Scalable Biosynthetic Production of Knotted Peptides Enables ADME and Thermodynamic Folding Studies.

Knotted peptides present a wealth of structurally diverse, biologically active molecules, with the inhibitor cystine knot/knottin class among the most ecologically common ones. Many of these natural products interact with extracellular targets such as voltage-gated ion channels with exquisite selectivity and potency, making them intriguing therapeutic modalities. Such compounds are often produced in low concentrations by intractable organisms, making structural and biological characterization challenging, which is frequently overcome by various expression strategies.

Design of Thioether Cyclic Peptide Scaffolds with Passive Permeability and Oral Exposure.

Advances in the design of permeable peptides and in the synthesis of large arrays of macrocyclic peptides with diverse amino acids have evolved on parallel but independent tracks. Less precedent combines their respective attributes, thereby limiting the potential to identify permeable peptide ligands for key targets. Herein, we present novel 6-, 7-, and 8-mer cyclic peptides (MW 774-1076 g·mol) with passive permeability and oral exposure that feature the amino acids and thioether ring-closing common to large array formats, including DNA- and RNA-templated synthesis.

Quantification of protonation in organic solvents using solution NMR spectroscopy: implication in salt formation.

Purpose: Investigate the use of solution NMR spectroscopy to evaluate whether the general ΔpKa rule is valid in organic solvents. Such information may be useful in evaluation of acid-base reactions and solvent selection for salt formation.

Methods: (1)H NMR chemical shift changes in model bases during titration with acids, and separately, on the addition of acids at a molar ratio of 1:1 were determined in water, dimethyl sulfoxide, and methanol.

Reverse micelles in integral membrane protein structural biology by solution NMR spectroscopy.

Integral membrane proteins remain a significant challenge to structural studies by solution NMR spectroscopy. This is due not only to spectral complexity, but also because the effects of slow molecular reorientation are exacerbated by the need to solubilize the protein in aqueous detergent micelles. These assemblies can be quite large and require deuteration for optimal use of the TROSY effect.

Cold denaturation of encapsulated ubiquitin.

Theoretical considerations suggest that protein cold denaturation can potentially provide a means to explore the cooperative substructure of proteins. Protein cold denaturation is generally predicted to occur well below the freezing point of water. Here NMR spectroscopy of ubiquitin encapsulated in reverse micelles dissolved in low viscosity alkanes is used to follow cold-induced unfolding to temperatures below -25 degrees C.

Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation.

The modern view of protein thermodynamics predicts that proteins undergo cold-induced unfolding. Unfortunately, the properties of proteins and water conspire to prevent the detailed observation of this fundamental process. Here we use protein encapsulation to allow cold denaturation of the protein ubiquitin to be monitored by high-resolution NMR at temperatures approaching -35 degrees C.

Preparation, characterization, and NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids.

Encapsulating a protein in a reverse micelle and dissolving it in a low-viscosity solvent can lower the rotational correlation time of a protein and thereby provides a novel strategy for studying proteins in a variety of contexts. The preparation of the sample is a key element in this approach and is guided by a number of competing parameters. Here we examine the applicability of several strategies for the preparation and characterization of encapsulated proteins dissolved in low viscosity fluids that are suitable for high performance NMR spectroscopy.

Characterization of the monomeric form of the transmembrane and cytoplasmic domains of the integrin beta 3 subunit by NMR spectroscopy.

We have characterized a membrane protein containing residues P688-T762 of the integrin beta3 subunit, encompassing its transmembrane and cytoplasmic domains, by nuclear magnetic resonance spectroscopy. Under conditions in which it is monomeric in dodecylphosphocholine micelles, the protein consists mainly of alpha-helical structures. An amino-terminal helix corresponding to the beta3 transmembrane helix extends into the membrane-proximal region of the cytoplasmic domain.

A simple and effective NMR cell for studies of encapsulated proteins dissolved in low viscosity solvents.

Application of triple-resonance and isotope-edited-NOE methods to the study of increasingly larger macromolecules and their complexes remains a central goal of solution NMR spectroscopy. The slow reorientational motion of larger molecules leads to rapid transverse relaxation and results in losses in both resolution and sensitivity of multidimensional-multinuclear solution NMR experiments. A recently described technique employs a physical approach to increase the tumbling rate of macromolecules in an attempt to preserve access to the full range of structural restraints available to studies of smaller systems.