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Abstract: Eukaryotic signal transduction involves the assembly of transient protein-protein complexes mediated by modular interaction domains. Specific Pro-rich sequences with the consensus core motif PxxP adopt the PPII helix conformation upon binding to SH3 domains. For short Pro-rich peptides, little or no ordered secondary structure is usually observed before binding interactions. The association of a Pro-rich peptide with the SH3 domain involves unfavorable binding entropy due to the loss of rotational freedom on forming the PPII helix. With the aim of stabilizing the PPII helix conformation in the Pro-rich HPKI decapeptide PPPLPPKPKF (P2), a series of P2 analogues was prepared, in which specific Pro positions were alternatively occupied by 4(S)- or 4(R)-4-fluoro-L-proline. The interactions of these peptides with the SH3 domain of the HPK1-binding partner HS1 were quantitatively analyzed by the NILIA-CD approach. A CD thermal analysis of the P2 analogues was performed to assess their propensity to adopt the PPII helix conformation. Contrary to our expectations, the K-d values of the analogues were lower than that of the parent peptide P2. These results clearly show that the induction of a stable PPII helix conformation in short Pro-rich peptides is not sufficient to increase their affinity toward the SH3 domain and that the effect of 4-fluoroproline strongly depends on the position of this residue in the sequence and the chirality of the substituent in the pyrrolidine ring. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

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Abstract: Grazing incidence x-ray fluorescence (GIXRF) analysis technique has the potential of being one of the most powerful and versatile methods for characterization of layered materials, as it combines features of both x-ray reflectivity (XRR) and x-ray fluorescence techniques. GIXRF technique allows non-destructive evaluation of layer thickness, interface roughness, interlayer formation and depth profiling for an impurity element inside a layer medium or in the substrates. A computer program CATGIXRF, has been developed for GIXRF characterization of thin film and surfaces. Methodology of the program and its various features has been discussed in detail. The program offers analysis of GIXRF and XRR data simultaneously. The utility of the program has been demonstrated by example calculations and by providing a few examples of XRR and GIXRF characterization of a variety of thin-layered materials.

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Abstract: The search for effective drug delivery systems is one of the major challenges in drug formulation especially for biopharmaceuticals Such as proteins, and peptide-based drugs and vaccines. A procedure has been developed whereby human serum albumin (HSA) call be Used as a delivery vehicle for these biomolecules using its role as main ratty acid carrier. Using essentially fatty acid free HSA (HSAff) it is possible to form stable complexes with lipidic chain compounds (lipo-compounds). Two lipo-compounds have been used to develop this system, a novel antimicrobial lipopeptide and gamma-amino-n-butyric acid, GABA. conjugated with an alkyl chain, lipo-GABA, in both cases C8 and C14 alkyl chain lengths were evaluated. The HAS-lipo compound complex had a mutual stabilizing effect on both the HSA and the lipo-compound. The protease enzyme study showed that the alkyl chains of these lipo-compounds bound to HSAff confer a similar if not greater biostability than caprylic acid shown by CD and importantly, the bound lipopeptide was stabilized by the HSA shown by mass spectrometry. Heat stability studies at 60 degrees C over 10 h also confirmed that the lipo-HSA complexes confer stability and provide a method of preparing sterile formulation for therapeutic use. No further increased in stability of the lipo-compounds when HSA containing fatty acid (HSAfa) Was used. With the antimicrobial lipopeptide, there was enhanced activity with HSAff formulation suggesting increased biostability and bioavailability of compounds. These finding allowed us to develop a simple and effective way of delivering lipo-compounds using fatty acid free HSA as the carrier.

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Abstract: Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 Å from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the ?-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower Km compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/cyclin A activity against a heptapeptide substrate (Ki = 0.83 nm) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (KD 0.4 ?m) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.