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). We uncover unique interactions between 9-1-1 and NEIL1, which suggest a possible inhibitory role of the disordered, phosphorylated C-terminal region of RAD9 in regulating NEIL1 activity in base excision repair through lack of physical association of 9-1-1 and NEIL1.

Parathyroid Hormone Senses Extracellular Calcium To Modulate Endocrine Signaling upon Binding to the Family B GPCR Parathyroid Hormone 1 Receptor.

Parathyroid hormone (PTH) binds to a family B G protein coupled receptor, parathyroid hormone 1 receptor (PTH1R). One of its functions is to regulate Ca2+ homeostasis in bone remodeling, during which Ca2+ can reach up to 40 mM. A truncated version of PTH, PTH(1-34), can fully activate PTH1R and has been used for osteoporosis treatments. Here, we used fluorescence anisotropy to examine the binding of PTH(1-34) to PTH1R purified in nanodiscs (PTH1R-ND) and found that the affinity increases 5-fold in the presence of 15 mM Ca2+. However, PTHrP(1-36), another truncated endogenous agonist for PTH1R, does not show this Ca2+ effect. Mutations of Glu19 and Glu22 in PTH(1-34) that are not conserved in PTHrP(1-36) largely abolished the Ca2+ effect. The results support that PTH(1-34) not only activates PTH1R but also uniquely senses Ca2+. This dual function of a peptide hormone is a novel observation that couples changes in extracellular environment with endocrine signaling. Understanding this can potentially reveal the complex role of PTH signaling in bone remodeling and improve the PTH(1-34) treatment for osteoporosis.

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

Peptides containing ß-amino acids are unique non-natural polymers known to assemble into protein-like tertiary and quaternary structures. When composed solely of ß-amino acids, the structures formed, defined assemblies of 14-helices called ß-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 ß-peptide bundles and provide an outlook on recent, unexpected discoveries that could influence research on ß-peptides and ß-peptide bundles (and ß-amino acid-containing proteins) for decades to come.

Design and high-resolution structure of a ß³-peptide bundle catalyst.

Despite the widespread exploration of α-peptides as catalysts, there are few examples of ß-peptides that alter the course of a chemical transformation. Our previous work demonstrated that a special class of ß(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 ß(3)-peptide bundles capable of both substrate binding and chemical catalysis--ester hydrolysis. A combination of kinetic and high-resolution structural analysis suggests an active site triad composed of residues from at least two strands of the octameric bundle structure.