WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/)P(p,g)PPpR, (p/)PPRgpPp, PPLPp, (p/)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified. For PPxY-recognizing domains, phospho-tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed. PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases.

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Experimental studies in animals and in vitro have shown the usefulness of photodynamic therapy (PDT) as an adjunct to periodontal treatment. The aim of this study was to evaluate the long-term clinical and microbiological effects of PDT associated with nonsurgical periodontal treatment. Three sites in each of 33 patients with chronic periodontitis were randomly allocated in a split-mouth design to a treatment group: (1) scaling and root planing (SRP group); (2) SRP and irrigation with toluidine blue O (TBO group); and (3) SRP, irrigation with TBO and low-level laser irradiation (PDT group). Clinical parameters including visible plaque index, bleeding gingival index, bleeding on probing, probing depth, gingival recession and clinical attachment level were measured at baseline, and after 60, 90 and 180 days. Additionally, subgingival plaque samples were collected for microbiological analysis by PCR. Intergroup and intragroup statistical analyses were performed. All treatment groups showed an improvement in all clinical parameters, and a significant reduction in the proportion of sites positive for periodontopathogens at 60, 90 and 180 days compared to baseline (p< 0.05). None of the periodontal parameters showed a significant difference among the groups (p > 0.05). At 180 days, PDT treatment led to a significant reduction in the percentage of sites positive for all bacteria compared to SRP alone (p < 0.05). Within the limits of this study it may be concluded that PDT as an adjunct to periodontal treatment produced statistically significant reductions in some of the key periodontal pathogens but produced no statistically significant benefit in terms of clinical outcome.

Obesity does not seem to play a negative role by interfering in the improvement of the periodontal clinical response or decreasing circulating proinflammatory cytokine levels after periodontal treatment.

Chemical synthesis allows the incorporation of nonnatural amino acids into proteins that may provide previously untried probes of their folding pathway and thermodynamic stability. We have used a flexible thioether linker as a loop mimetic in the human yes kinase-associated protein (YAP 65) WW domain, a three-stranded, 44-residue, ␤-sheet protein. This linkage avoids problems of incorporating sequences that constrain loops to the extent that they significantly change the nature of the denatured state with concomitant effects on the folding kinetics. An NMR solution structure shows that the thioether linker had little effect on the global fold of the domain, although the loop is apparently more dynamic. The thioether variants are destabilized by up to 1.4 kcal͞mol (1 cal ‫؍‬ 4.18 J). Preliminary ⌽-value analysis showed that the first loop is highly structured in the folding transition state, and the second loop is essentially unstructured. These data are consistent with results from simulated unfolding and detailed protein-engineering studies of structurally homologous WW domains. Previously, ⌽-value analysis was limited to studying side-chain interactions. The linkers used here extend the protein engineering method directly to secondary-structure interactions. U nderstanding how proteins spontaneously fold to a stable three-dimensional structure is exceptionally difficult. This difficulty applies especially to ␤-sheet proteins, whose secondary structure is stabilized more by long-range sequence-distant interactions than is that in ␣-helical proteins. Small ␤-sheet proteins are useful paradigms for understanding the search problem. There is increasing evidence of the importance of ␤-hairpins and loops in driving ␤-sheet folding (1-6). The stability of these loops may also influence the overall stability of ␤-sheet proteins in two ways. One way is by contributing to the overall thermodynamic stability of the system (7-9). The other way is in the effect on the folding pathway (2, 3), such that folding may or may not be facilitated by loop or hairpin formation, with concomitant effects on the folding rate and, thus, on stability.The WW domains are a family of small, triple-stranded, antiparallel ␤-sheet proteins (typically, of 34-44 amino acids) with a small hydrophobic core involving residues from both the N and C termini (10). The loop structures and overall bend of the ␤-sheet are well conserved in the different members of this family (11-13). A number of these domains are well suited for biophysical studies of ␤-sheet folding and loop formation as they reversibly denature, are amenable to mutagenesis, and are easily synthesized by using both recombinant and organic chemistry techniques (12,14,15). Thus, these domains can be synthesized with both natural and nonnatural amino acids in their sequences, allowing for sophisticated experiments that cannot easily be performed with recombinant proteins. The effects of substitutions on the stabilities of WW domains have been studied in the context of a designed WW-domain...

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single ␣-helical globular domain, microplusin consists of five ␣-helices: ␣1 (residues Gly-9 to Arg-21), ␣2 (residues Glu-27 to Asn-40), ␣3 (residues Arg-44 to Thr-54), ␣4 (residues Leu-57 to Tyr-64), and ␣5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

This paper investigates the structure of the products obtained from the polymerization of aniline with ammonium persulfate in a citrate/phosphate buffer solution at pH 3 by resonance Raman, NMR, FTIR, and UV-vis-NIR spectroscopies. All the spectroscopic data showed that the major product presented segments that were formed by a 1,4-Michael reaction between aniline and p-benzoquinone monoimine, ruling out the formation of polyazane structure that has been recently proposed. The characterization of samples obtained at different stages of the reaction indicated that, as the reaction progressed, phenazine units were formed and 1,4-Michael-type adducts were hydrolyzed/oxidized to yield benzoquinone. Raman mapping data suggested that phenazine-like segments could be related to the formation of the microspheres morphology.

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