Fluorinated analogues of tyrosine can be used to manipulate the electronic environments of protein active sites. The ability to selectively mutate tyrosine residues to fluorotyrosines is limited, however, and can currently only be achieved through the total synthesis of proteins. As a general solution to this problem, we genetically encoded the unnatural amino acids o-nitrobenzyl-2-fluorotyrosine, -3-fluorotyrosine, and -2,6-difluorotyrosine in Escherichia coli. These amino acids are disguised from recognition by the endogenous protein biosynthetic machinery, effectively preventing global incorporation of fluorotyrosine into proteins.
Abstract Chromatin remodelling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair and transcription. A key question is how these complexes find their target sites on chromatin. Here, we use genetically encoded photo-crosslinker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC (remodels the structure of chromatin) complex, on the nucleosome in living yeast. We find that the interaction of the Sth1 bromodomain with the H3 tail depends on K14 acetylation by Gcn5. This modification does not recruit RSC to chromatin but mediates its interaction with neighbouring nucleosomes. We observe a preference of RSC for H2B SUMOylated nucleosomes in vivo and show that this modification moderately enhances RSC binding to nucleosomes in vitro . Furthermore, RSC is not ejected from chromatin in mitosis, but its mode of nucleosome binding differs between interphase and mitosis. In sum, our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications most likely in combination with other components such as histone variants and transcription factors. Key Points In vivo photo-crosslinking reveals the footprint of the ATPase subunit of RSC on the nucleosome. RSC binds to H3 K14ac nucleosomes via the C-terminal bromodomain of its ATPase-subunit Sth1. RSC preferentially localizes to H2B-SUMOylated nucleosomes.
In eukaryotes, genetic information is stored as chromatin. Within the chromatin, DNA is wound around octameric units of histone proteins. The histone‐DNA complex forms the nucleosome and represents the most basic level of DNA compaction. A class of proteins known as chromatin remodelers physically reposition nucleosomes along the chromatin fiber. This can directly affect and mediate many important processes such as DNA replication, repair, and transcription. Albeit, due to their large sizes and numerous auxiliary subunits, chromatin remodeler interactions at the nucleosome have yet to be fully characterized and remain relatively obscure. The RSC complex is the most abundant remodeling complex of budding yeast, with homologs in both animals and plants. In this work, we use synthetic biology to trap histone‐RSC remodeler interactions in living cells using the unnatural amino acid p‐benzoylphenylalanine (pBPA). We site‐specifically insert pBPA into histones and identify remodeler binding via the addition of short peptide fusion tags to our interaction target protein. This system has the potential to illuminate chromatin remodeler complex interactions which can then be used to identify and assign biologically important structures. By properly implementing the system, we exemplify the potential of this technology for creating crosslinking maps for RSC. This conclusion is supported by Western blot analysis and a clearly identified crosslink between a myc‐tagged subunit of RSC (Sth1 protein) with histone H2A and histone H3. In addition, we show that binding to H3 (but not H2A) is dependent upon PTMs and a defined acetylation event at lysine 14 on histone H3. In cells that lack H3 K14ac, Sth1 can no longer be sequestered to the nucleosome providing in vivo mechanistic details about RSC‐nucleosomal function/structure relationships. Support or Funding Information NIH R15 Academic Research Enhancement Award (AREA) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
When isotopically labelled photo-crosslinking amino acids are site-specifically incorporated into proteins, in combination with the corresponding non-labeled analogue, cross-linked tryptic peptides are easily identified in mass spectra via characteristic "doublet" patterns.
The utilization of an expanded genetic code and in vivo unnatural amino acid crosslinking has grown significantly in the past decade, proving to be a reliable system for the examination of protein-protein interactions. Perhaps the most utilized amino acid crosslinker, p-benzoyl-(l)-phenylalanine (pBPA), has delivered a vast compendium of structural and mechanistic data, placing it firmly in the upper echelons of protein analytical techniques. pBPA contains a benzophenone group that is activated with low energy radiation (~365 nm), initiating a diradical state that can lead to hydrogen abstraction and radical recombination in the form of a covalent bond to a neighboring protein. Importantly, the expanded genetic code system provides for site-specific encoding of the crosslinker, yielding spatial control for protein surface mapping capabilities. Paired with UV-activation, this process offers a practical means for spatiotemporal understanding of protein-protein dynamics in the living cell. The chromatin field has benefitted particularly well from this technique, providing detailed mapping and mechanistic insight for numerous chromatin-related pathways. We provide here a brief history of unnatural amino acid crosslinking in chromatin studies and outlooks into future applications of the system for increased spatiotemporal resolution in chromatin related research.
Inspired by “One La Salle,” a global initiative for collaboration among Lasallian institutions to strengthen the effectiveness of our mission, members from Manhattan College in New York City and Universidad de La Salle in Bogota and Yopal, Colombia, are collaborating to strengthen our joint mission in research and teaching opportunities connected to social justice. In this article, we summarize the various collaboration projects and their connections to social justice. We illustrate the diversity of exciting projects that can arise through global partnerships within the One La Salle network. The projects are proposed by economics, mechanical engineering, civil engineering, education, and science faculty members. In addition to describing each project, we include research questions and hypotheses that will be studied. We conclude with recommendations for other Lasallian institutions that may embark on a similar quest to strengthen our Lasallian missions within the global Lasallian network.
Post-translational modifications of proteins are important modulators of protein function. In order to identify the specific consequences of individual modifications, general methods are required for homogeneous production of modified proteins. The direct installation of modified amino acids by genetic code expansion facilitates the production of such proteins independent of the knowledge and availability of the enzymes naturally responsible for the modification. The production of recombinant histone H4 with genetically encoded modifications has proven notoriously difficult in the past. Here, we present a general strategy to produce histone H4 with acetylation, propionylation, butyrylation, and crotonylation on lysine residues. We produce homogeneous histone H4 containing up to four simultaneous acetylations to analyze the impact of the modifications on chromatin array compaction. Furthermore, we explore the ability of antibodies to discriminate between alternative lysine acylations by incorporating these modifications in recombinant histone H4.
There are limitations on the current methods used to diversify protein sequences, such as the inability to scan mutations throughout an entire protein. We have developed a novel method, “Codon Scanning Mutagenesis”, which allows for rapid and complete scanning of a protein sequence with the use of elementary molecular biology techniques. This method is made possible by a modified transposon mutagenesis approach that randomly targets DNA sequences. This transposon allows for random insertion of a NotI restriction site, which is unique to the DNA sequence to be mutated. The randomized insertion of this restriction site thus allows for the insertion of a linker with strategically placed type II restriction sites. The properties of the type II restriction sites allow for a codon to be replaced with a new codon. We are specifically interested in the random insertion of TAG stop codons which code for unnatural amino acids carrying photoaffinity labels that can be used to probe protein-protein interactions. Finally, we show that the detection of photo-crosslinked protein-protein interactions can easily be done by mass spectrometry studies when the deuterated p-benzoylphenylalanine is incorporated as an isotopic label. This method will allow for obtaining structural information on multi-component protein complexes, provide insight into the active site residues, or even trap receptor ligands at the cell surface.
Abstract Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1β is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1β bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1β genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin.
