We propose to continue our research program to study and exploit intein-mediated protein splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, becomes excised from a host protein, the extein, in an autocatalytic manner. In protein trans-splicing the intein is split into two pieces ad splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all three phylogenetic domains, including several pathogens such as Mycobacterium tuberculosis (Mtb) where their splicing activity is required for DNA replication. Work from many laboratories, including our own, has painted a picture of protein splicing as being a slow, often inefficient, process taking several hours to reach completion. Very recent work has, however, caused us to drastically re-evaluate this dogma. This is because of a remarkable split intein from the cyanobacterium, Nostoc punctiforme (Npu intein) that supports protein splicing orders of magnitude faster than anything seen before (half-life of less than a minute). This finding raises several fascinating questions that form the core of this renewal application. First and foremost, how does this intein splice so fast? Are there other uncharacterized split inteins that splice as fast or even faster? Do these split proteins retain an structure prior to association and what is the mechanism of fragment complementation? How can we harness the ultra-fast kinetics of Npu (and other family members) for new applications? We will take a multi-pronged approach to study catalysis (Aim 1) and folding (Aim 2) of the Npu intein, as well as other as yet uncharacterized split inteins. For this we will employ the tools of enzymology, biophysics, bioinformatics and structural biology. All of these studies will be aided by our ability to incorporate a broad range of biophysical and biochemical probes (unnatural amino acids) into the proteins using chemical synthesis.
In Aim 3, new technologies will be developed that exploit the properties of these new split inteins for both in vitro and in vivo protein chemistry applications. This includes developing functionally orthogonal split inteins, conditional split inteins and split inteins for semi-synthesis in cells. While expected to be broady applicable, these tools will be applied to a specific set of problems in chromatin biology of ongoing interest to my laboratory.
Split inteins are Nature's protein ligases;they mediate the traceless ligation of any polypeptide chains to which they are appended. The research program focuses on a recently discovered family of split inteins that mediate this ligation process with unprecedented speed and fidelity. These proteins have enormous potential in protein biotechnology with applications ranging from basic molecular and cell biology to the preparation of protein therapeutics. We will determine how these proteins catalyze the ligation reaction so efficiently and then use this information to develop new technologies for manipulating the structure and function of proteins, with immediate applications to epigenetics.
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|Zhou, Linjiao; Holt, Matthew T; Ohashi, Nami et al. (2016) Evidence that ubiquitylated H2B corrals hDot1L on the nucleosomal surface to induce H3K79 methylation. Nat Commun 7:10589|
|David, Yael; Vila-PerellÃ³, Miquel; Verma, Shivam et al. (2015) Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins. Nat Chem 7:394-402|
|Holt, Matthew T; David, Yael; Pollock, Sam et al. (2015) Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin. Proc Natl Acad Sci U S A 112:10365-70|
|MÃ¼ller, Manuel M; Muir, Tom W (2015) Histones: at the crossroads of peptide and protein chemistry. Chem Rev 115:2296-349|
|Brown, Zachary Z; MÃ¼ller, Manuel M; Kong, Ha Eun et al. (2015) Targeted Histone Peptides: Insights into the Spatial Regulation of the Methyltransferase PRC2 by using a Surrogate of Heterotypic Chromatin. Angew Chem Int Ed Engl 54:6457-61|
|Brown, Zachary Z; MÃ¼ller, Manuel M; Jain, Siddhant U et al. (2014) Strategy for ""detoxification"" of a cancer-derived histone mutant based on mapping its interaction with the methyltransferase PRC2. J Am Chem Soc 136:13498-501|
|Nguyen, Uyen T T; Bittova, Lenka; MÃ¼ller, Manuel M et al. (2014) Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries. Nat Methods 11:834-40|
|Shah, Neel H; Muir, Tom W (2014) Inteins: Nature's Gift to Protein Chemists. Chem Sci 5:446-461|
|Eryilmaz, Ertan; Shah, Neel H; Muir, Tom W et al. (2014) Structural and dynamical features of inteins and implications on protein splicing. J Biol Chem 289:14506-11|
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