd. the crystal structure of SsEF-1a×GDP in the presence of a nonphysiol. concn. (100 mM) of Mg2+. The anal. of the coordination of Mg2+ unveils the structural bases for the marginal role played by the ion in the nucleotide exchange process. Furthermore, nucleotide exchange expts. carried out on a truncated form of SsEF-1a, consisting only of the nucleotide binding domain, demonstrate that the low affinity of SsEF-1a×GDP for Mg2+ is due to the local architecture of the active site and does not depend on the presence of the other two domains. Finally, considering the available structures of EF-1a, a detailed mechanism for the nucleotide exchange process has been traced. Notably, this mechanism involves residues such as His14, Arg95, Gln131, and Glu134, which are strictly conserved in all archaea and eukarya EF-1a sequences hitherto reported.

Valine 114 in the D(109)AAILVVA sequence of elongation factor 1alpha from the archaeon Sulfolobus solfataricus (SsEF-1alpha) was substituted with an acidic (V114E), basic (V114K), or cavity-forming (V114A) residue, and the effects on the biochemical properties of the factor were investigated. This sequence is well-conserved among most of eukaryal and eubacterial counterparts, and in the three-dimensional structure of SsEF-1alpha, V114 is located in a hydrophobic pocket near the first GDP-binding consensus sequence G(13)XXXXGK[T,S] [Vitagliano, L., Masullo, M., Sica, F., Zagari, A., and Bocchini, V. (2001) EMBO J. 20, 5305-5311]. These mutants displayed functions absent in the wild-type factor. In fact, although they exhibited a rate in poly(Phe) incorporation almost identical to that of SsEF-1alpha, V114K and V114A exhibited an affinity for GDP and GTP higher and a capability to bind heterologous aa-tRNA stronger than that elicited by SsEF-1alpha but similar to that of eubacterial EF-Tu. V114E instead displayed not only a weaker binding capability for aa-tRNA but also a lower affinity for GDP. The intrinsic GTPase activity of V114E was drastically reduced compared to those of SsEF-1alpha, V114K, and V114A. Interestingly, the decreased intrinsic GTPase activity of V114E was partially restored by kirromycin, an effect already observed for the G13A mutant of SsEF-1alpha [Masullo, M., Cantiello, P., de Paola, B., Catanzano, F., Arcari, P., and Bocchini, V. (2002) Biochemistry 41, 628-633]. Finally, the V114A substitution showed only a marginal effect on both the thermostability and thermophilicity of SsEF-1alpha, whereas V114K and V114E replacements strongly destabilized the molecule.