Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9-RAD1-HUS1 (9-1-1) Complex.

Affinity pulldown is a powerful technique to discover novel interaction partners and verify a predicted physical association between two or more proteins. Pulldown assays capture a target protein fused with an affinity tag and analyze the complexed proteins. Here, we detail methods of pulldown assays for two high-affinity peptide fusion tags, Flag tag (DYKDDDDK) and hexahistidine tag (6xHis), to study protein-protein interactions of human NEIL1 glycosylase and the checkpoint protein complex RAD9-RAD1-HUS1 (9-1-1).

β-Peptide bundles: Design. Build. Analyze. Biosynthesize.

Peptides containing β-amino acids are unique non-natural polymers known to assemble into protein-like tertiary and quaternary structures. When composed solely of β-amino acids, the structures formed, defined assemblies of 14-helices called β-peptide bundles, fold cooperatively in water solvent into unique and discrete quaternary assemblies that are highly thermostable, bind complex substrates and metal ion cofactors, and, in certain cases, catalyze chemical reactions. In this Perspective, we recount the design and elaboration of β-peptide bundles and provide an outlook on recent, unexpected discoveries that could influence research on β-peptides and β-peptide bundles (and β-amino acid-containing proteins) for decades to come.

Design and high-resolution structure of a β³-peptide bundle catalyst.

Despite the widespread exploration of α-peptides as catalysts, there are few examples of β-peptides that alter the course of a chemical transformation. Our previous work demonstrated that a special class of β(3)-peptides spontaneously self-assembles in water into discrete protein-like bundles possessing unique quaternary structures and exceptional thermodynamic stability. Here we describe a series of β(3)-peptide bundles capable of both substrate binding and chemical catalysis--ester hydrolysis.

Relationship between side-chain branching and stoichiometry in β(3)-peptide bundles.

The stability and stoichiometry of β(3)-peptide bundles is influenced by side-chain identity. β(3)-peptides containing β(3)-homoleucine on one helical face assemble into octamers, whereas those containing β(3)-homovaline form tetramers. From a structural perspective, the side chains of β(3)-homoleucine and β(3)-homovaline differ in terms of both side-chain length and γ-carbon branching.