Investigating local protein CO-translational folding in situ with high spatiotemporal resolution

The National Institute of General Medical Sciences (NIGMS) awarded University of Colorado-Denver a $1,998,288 Project Grant under the Biomedical Research and Research Training program (CFDA 93.859) effective September 1, 2025, through August 31, 2029. The award funds investigator-initiated research to develop and apply Co-Translational Folding Tracking (COTFT), a novel technology platform designed to visualize and quantify protein co-translational folding kinetics in living cells with single messenger RNA (mRNA) precision and sub-second temporal resolution. The research addresses a critical gap in understanding how protein folding occurs during translation in diverse intracellular environments, including the cytosol and organelle surfaces, and investigates how translation elongation rate alterations affect folding outcomes.

The project deliverables include the development of a novel methodology to specifically label and isolate short-lived co-translational folding complexes, comprehensive quantitative analyses of co-translational folding heterogeneity across different cellular locations, investigation of how translation kinetics modulate protein folding, and characterization of the interactome associated with co-translational folding complexes. These research outputs are intended to advance fundamental understanding of protein folding mechanisms relevant to human health and disease prevention by elucidating the cellular factors and ribosome-associated proteins that orchestrate proper protein folding during translation.

Abstract

The folding of many proteins occurs CO-translationally and involves the orchestration of numerous cellular factors, including a large set of ribosome-associated proteins and chaperones. In the CO-translational folding process, the folding is intimately coupled to translation. If the ribosome elongates too fast or stalls for too long at the wrong place and time, folding kinetics can be severely perturbed, leading to protein misfolding and/or aggregation. In extreme cases, this can lead to diseases. A better understanding of protein CO-translational folding kinetics in the native context of translation is therefore critical to human health. The major challenge in the field is the lack of spatiotemporal resolution needed to track and quantify CO-translational folding in a living intracellular environment. To address this challenge, we have developed a novel CO-translational folding tracking (cotft) technology that enables US to directly visualize CO-translational folding with single mrna precision and sub- second resolution in living cells. With cotft, we will focus on investigating CO-translational folding in living cells. Specifically, we will quantify the kinetics of CO-translational folding occurring in diverse intracellular environments, such as in cytosol and on the surfaces of organelles, to identify any location heterogeneity in CO-translational folding as well as heterogeneity in mrnas in each location. Then we will further investigate an ongoing question in the protein CO-translational folding field - how alterations in translation elongation rate affects folding. Moreover, we will develop a novel method that specifically labels and isolates the short-lived CO-translational folding complexes with high spatiotemporal resolution, which is challenging to achieve with current technologies. We will apply this method to investigate the interactome that is highly specific for CO-translational folding in diverse subcellular environments. For discovered novel interactors, we will study their impact on CO-translational folding using our established cotft technology. The discoveries from this study will help US further understand CO- translational folding in a native intracellular environment. In addition, our developed technologies are highly adaptable. In the long run, we expect to adapt our technologies to study a group of disease related transmembrane proteins and discover general as well as target-specific regulation mechanisms of CO- translational folding. The discovered novel regulation mechanisms have the potential to lead to new therapeutics that have never been explored before for protein misfolding-related diseases.