Prediction of functionally important residues in globular proteins from unusual central distances of amino acids

<p>Abstract</p> <p>Background</p> <p>Well-performing automated protein function recognition approaches usually comprise several complementary techniques. Beside constructing better consensus, their predictive power can be improved by either adding or refining independent modules that explore orthogonal features of proteins. In this work, we demonstrated how the exploration of global atomic distributions can be used to indicate functionally important residues.</p> <p>Results</p> <p>Using a set of carefully selected globular proteins, we parametrized continuous probability density functions describing preferred central distances of individual protein atoms. Relative preferred burials were estimated using mixture models of radial density functions dependent on the amino acid composition of a protein under consideration. The unexpectedness of extraordinary locations of atoms was evaluated in the information-theoretic manner and used directly for the identification of key amino acids. In the validation study, we tested capabilities of a tool built upon our approach, called SurpResi, by searching for binding sites interacting with ligands. The tool indicated multiple candidate sites achieving success rates comparable to several geometric methods. We also showed that the unexpectedness is a property of regions involved in protein-protein interactions, and thus can be used for the ranking of protein docking predictions. The computational approach implemented in this work is freely available via a Web interface at <url>http://www.bioinformatics.org/surpresi</url>.</p> <p>Conclusions</p> <p>Probabilistic analysis of atomic central distances in globular proteins is capable of capturing distinct orientational preferences of amino acids as resulting from different sizes, charges and hydrophobic characters of their side chains. When idealized spatial preferences can be inferred from the sole amino acid composition of a protein, residues located in hydrophobically unfavorable environments can be easily detected. Such residues turn out to be often directly involved in binding ligands or interfacing with other proteins.</p

Development of multi-probe fluorescence-based assay for boNTA detection

Botulinum neurotoxins (BoNTs) cause the lethal disease botulism through the inhibition of acetyl choline secretion by the cleavage of crucial SNARE proteins. Determination of critical residues in the protein sequence of BoNT serotype A was the primary step to identify novel fluorescence recognition agents for BoNTA. Computational and experimental studies were employed to identify paclitaxel as a new inhibitor (IC50 equal to 5.2 μM) for the proteolytic activity of BoNTA light chain (LC) using Fluorescence Resonance Energy Transfer (FRET) assay. A fluorescent derivative of paclitaxel (PAC-BDP) exhibited binding to complex BoNTA. A Primary Amines Database comprised of 1,153 compounds suitable for fluorescent labeling was computationally screened to select 6-aminofluorescein (6-AFLU) and aspartame (APM) as recognition agent candidates. Fluorescent labeled APM (APM-BDP) was synthesised and the purity of the compound was confirmed using liquid chromatography mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). 6-AFLU exhibited good binding affinity to BoNTA heavy chain (HC) with an EC50 of 546 ± 60nM, whereas APM-BDP displayed binding to BoNTA LC with an EC50 of 20.96 ± 10 nM, as determined by fluorescence polarization (FP) assay. APM was shown to compete with APM-BDP for the same binding site in BoNTA LC, but showed no binding to BoNTA HC in FP competition assay. Also, aminopterin (AMN) and 6-AFLU exhibited binding to the same site of BoNTA HC, whereas desmosine (DES) showed affinity to a different binding site in BoNTA HC. Additionally, PAC exhibited binding to BoNTA LC, however paclitaxel (PAC) did not compete with APM-BDP for the same binding region. PAC-BDP showed binding to both BoNTA LC and BoNTA HC and did not compete with APM-BDP for the same binding site in BoNTA LC. A library consisting of 1,624 commercially available radiolabeled ligands were screened computationally to select the ligands with binding affinity against BoNTA LC and HC. The binding of [3H] Aminopterin and [3H] desmosine was shown to be concentration-dependent with EC50of 703 ± 98 nM and 1.6 ± 0.3μM, respectively, against BoNTA HC using scintillation proximity assay (SPA). [3H]Solanesyl pyrophosphate (Solanesyl PP) exhibited high binding to both BoNTA LC and BoNTA HC. However its related compound, [3H]Solanesol, show no binding against BoNTA LC or BoNTA HC using SPA assay. The development of a fast, simple, reliable assay for BoNTA detection is essential since mouse lethality assay (MLA), the only trustable assay, is a costly, time consuming and complicated assay. In addition, detection of BoNTA in the initial steps of contamination is critical for successful treatment. This study demonstrated that FP can be used as a platform for BoNTA detection and that PAC-BDP, APM-BDP and 6-AFLU can be used simultaneously since they bind to different binding regions of BoNTA. The identified recognition agents can potentially be used in a multi-probe FP assay against the whole BoNTA complex

フルタミドの活性に影響を与える酵素的・非酵素的反応についての物理化学的研究

13301甲第4373号博士(薬学)金沢大学博士論文本文Ful

Molecular recognition from atomic interactions: insights into drug discovery

The failure of the pharmaceutical industry to increase the delivery of new drugs into the market is driving a re-assessment of practices and methods in drug discovery and development. In particular alternative strategies are being pursued to find therapeutics that are more selective, including small molecules that target protein-protein interactions. However, success depends on improving our understanding of the recognition of small molecules by interfaces in order to develop better methods for maximising their affinity and selectivity, whilst trying to confer an appropriate therapeutic profile. This thesis starts with the description of the creation of TIMBAL, a database that holds small molecules disrupting protein-protein interactions. The thesis then focuses on the analysis of these molecules and their interactions in a medicinal chemistry and structural biology context. TIMBAL molecules are profiled against other sets of molecules (drugs, drug-like and screening compounds) in terms of molecular properties. Using the structural databases in the Blundell group, the atomic detail of the interaction patterns of TIMBAL molecules with their protein targets are compared with other molecules interacting with proteins, comprising natural molecules, small peptides, synthetic small molecules (including drug-like and drugs) and other proteins. The structural features and composition of the binding sites of these complexes are also analysed. Keeping in mind that current drug candidates are somewhat too lipophilic to succeed, these interaction profiles are defined in terms of polar and apolar contacts, with the aim of migrating natural patterns into the design of new therapeutics.UCB and BBSR