Project description:Class II viral fusion proteins are present on the envelope of flaviviruses and togaviruses, viruses that often cause tropical and subtropical diseases. These proteins use a second membrane protein as a molecular chaperone to assist their folding and to ensure proper function during viral assembly, maturation, and infection. Recent progress in structural studies of dengue viruses has revealed how the chaperone pre-membrane (prM) protein guides viral maturation and how pH is sensed in both the maturation and infection processes. Drastic conformation changes and reorganization of these viral membrane proteins occur during the transition from their metastable to stable structural states in a unidirectional, entropy-driven process.
Project description:?-Lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid?II processing and cell wall biosynthesis through inactivation of penicillin-binding proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid?II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid?II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP-Lipid?II association has not been established in live cells. To determine this key interaction, we designed an unnatural d-amino acid dipeptide that is metabolically incorporated into Lipid?II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid?II, thereby allowing, for the first time, demonstration of PBP interactions in?vivo with Lipid?II.
Project description:This article describes recent attempts to understand the biological chemistry of lead using a synthetic biology approach. Lead binds to a variety of different biomolecules ranging from enzymes to regulatory and signaling proteins to bone matrix. We have focused on the interactions of this element in thiolate-rich sites that are found in metalloregulatory proteins such as Pbr, Znt, and CadC and in enzymes such as δ-aminolevulinic acid dehydratase (ALAD). In these proteins, Pb(II) is often found as a homoleptic and hemidirectic Pb(II)(SR)3- complex. Using first principles of biophysics, we have developed relatively short peptides that can associate into three-stranded coiled coils (3SCCs), in which a cysteine group is incorporated into the hydrophobic core to generate a (cysteine)3 binding site. We describe how lead may be sequestered into these sites, the characteristic spectral features may be observed for such systems and we provide crystallographic insight on metal binding. The Pb(II)(SR)3- that is revealed within these α-helical assemblies forms a trigonal pyramidal structure (having an endo orientation) with distinct conformations than are also found in natural proteins (having an exo conformation). This structural insight, combined with 207Pb NMR spectroscopy, suggests that while Pb(II) prefers hemidirected Pb(II)(SR)3- scaffolds regardless of the protein fold, the way this is achieved within α-helical systems is different than in β-sheet or loop regions of proteins. These interactions between metal coordination preference and protein structural preference undoubtedly are exploited in natural systems to allow for protein conformation changes that define function. Thus, using a design approach that separates the numerous factors that lead to stable natural proteins allows us to extract fundamental concepts on how metals behave in biological systems.
Project description:The complexity of proteins is substantially simplified by regarding them as archetypical examples of self-organized criticality (SOC). To test this idea and to elaborate it, this article applies the Moret-Zebende (MZ) SOC hydrophobicity scale to transport repeat proteins of the HEAT superfamily, importin beta, and transportin, as well as the export protein Cse1p, and their ubiquitous cargo manager Ran. The difference between the MZ scale and conventional hydrophobicity scales reflects long-range conformational forces that are central to protein functionality. These compete with long-range Coulomb forces associated with cationic and anionic side chains in a revealing way.
Project description:This tutorial review presents descriptions of two amyloidogenic proteins, amyloid-? (A?) peptides and islet amyloid polypeptide (IAPP), whose misfolding propensities are implicated in Alzheimer's disease (AD) and type II diabetes, respectively. Protein misfolding diseases share similarities, as well as some unique protein-specific traits, that could contribute to the initiation and/or development of their associated conditions. A? and IAPP are representative amyloidoses and are used to highlight some of the primary considerations for studying misfolded proteins associated with human diseases in this review. Among these factors, their physiological formation, aggregation, interactions with metal ions and other protein partners, and toxicity are presented. Small molecules that target and modulate the metal-A? interaction and neurotoxicity are included to illustrate one of the current approaches for uncovering the complexities of protein misfolding at the molecular level.
Project description:Class II human leukocyte antigens (HLA II) are proteins involved in the human immunological adaptive response by binding and exposing some pre-processed, non-self peptides in the extracellular domain in order to make them recognizable by the CD4+ T lymphocytes. However, the understanding of HLA-peptide binding interaction is a crucial step for designing a peptide-based vaccine because the high rate of polymorphisms in HLA class II molecules creates a big challenge, even though the HLA II proteins can be grouped into supertypes, where members of different class bind a similar pool of peptides. Hence, first we performed the supertype classification of 27 HLA II proteins using their binding affinities and structural-based linear motifs to create a stable group of supertypes. For this purpose, a well-known clustering method was used, and then, a consensus was built to find the stable groups and to show the functional and structural correlation of HLA II proteins. Thus, the overlap of the binding events was measured, confirming a large promiscuity within the HLA II-peptide interactions. Moreover, a very low rate of locus-specific binding events was observed for the HLA-DP genetic locus, suggesting a different binding selectivity of these proteins with respect to HLA-DR and HLA-DQ proteins. Secondly, a predictor based on a support vector machine (SVM) classifier was designed to recognize HLA II-binding peptides. The efficiency of prediction was estimated using precision, recall (sensitivity), specificity, accuracy, F-measure, and area under the ROC curve values of random subsampled dataset in comparison with other supervised classifiers. Also the leave-one-out cross-validation was performed to establish the efficiency of the predictor. The availability of HLA II-peptide interaction dataset, HLA II-binding motifs, high-quality amino acid indices, peptide dataset for SVM training, and MATLAB code of the predictor is available at http://sysbio.icm.edu.pl/HLA .
Project description:We developed a process to produce novel interactions between two previously unrelated proteins. This process selects protein scaffolds and designs protein interfaces that bind to a surface patch of interest on a target protein. Scaffolds with shapes complementary to the target surface patch were screened using an exhaustive computational search of the human proteome and optimized by directed evolution using phage display. This method was applied to successfully design scaffolds that bind to epidermal growth factor receptor (EGFR) domain II, the interface of EGFR dimerization, with high reactivity toward the target surface patch of EGFR domain II. One potential application of these tailor-made protein interactions is the development of therapeutic agents against specific protein targets.
Project description:IntroductionGene therapies are using Adeno-associated viruses (AAVs) as vectors, but immune responses against the capsids pose challenges to their efficiency and safety. Helper T cell recognition of capsid-derived peptides bound to human leukocyte antigen (HLA) class II molecules is an essential step in the AAV-specific adaptive immunity.MethodsUsing MHC-associated peptide proteomics, we identified the HLA-DR and HLA-DQ immunopeptidomes of the capsid proteins of three different AAV serotypes (AAV2, AAV6, and AAV9) from a panel of healthy donors selected to represent a majority of allele usage.ResultsThe identified sequences span the capsids of all serotypes, with AAV2 having the highest peptide count. For all the serotypes, multiple promiscuous peptides were identified and displayed by both HLA-DR and -DQ. However, despite high sequence homology, there were few identical peptides among AAV2, AAV6, and AAV9 immunopeptidomes, and none were promiscuous.DiscussionResults from this work represent a comprehensive immunopeptidomics research of potential CD4+ T cell epitopes and provide the basis for immunosurveillance efforts for safer and more efficient AAV-based gene therapies.
Project description:MotivationRecent research underlines the importance of finegrained knowledge on protein localization. In particular, subcompartmental localization in the Golgi apparatus is important, for example, for the order of reactions performed in glycosylation pathways or the sorting functions of SNAREs, but is currently poorly understood.ResultsWe assemble a dataset of type II transmembrane proteins with experimentally determined sub-Golgi localizations and use this information to develop a predictor based on the transmembrane domain of these proteins, making use of a dedicated proteinstructure based kernel in an SVM. Various applications demonstrate the power of our approach. In particular, comparison with a large set of glycan structures illustrates the applicability of our predictions on a 'glycomic' scale and demonstrates a significant correlation between sub-Golgi localization and the ordering of different steps in glycan biosynthesis.Supplementary informationSupplementary data are available at Bioinformatics online.
Project description:Background: The polyproline II helix (PPIIH) is an extended protein left-handed secondary structure that usually but not necessarily involves prolines. Short PPIIHs are frequently, but not exclusively, found in disordered protein regions, where they may interact with peptide-binding domains. However, no readily usable software is available to predict this state. Results: We developed PPIIPRED to predict polyproline II helix secondary structure from protein sequences, using bidirectional recurrent neural networks trained on known three-dimensional structures with dihedral angle filtering. The performance of the method was evaluated in an external validation set. In addition to proline, PPIIPRED favours amino acids whose side chains extend from the backbone (Leu, Met, Lys, Arg, Glu, Gln), as well as Ala and Val. Utility for individual residue predictions is restricted by the rarity of the PPIIH feature compared to structurally common features. Conclusion: The software, available at http://bioware.ucd.ie/PPIIPRED, is useful in large-scale studies, such as evolutionary analyses of PPIIH, or computationally reducing large datasets of candidate binding peptides for further experimental validation.