Here, we compare the distributions of main chain (⌽,⌿) angles (i.e., Ramachandran maps) of the 20 naturally occurring amino acids in three contexts: (i) molecular dynamics (MD) simulations of GlyGly-X-Gly-Gly pentapeptides in water at 298 K with exhaustive sampling, where X ‫؍‬ the amino acid in question; (ii) 188 independent protein simulations in water at 298 K from our Dynameomics Project; and (iii) static crystal and NMR structures from the Protein Data Bank. The GGXGG peptide series is often used as a model of the unstructured denatured state of proteins. The sampling in the peptide MD simulations is neither random nor uniform. Instead, individual amino acids show preferences for particular conformations, but the peptide is dynamic, and interconversion between conformers is facile. For a given amino acid, the (⌽,⌿) distributions in the protein simulations and the Protein Data Bank are very similar and often distinct from those in the peptide simulations. Comparison between the peptide and protein simulations shows that packing constraints, solvation, and the tendency for particular amino acids to be used for specific structural motifs can overwhelm the ''intrinsic propensities'' of amino acids for particular (⌽,⌿) conformations. We also compare our helical propensities with experimental consensus values using the host-guest method, which appear to be determined largely by context and not necessarily the intrinsic conformational propensities of the guest residues. These simulations represent an improved coil library free from contextual effects to better model intrinsic conformational propensities and provide a detailed view of conformations making up the ''random coil'' state.coil library ͉ Dynameomics ͉ molecular dynamics ͉ protein folding ͉ host-guest P rotein secondary structure was predicted before the atomic structures of protein were determined (1-3). Conformational preferences of the amino acids were also estimated very early on, beginning with Ramachandran's ''map'' in 1963, ''based solely on repulsive van der Waals'' forces in dipeptides (4, 5). Remarkably, these predictions regarding structure and conformational preferences were later largely validated in protein crystal structures (6-8).In the protein folding field, these preferences are seen as both means of excluding regions of conformational space and as driving forces for the formation of secondary structure, both of which limit and bias the necessary search of conformational space required during protein folding.(⌽,⌿) dihedral angle distributions are increasingly used to check the validity of structures. Although there can be no doubt about the general tendency of amino acids in globular proteins to populate some regions of (⌽,⌿) space relative to others, the use of such distributions to judge and refine structures leads to dangerous circular reasoning. That is, (⌽,⌿) preferences are used as tests of crystal structures, and those very crystal structures are then used to define and support the Ramachandran (⌽,⌿) angle distributions.Many exper...

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1-Nrf2 protein-protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure-activity relationships support its use as a lead for our ongoing optimization.

Here, we present a natural product discovery approach whereby structures are bioinformatically predicted from primary sequence and produced by chemical synthesis (synthetic-bioinformatic natural products, syn-BNPs), circumventing the need for bacterial culture and gene expression. When applied to nonribosomal peptide synthetase gene clusters from human-associated bacteria we identified the humimycins. These antibiotics inhibit lipid II flippase and potentiate β-lactam activity against methicillin-resistant Staphylococcus aureus in mice, potentially providing a new treatment regimen.

Activation of the antioxidant response element (ARE) up-regulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein, Keap1, and the transcription factor, Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small molecule inhibitors of Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance (SPR) assay. The FP assay exhibits considerable tolerance towards DMSO and produced a Z'-factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high throughput screening (HTS) assay to discover small molecule inhibitors of Keap1-Nrf2 interaction.

The Keap1-Nrf2 interaction plays important roles in regulation of Nrf2 activity and induction of chemopreventive enzymes. To better understand the interaction and to determine the minimal Nrf2 sequence required for Keap1 binding, we synthesized a series of Nrf2 peptides containing ETGE motif and determined their binding affinities to the Kelch domain of Keap1 in solution using a surface plasmon resonance (SPR)-based competition assay. The equilibrium dissociation constant for the interaction between 16mer Nrf2 peptide and Keap1 Kelch domain in solution (KDsolution) was found to be 23.9 nM, which is 10× lower than the surface binding constant (KDsurface) of 252 nM obtained for the direct binding of Keap1 Kelch domain to the immobilized 16mer Nrf2 peptide on a SPR sensor chip surface. The binding affinity of Nrf2 peptides to Keap1 Kelch domain was not lost until after deletion of 8 residues from the N-terminus of the 16mer Nrf2 peptide. The 9mer Nrf2 peptide has a moderate binding affinity with a KDsolution of 352 nM and the affinity was increased 15× upon removal of the positive charge at the peptide N-terminus by acetylation. These results suggest that the minimal Nrf2 peptide sequence required for Keap1 binding is the 9mer sequence of LDEETGEFL.