Sortase mediated protein ubiquitination with defined chain length and topology.

Ubiquitination is a key post-translational modification on protein lysine sidechains known to impact protein stability, signal transduction cascades, protein-protein interactions, and beyond. Great strides have been made towards developing new methods to generate discrete chains of polyubiquitin and conjugate them onto proteins site-specifically, with methods ranging from chemical synthetic approaches, to enzymatic approaches and many in between. Previous work has demonstrated the utility of engineered variants of the bacterial transpeptidase enzyme sortase (SrtA) for conjugation of ubiquitin site-specifically onto target proteins. In this manuscript, we've combined the classical E1/E2-mediated polyubiquitin chain extension approach with sortase-mediated ligation and click chemistry to enable the generation of mono, di, and triubiquitinated proteins sfGFP and PCNA. We demonstrate the utility of this strategy to generate both K48-linked and K63-linked polyubiquitins and attach them both N-terminally and site-specifically to the proteins of interest. Further, we highlight differential activity between two commonly employed sortase variants, SrtA 5M and 7M, and demonstrate that while SrtA 7M can be used to conjugate these ubiquitins to substrates, SrtA 5M can be employed to release the ubiquitin from the substrates as well as to cleave C-terminal tags from the ubiquitin variants used. Overall, we envision that this approach is broadly applicable to readily generate discrete polyubiquitin chains of any linkage type that is accessible via E1/E2 systems and conjugate site-specifically onto proteins of interest, thus granting access to bespoke ubiquitinated proteins that are not currently possible.

Direct Measurements of FLASH-Induced Changes in Intracellular Oxygenation.

PURPOSE: The goal of our study was to characterize the dynamics of intracellular oxygen during application of radiation at conventional (CONV) and FLASH dose rates and obtain evidence for or against the oxygen depletion hypothesis as a mechanism of the FLASH effect. METHODS AND MATERIALS: The measurements were performed by the phosphorescence quenching method using probe Oxyphor PtG4, which was delivered into the cellular cytosol by electroporation. RESULTS: Intracellular radiochemical oxygen depletion (ROD) g-value for a dose rate of 100 Gy/s in the normoxic range was found to be 0.58 ± 0.03 µM/Gy. Intracellular ROD g-values for FLASH and CONV dose rates in the normoxic range were found to be nearly equal. As in solution-based studies, intracellular ROD was found to exhibit strong dependence on oxygen concentration in the range of 0 to ∼40 µM [O2]. CONCLUSIONS: Depletion of oxygen in cells in vitro by a clinical dose of proton radiation delivered as FLASH is unable to produce a transient state of hypoxia and, therefore, unable to induce radioprotection. The difference between ROD g-values for FLASH and CONV dose rates, detected previously in solutions-based experiments, disappears when measurements are conducted inside cells. Understanding this phenomenon should provide additional insight into the role of oxygen in FLASH radiation therapy and help to decipher the mechanism of the FLASH effect.

Advances and Opportunities in Epigenetic Chemical Biology.

The study of epigenetics has greatly benefited from the development and application of various chemical biology approaches. In this review, we highlight the key targets for modulation and recent methods developed to enact such modulation. We discuss various chemical biology techniques to study DNA methylation and the post-translational modification of histones as well as their effect on gene expression. Additionally, we address the wealth of protein synthesis approaches to yield histones and nucleosomes bearing epigenetic modifications. Throughout, we highlight targets that present opportunities for the chemical biology community, as well as exciting new approaches that will provide additional insight into the roles of epigenetic marks.

Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase.

Genetic code expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA-protein yields, genomically recoded strains of Escherichia coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a second-generation amino-acyl tRNA synthetase (aaRS)/tRNACUA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~2-fold greater efficiency, and remarkably, this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues.