An albumin-derived peptide scaffold capable of binding and catalysis.

We have identified a 101-amino-acid polypeptide derived from the sequence of the IIA binding site of human albumin. The polypeptide contains residues that make contact with IIA ligands in the parent protein, and eight cysteine residues to form disulfide bridges, that stabilize the polypeptide structure. Seventy-four amino acids are located in six α-helical regions, while the remaining thirty-seven amino acids form six connecting coil/loop regions. A soluble GST fusion protein was expressed in E. coli in yields as high as 4 mg/l. This protein retains the IIA fragment's capacity to bind typical ligands such as warfarin and efavirenz and other albumin's functional properties such as aldolase activity and the ability to direct the stereochemical outcome of a diketone reduction. This newly cloned polypeptide thus represents a valuable starting point for the construction of libraries of binders and catalysts with improved proficiency.

Designing Short Peptides with High Affinity for Organic Molecules: A Combined Docking, Molecular Dynamics, And Monte Carlo Approach.

We present a method for designing artificial receptors capable of binding with high affinity to a chosen target organic molecule. The primary sequence of the peptide is optimized to maximize its binding affinity. Our algorithm builds on a combination of molecular dynamics, semiflexible docking, and replica exchange Monte Carlo and performs simultaneous sampling in sequence and conformational spaces carefully selecting the degree of flexibility in the mutated peptides. The approach is used to design a decapeptide able to bind efavirenz. The calculated binding energy of the designed peptide (approximately -12 kcal/mol) was confirmed experimentally by fluorescence measurements. NMR spectroscopy confirmed the interactions between the peptide and the efavirenz molecule predicted by the algorithm.

Development and evaluation of an immunoassay for the monitoring of the anti-HIV drug amprenavir.

An assay for routine therapeutic drug monitoring of anti-HIV HAART drugs in clinical use is highly desirable, in order to rapidly measure the pharmacokinetic parameters on single patients. We have started a project to develop a panel of enzyme-linked immunosorbent assays (ELISA) for the whole set of HAART drugs, and the development, performance and evaluation of the assay for amprenavir is described here. A diazo conjugate of amprenavir has been used in order to raise polyclonal anti-amprenavir antibodies in rabbits. Antisera have been used to set up a quantitative and rapid competitive assay. Plasma samples are simply diluted in the assay buffer after thermal inactivation, before running the assay. The assay allows the detection of amprenavir in the quantification range 400-5000 ng/ml, in a diluted plasma sample. The assay has been compared with an HPLC reference technique, on 27 samples from treated patients. Within the quantification range, the ELISA data are well correlated with the HPLC results by a regression line close to the identity, and a Bland-Altman analysis shows the agreement between the two methods.

Low variability of the protein species synthesized byDrosophila melanogaster embryos.

Stainable proteins as well as newly synthesized polypeptide chains of proteins extracted fromDrosophila melanogaster embryos were analyzed by two-dimensional gel electrophoresis. The following developmental stages were studied: unfertilized eggs, early nuclear multiplication (25 min average age), late nuclear multiplication (105 min), cellular blastoderm (165 min), gastrula (4 h), mesodermal segmentation (6 h) and muscleattachment (8 h). One hundred and fifty stainable spots were present at all developmental stages and were all also synthesized during development, with the exception of 5 unkown proteins and the three yolk proteins. Out of 400 proteins which were labelled by35S-methionine, only 5% showed a reproducible pattern of variable synthesis. Three proteins appeared upon fertilization. The early nuclear multiplication stage showed the largest number of labelled spots while the lowest number of labelled spots was observed at blastoderm formation. The pattern of synthesis of a few specific proteins was also followed. Actin I was synthesized only at 8 h, actin II and actin III were synthesized at all stages. ß-tubulin was synthesized at all stages, while we observed a reduction, if not a cessation, of synthesis of α-tubulin at 105 min, 165 min and 4 h of development. Non heat-shock embryos synthesized a large amount of heat-shock protein (hsp) 84 at 25 min while hsp 70 and 68 were first detected after 4 h of development. Though it is generally accepted that the embryonic genome is activated at blastoderm formation we did not observe a parallel increase in protein species. It is possible that protein synthesis on the new transcripts is below the detection limit of the technique. Alternatively the embryonic messages may gradually substitute preexisting maternal messages or only become available for translation some time after they are transcribed.