An evolutionary approach for structure-based design of natural and non-natural peptidic ligands.

A new computational approach (PEP) is presented for the structure-based design of linear polymeric ligands consisting of any type of amino acid. Ligands are grown from a seed by iteratively adding amino acids to the growing construct. The search in chemical space is performed by a build-up approach which employs all of the residues of a user-defined library. At every growing step, a genetic algorithm is used for conformational optimization of the last added monomer inside the binding site of a rigid target protein. The binding energy with electrostatic solvation is evaluated to select sequences for further growing. PEP is tested on the peptide substrate binding site of the insulin receptor tyrosine kinase and farnesyltransferase. In both test cases, the peptides designed by PEP correspond to the sequence motifs of known substrates. For tyrosine kinase, tyrosine residues are suggested at position P and P+2. While the tyrosine at P is in agreement with the experimental data, the one at P+2 is a prediction which awaits experimental validation. For farnesyltransferase, it is shown that electrostatic solvation is necessary for the correct design of peptidic inhibitors.

Real-time multichannel abdominal fetal ECG monitor using digital signal coprocessor.

A real-time multichannel fetal ECG monitor based on a personal computer (PC) and a MOTOROLA DSP56001 Digital Signal CoProcessor (DSP) is introduced. The DSP board is plugged into the PC, which functions as a HOST computer. An analog 8 Leads Interface and Analog to Digital circuits module is connected to the DSP through a synchronous, optical-isolated communication channel. The fetal ECG detection is based on a cross-correlation technique. An averaged maternal ECG waveform is generated using a cross-correlation alignment procedure and a user-defined template. The fetal ECG signals present in the maternal waveform is suppressed during the averaging procedure, since both are uncorrelated. The average maternal ECG waveform is then subtracted from the abdominal real time signals, and maternal-free fetal ECGs signals are obtained, including fetal QRS complexes that coincide with maternal ones. Using the abdominal ECGs signals after subtraction, an averaged fetal waveform is generated. The maternal and the fetal heart rate are calculated during the process. The algorithm described above can be performed in real time on up to eight abdominal ECG traces by the DSP, and the desired results are passed to the HOST PC, to be stored and displayed. Electrodes positioning procedures for detecting the fetal QRS complexes with the best signal to noise ratio are not needed. Using the multichannel system, the user can select the best channel for fetal QRS detection, and accurate results for the heart rate signal are obtained. Averaged fetal waveforms are obtained from all the leads.

Fragment-Based flexible ligand docking by evolutionary optimization.

A new computational approach for the efficient docking of flexible ligands in a rigid protein is presented. It exploits the binding modes of functional groups determined by an exhaustive search with solvation. The search in ligand conformational space is performed by a genetic algorithm whose scoring function approximates steric effects and intermolecular hydrogen bonds. Ligand conformations generated by the genetic algorithm are docked in the protein binding site by optimizing the fit of their fragments to optimal positions of chemically related functional groups. We show that the use of optimal binding modes of molecular fragments allows to dock known inhibitors with about ten rotatable bonds in the active site of the uncomplexed and complexed conformations of thrombin and HIV-1 protease.

Generation of proteoliposomes from subcellular fractions.

Intracellular membranes are highly dynamic, yet they retain their identity and functional characteristics. Integral membrane proteins, which must confer this specific membrane identity, remain poorly characterized at the biochemical level, largely because detergent-mediated solubilization is required for purification and analysis, and several properties of integral membrane proteins can only be investigated when the molecule is properly embedded in a lipid bilayer. We present a method for the efficient reconstitution into proteoliposomes of integral membrane proteins from subcellular fractions. Integral membrane proteins were identified on high-resolution two-dimensional gels after selective extraction of soluble and peripheral membrane proteins; they accounted for 8% of the number of resolved polypeptides. A reconstitution procedure based on membrane solubilization with dodecyl-octaoxyethylene (C12E8) and subsequent detergent removal with BioBeads SM-2 resulted in the efficient reconstitution of several membrane proteins into proteoliposomes of uniform density. The generated proteoliposomes strongly resemble the starting membrane fraction in protein composition. This reconstitution allows the functional characterization of integral membrane proteins after enrichment and/or specific (immuno)depletion.