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Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

ABSTRACT: The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here, we deploy a proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from pre-made parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions BCR/ABL and DNAJB1/PRKACA where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as novel therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

SUBMITTER: Lee G

PROVIDER: S-EPMC10369872 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

Lee Gihoon G Muir Tom W TW

bioRxiv : the preprint server for biology 20230710

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here, we deploy a proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from pre-made parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the ...[more]

PMID: 37503273

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Project description:One of the most important questions in biological science is how a protein functions. When a protein performs its function, it undergoes regulated structural transitions. In this regard, to better understand the underlying principle of a protein function, it is desirable to monitor the dynamic evolution of the protein structure in real time. To probe fast and subtle motions of a protein in physiological conditions demands an experimental tool that is not only equipped with superb spatiotemporal resolution but also applicable to samples in solution phase. Time-resolved X-ray solution scattering (TRXSS), discussed in this Account, fits all of those requirements needed for probing the movements of proteins in aqueous solution. The technique utilizes a pump-probe scheme employing an optical pump pulse to initiate photoreactions of proteins and an X-ray probe pulse to monitor ensuing structural changes. The technical advances in ultrafast lasers and X-ray sources allow us to achieve superb temporal resolution down to femtoseconds. Because X-rays scatter off all atomic pairs in a protein, an X-ray scattering pattern provides information on the global structure of the protein with subangstrom spatial resolution. Importantly, TRXSS is readily applicable to aqueous solution samples of proteins with the aid of theoretical models and therefore is well suited for investigating structural dynamics of protein transitions in physiological conditions. In this Account, we demonstrate that TRXSS can be used to probe real-time structural dynamics of proteins in solution ranging from subtle helix movement to global conformational change. Specifically, we discuss the photoreactions of photoactive yellow protein (PYP) and homodimeric hemoglobin (HbI). For PYP, we revealed the kinetics of structural transitions among four transient intermediates comprising a photocycle and, by applying structural analysis based on ab initio shape reconstruction, showed that the signaling of PYP involves the protrusion of the N-terminus with significant increase of the overall protein size. For HbI, we elucidated the dynamics of complex allosteric transitions among transient intermediates. In particular, by applying structural refinement analysis based on rigid-body modeling, we found that the allosteric transition of HbI accompanies the rotation of quaternary structure and the contraction between two heme domains. By making use of the experimental and analysis methods presented in this Account, we envision that the TRXSS can be used to probe the structural dynamics of various proteins, allowing us to decipher the working mechanisms of their functions. Furthermore, when combined with femtosecond X-ray pulses generated from X-ray free electron lasers, TRXSS will gain access to ultrafast protein dynamics on sub-picosecond time scales.

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Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

Publications

Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

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