Role of Nucleic Acids as Chaperones in Protein Folding

Nucleotide-binding motifs nucleated folding of the first enzymes

It is concluded that the early emergence of the Rossmann fold reflects the chemical and physical constraints of protein folding, explaining both its profound antiquity and sustained longevity.

The initiation of de novo protein folding on the ribosome

A mechanistic model of de novo folding initiation during biosynthesis is infer and a complete atomistic description of a co-translational folding pathway is provided by linking the folding nucleus to downstream partially structured intermediates and the native state is provided.

Direct single-molecule visualization of Hsp90-mediated relief of an Hsp70-folding block

Heat shock protein 70 (Hsp70) and Hsp90 are essential molecular chaperones that cooperate to fold diverse client proteins, yet how their activities are coordinated to remodel clients remains unclear. To address this, we used a combination of single-molecule fluorescence resonance energy transfer and total internal reflection fluorescence microscopy to observe individual firefly luciferase proteins during sequential engagement with Escherichia coli Hsp70 (DnaK) and Hsp90 (HtpG). We show that HtpG reduces rebinding of DnaK to folding intermediates while still allowing engagement with misfolded clients, enabling productive refolding in the presence of typically inhibitory concentrations of DnaK. HtpG couples adenosine 5′-triphosphate binding and hydrolysis to promote progressive folding through localized compaction across multiple regions of the client, reducing misfolding and establishing native interdomain contacts. Kinetic simulations support a model whereby heterogeneous DnaK binding generates region-specific folding kinetics and conformational dynamics. This enables efficient subdomain folding by DnaK/HtpG and suggests that the number and position of DnaK binding sites on clients provide a mechanism by which proteins can harness chaperone promiscuity for optimal folding.

Chaperoning through time - defining DNAJA2 co-chaperone client selectivity via pulse-SILAC and BioID

Rho (𝛒) Analysis to Dissect RNA Folding and Assembly Pathways

The results establish ρ analysis as a general framework to probe RNA conformational pathways and function and uncover multiple folding pathways and modulation in pathway flux that are readily understood from the energetic properties of the constituent RNA motifs.

Chemical Boosting of Foldase Condensates Accelerates Oxidative Protein Folding

Dynamic biomolecular condensates play crucial roles in intracellular compartmentalization and physiological functions. While engineering tools for compartmentalization have expanded add-on functionalities, directly amplifying the inherent catalytic machinery within biological phase-separated droplets has remained elusive. Herein, we developed a phase-separated oxidative folding reaction chamber based on protein disulfide isomerase A6 (PDIA6) by chemically targeting its active site CxxC motif to enhance enzymatic activity within PDIA6 droplets. A para-substituted N-methylated pyridinylmethanethiol (pMePySH) enhanced the catalytic oxidative folding of bovine pancreatic trypsin inhibitor, proinsulin, and antibody up to 12-fold within in vitro PDIA6 droplets. Furthermore, pMePySH targeted PDIA6 foci within the endoplasmic reticulum, significantly promoting insulin secretion. These findings offer a powerful platform for the spatiotemporal manipulation of protein folding, with profound implications for the scalable manufacturing of therapeutic antibodies and other complex biopharmaceuticals.