Tom W. Muir - Cochemist

Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis

Co-reporter:Josef A. Gramespacher, Adam J. Stevens, Duy P. Nguyen, Jason W. Chin, and Tom W. Muir

Journal of the American Chemical Society June 21, 2017 Volume 139(Issue 24) pp:8074-8074

Publication Date(Web):May 31, 2017

DOI:10.1021/jacs.7b02618

Naturally split inteins have found widespread use in chemical biology due to their ability to drive the ligation of separately expressed polypeptides through a process termed protein trans-splicing (PTS). In this study, we harness PTS by rendering association of split intein fragments conditional upon the presence of a user-defined protease. We show that these intein “zymogens” can be used to create protein sensors and actuators that respond to the presence of various stimuli, including bacterial pathogens, viral infections, and light. We also show that this design strategy is compatible with several orthogonal split intein pairs, thereby opening the way to the creation of multiplexed sensor systems.

Emerging Chemistry Strategies for Engineering Native Chromatin

Co-reporter:Yael David and Tom W. Muir

Journal of the American Chemical Society July 12, 2017 Volume 139(Issue 27) pp:9090-9090

Publication Date(Web):June 21, 2017

DOI:10.1021/jacs.7b03430

Chromosomes present one of most challenging of all substrates for biochemical study. This is because genomic DNA is physically associated with an astonishing collection of nuclear factors, which serve to not only store the nucleic acid in a stable form, but also grant access to the information it encodes when needed. Understanding this complex molecular choreography is central to the field of epigenetics. One of the great challenges in this area is to move beyond correlative type information, which is now in abundant supply, to the point where we can truly connect the dots at the molecular level. Establishing such causal relationships requires precise manipulation of the covalent structure of chromatin. Tools for this purpose are currently in short supply, creating an opportunity that, as we will argue in this Perspective, is well suited to the sensibilities of the chemist.

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Co-reporter:Glen P. Liszczak;Zachary Z. Brown;Rob C. Oslund;Samuel H. Kim;Yael David

PNAS 2017 Volume 114 (Issue 4 ) pp:681-686

Publication Date(Web):2017-01-24

DOI:10.1073/pnas.1615723114

Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

Functional Plasticity of the AgrC Receptor Histidine Kinase Required for Staphylococcal Virulence

Co-reporter:Boyuan Wang, Aishan Zhao, Qian Xie, Paul Dominic Olinares, ... Tom W. Muir

Cell Chemical Biology 2017 Volume 24, Issue 1(Volume 24, Issue 1) pp:

Publication Date(Web):19 January 2017

DOI:10.1016/j.chembiol.2016.12.008

•AgrC activity correlates with the timing of agr induction among S. aureus subgroups•Single mutations in AgrC produce constitutive mutants by modifying the response curve•AgrC constitutive mutants differ in phospho-transfer rate with non-cognate AIP boundStaphylococcus aureus employs the receptor histidine kinase (RHK), AgrC, to detect quorum-sensing (QS) pheromones, the autoinducer peptides (AIPs), which regulate the virulence of the bacterium. Variation in the QS circuit divides S. aureus into four subgroups, each producing a specific AIP-AgrC pair. While the timing of QS induction is known to differ among these subgroups, the molecular basis of this phenomenon is unknown. Here, we report the successful reconstitution of several AgrC variants and show that the agonist-induced activity of the receptors varies in a manner that accounts for these temporal differences in QS induction. Our studies also reveal a key regulatory hotspot on AgrC that controls the basal activity of RHK as well as the responsiveness of the system to ligand inputs. Collectively, these studies offer insights into the capacity of the RHK for adaptive evolution.Download high-res image (188KB)Download full-size image

Design of a Split Intein with Exceptional Protein Splicing Activity

Co-reporter:Adam J. Stevens; Zachary Z. Brown; Neel H. Shah; Giridhar Sekar; David Cowburn

Journal of the American Chemical Society 2016 Volume 138(Issue 7) pp:2162-2165

Publication Date(Web):February 8, 2016

DOI:10.1021/jacs.5b13528

Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques.

Histones: At the Crossroads of Peptide and Protein Chemistry

Co-reporter:Manuel M. Müller and Tom W. Muir

Chemical Reviews 2015 Volume 115(Issue 6) pp:2296

Publication Date(Web):October 20, 2014

DOI:10.1021/cr5003529

A Second-Generation Phosphohistidine Analog for Production of Phosphohistidine Antibodies

Co-reporter:Jung-Min Kee, Rob C. Oslund, Anthony D. Couvillon, and Tom W. Muir

Organic Letters 2015 Volume 17(Issue 2) pp:187-189

Publication Date(Web):December 22, 2014

DOI:10.1021/ol503320p

Protein histidine phosphorylation plays a crucial role in cell signaling and central metabolism. However, its detailed functions remain elusive due to technical challenges in detecting and isolating proteins bearing phosphohistidine (pHis), a labile posttranslational modification (PTM). To address this issue, we previously developed the first pHis-specific antibodies using stable, synthetic triazole-based pHis analogs. A second-generation, pyrazole-based pHis analog that enabled the development of a pan-pHis antibody with much improved pHis specificity is now reported.

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Co-reporter:Yael David;Sam Pollock;Zhanyun Tang;Matthew T. Holt;Jongcheol Jeon;Jaehoon Kim;Robert G. Roeder

PNAS 2015 Volume 112 (Issue 33 ) pp:10365-10370

Publication Date(Web):2015-08-18

DOI:10.1073/pnas.1504483112

Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure–activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub–induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone posttranslational modification.

Targeted Histone Peptides: Insights into the Spatial Regulation of the Methyltransferase PRC2 by using a Surrogate of Heterotypic Chromatin

Co-reporter:Dr. Zachary Z. Brown;Dr. Manuel M. Müller;Ha Eun Kong;Dr. Peter W. Lewis;Dr. Tom W. Muir

Angewandte Chemie 2015 Volume 127( Issue 22) pp:6557-6561

Publication Date(Web):

DOI:10.1002/ange.201500085

Abstract

Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native chromatin, each nucleosome can differ from its neighbors as a result of covalent modifications to both the DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation. A chemical approach to the production of heterotypic chromatin that can be used in such studies is introduced. This method involves the attachment of a user-defined modified histone peptide to a designated nucleosome within the polymer by using a peptide nucleic acid (PNA) targeting compound. This strategy was applied to dissect the effect of chromatin context on the activity of the histone methyltransferase PRC2. The results show that PRC2 can be stimulated to produce histone H3 methylation from a defined nucleation site.

Increasing AIP Macrocycle Size Reveals Key Features of agr Activation in Staphylococcus aureus

Co-reporter:Jeffrey G. Johnson;Boyuan Wang;Dr. Galia T. Debelouchina; Richard P. Novick; Tom W. Muir

ChemBioChem 2015 Volume 16( Issue 7) pp:1093-1100

Publication Date(Web):

DOI:10.1002/cbic.201500006

Abstract

The agr locus in the commensal human pathogen, Staphylococcus aureus, is a two-promoter regulon with allelic variability that produces a quorum-sensing circuit involved in regulating virulence within the bacterium. Secretion of unique autoinducing peptides (AIPs) and detection of their concentrations by AgrC, a transmembrane receptor histidine kinase, coordinates local bacterial population density with global changes in gene expression. The finding that staphylococcal virulence can be inhibited through antagonism of this quorum-sensing pathway has fueled tremendous interest in understanding the structure–activity relationships underlying the AIP–AgrC interaction. The defining structural feature of the AIP is a 16-membered, thiolactone-containing macrocycle. Surprisingly, the importance of ring size on agr activation or inhibition has not been explored. In this study, we address this deficiency through the synthesis and functional analysis of AIP analogues featuring enlarged and reduced macrocycles. Notably, this study is the first to interrogate AIP function by using both established cell-based reporter gene assays and newly developed in vitro AgrC-I binding and autophosphorylation activity assays. Based on our data, we present a model for robust agr activation involving a cooperative, three-points-of-contact interaction between the AIP macrocycle and AgrC.

Targeted Histone Peptides: Insights into the Spatial Regulation of the Methyltransferase PRC2 by using a Surrogate of Heterotypic Chromatin

Co-reporter:Dr. Zachary Z. Brown;Dr. Manuel M. Müller;Ha Eun Kong;Dr. Peter W. Lewis;Dr. Tom W. Muir

Angewandte Chemie International Edition 2015 Volume 54( Issue 22) pp:6457-6461

Publication Date(Web):

DOI:10.1002/anie.201500085

Abstract

Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native chromatin, each nucleosome can differ from its neighbors as a result of covalent modifications to both the DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation. A chemical approach to the production of heterotypic chromatin that can be used in such studies is introduced. This method involves the attachment of a user-defined modified histone peptide to a designated nucleosome within the polymer by using a peptide nucleic acid (PNA) targeting compound. This strategy was applied to dissect the effect of chromatin context on the activity of the histone methyltransferase PRC2. The results show that PRC2 can be stimulated to produce histone H3 methylation from a defined nucleation site.

Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence

Co-reporter:Boyuan Wang;Aishan Zhao;Richard P. Novick

PNAS 2015 Volume 112 (Issue 34 ) pp:10679-10684

Publication Date(Web):2015-08-25

DOI:10.1073/pnas.1506030112

Staphylococci produce autoinducing peptides (AIPs) as quorum-sensing signals that regulate virulence. These AIPs feature a thiolactone macrocycle that connects the peptide C terminus to the side chain of an internal cysteine. AIPs are processed from ribosomally synthesized precursors [accessory gene regulator D (AgrD)] through two proteolytic events. Formation of the thiolactone is coupled to the first of these and involves the activity of the integral membrane protease AgrB. This step is expected to be thermodynamically unfavorable, and therefore, it is unclear how AIP-producing bacteria produce sufficient amounts of the thiolactone-containing intermediate to drive quorum sensing. Herein, we present the in vitro reconstitution of the AgrB-dependent proteolysis of an AgrD precursor from Staphylococcus aureus. Our data show that efficient thiolactone production is driven by two unanticipated features of the system: (i) membrane association of the thiolactone-containing intermediate, which stabilizes the macrocycle, and (ii) rapid degradation of the C-terminal proteolysis fragment AgrDC, which affects the reaction equilibrium position. Cell-based studies confirm the intimate link between AIP production and intracellular AgrDC levels. Thus, our studies explain the chemical principles that drive AIP production, including uncovering a hitherto unknown link between quorum sensing and peptide turnover.

Strategy for “Detoxification” of a Cancer-Derived Histone Mutant Based on Mapping Its Interaction with the Methyltransferase PRC2

Co-reporter:Zachary Z. Brown ; Manuel M. Müller ; Siddhant U. Jain ; C. David Allis ; Peter W. Lewis

Journal of the American Chemical Society 2014 Volume 136(Issue 39) pp:13498-13501

Publication Date(Web):September 2, 2014

DOI:10.1021/ja5060934

The histone methyltransferase PRC2 plays a central role in genomic stability and cellular development. Consequently, its misregulation has been implicated in several cancers. Recent work has shown that a histone H3 mutant, where the PRC2 substrate residue Lys27 is replaced by methionine, is also associated with cancer phenotypes and functions as an inhibitor of PRC2. Here we investigate the mechanism of this PRC2 inhibition through kinetic studies and photo-cross-linking. Efficient inhibition is dependent on (1) hydrophobic lysine isosteres blocking the active site, (2) proximal residues, and (3) the H3 tail forming extensive contacts with the EZH2 subunit of PRC2. We further show that naturally occurring post-translational modifications of the same H3 tail, both proximal and distal to K27M, can greatly diminish the inhibition of PRC2. These results suggest that this potent gain of function mutation may be “detoxified” by modulating alternate chromatin modification pathways.

A Phosphohistidine Proteomics Strategy Based on Elucidation of a Unique Gas-Phase Phosphopeptide Fragmentation Mechanism

Co-reporter:Rob C. Oslund ; Jung-Min Kee ; Anthony D. Couvillon ; Vivek N. Bhatia ; David H. Perlman

Journal of the American Chemical Society 2014 Volume 136(Issue 37) pp:12899-12911

Publication Date(Web):August 25, 2014

DOI:10.1021/ja507614f

Protein histidine phosphorylation is increasingly recognized as a critical posttranslational modification (PTM) in central metabolism and cell signaling. Still, the detection of phosphohistidine (pHis) in the proteome has remained difficult due to the scarcity of tools to enrich and identify this labile PTM. To address this, we report the first global proteomic analysis of pHis proteins, combining selective immunoenrichment of pHis peptides and a bioinformatic strategy based on mechanistic insight into pHis peptide gas-phase fragmentation during LC–MS/MS. We show that collision-induced dissociation (CID) of pHis peptides produces prominent characteristic neutral losses of 98, 80, and 116 Da. Using isotopic labeling studies, we also demonstrate that the 98 Da neutral loss occurs via gas-phase phosphoryl transfer from pHis to the peptide C-terminal α-carboxylate or to Glu/Asp side chain residues if present. To exploit this property, we developed a software tool that screens LC–MS/MS spectra for potential matches to pHis-containing peptides based on their neutral loss pattern. This tool was integrated into a proteomics workflow for the identification of endogenous pHis-containing proteins in cellular lysates. As an illustration of this strategy, we analyzed pHis peptides from glycerol-fed and mannitol-fed Escherichia coli cells. We identified known and a number of previously speculative pHis sites inferred by homology, predominantly in the phosphoenolpyruvate:sugar transferase system (PTS). Furthermore, we identified two new sites of histidine phosphorylation on aldehyde-alcohol dehydrogenase (AdhE) and pyruvate kinase (PykF) enzymes, previously not known to bear this modification. This study lays the groundwork for future pHis proteomics studies in bacteria and other organisms.

Inteins: nature's gift to protein chemists

Co-reporter:Neel H. Shah and Tom W. Muir

Chemical Science 2014 vol. 5(Issue 2) pp:446-461

Publication Date(Web):10 Dec 2013

DOI:10.1039/C3SC52951G

Inteins are auto-processing domains found in organisms from all domains of life. These proteins carry out a process known as protein splicing, which is a multi-step biochemical reaction comprised of both the cleavage and formation of peptide bonds. While the endogenous substrates of protein splicing are specific essential proteins found in intein-containing host organisms, inteins are also functional in exogenous contexts and can be used to chemically manipulate virtually any polypeptide backbone. Given this, protein chemists have exploited various facets of intein reactivity to modify proteins in myriad ways for both basic biological research as well as potential therapeutic applications. Here, we review the intein field, first focusing on the biological context and phylogenetic diversity of inteins, followed by a description of intein structure and biochemical function. Finally, we discuss prevalent intein-based technologies, focusing on their applications in chemical biology, followed by persistent caveats of intein chemistry and approaches to alleviate these shortcomings. The findings summarized herein describe two and a half decades of research, leading from a biochemical curiosity to the development of powerful protein engineering tools.

Structure of the branched intermediate in protein splicing

Co-reporter:Zhihua Liu;Silvia Frutos;Matthew J. Bick;Miquel Vila-Perelló;Galia T. Debelouchina;Seth A. Darst

PNAS 2014 111 (23 ) pp:8422-8427

Publication Date(Web):2014-06-10

DOI:10.1073/pnas.1402942111

Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use protein-engineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogen-bond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site.

Structure of the branched intermediate in protein splicing

Co-reporter:Zhihua Liu;Silvia Frutos;Matthew J. Bick;Miquel Vila-Perelló;Galia T. Debelouchina;Seth A. Darst

PNAS 2014 111 (23 ) pp:8422-8427

Publication Date(Web):2014-06-10

DOI:10.1073/pnas.1402942111

Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use protein-engineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogen-bond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site.

Naturally Split Inteins Assemble through a “Capture and Collapse” Mechanism

Co-reporter:Neel H. Shah ; Ertan Eryilmaz ; David Cowburn

Journal of the American Chemical Society 2013 Volume 135(Issue 49) pp:18673-18681

Publication Date(Web):November 15, 2013

DOI:10.1021/ja4104364

Split inteins are a class of naturally occurring proteins that carry out protein splicing in trans. The chemical mechanism of protein trans-splicing is well-understood and has been exploited to develop several powerful protein engineering technologies. Split intein chemistry is preceded by efficient molecular recognition between two protomers that become intertwined in their bound state. It is currently unclear how this unique topology is achieved upon fragment association. Using biophysical techniques in conjunction with protein engineering methods, including segmental isotopic labeling, we show that one split intein fragment is partly folded, while the other is completely disordered. These polypeptides capture each other through their disordered regions and form an ordered intermediate with native-like structure at their interface. This intermediate then collapses into the canonical intein fold. This mechanism provides insight into the evolutionary constraints on split intein assembly and should enhance the development of split intein-based technologies.

Extein Residues Play an Intimate Role in the Rate-Limiting Step of Protein Trans-Splicing

Co-reporter:Neel H. Shah ; Ertan Eryilmaz ; David Cowburn

Journal of the American Chemical Society 2013 Volume 135(Issue 15) pp:5839-5847

Publication Date(Web):March 18, 2013

DOI:10.1021/ja401015p

Split inteins play an important role in modern protein semisynthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N- and C-exteins) are strongly preferred, while other sequences cause a dramatic reduction in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here we present a thorough kinetic analysis of the relationship between C-extein composition and split intein activity. The results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, our structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.

Ultrafast Protein Splicing is Common among Cyanobacterial Split Inteins: Implications for Protein Engineering

Co-reporter:Neel H. Shah ; Geoffrey P. Dann ; Miquel Vila-Perelló ; Zhihua Liu

Journal of the American Chemical Society 2012 Volume 134(Issue 28) pp:11338-11341

Publication Date(Web):June 26, 2012

DOI:10.1021/ja303226x

We describe the first systematic study of a family of inteins, the split DnaE inteins from cyanobacteria. By measuring in vivo splicing efficiencies and in vitro kinetics, we demonstrate that several inteins can catalyze protein trans-splicing in tens of seconds rather than hours, as is commonly observed for this autoprocessing protein family. Furthermore, we show that when artificially fused, these inteins can be used for rapid generation of protein α-thioesters for expressed protein ligation. This comprehensive survey of split inteins provides indispensable information for the development and improvement of intein-based tools for chemical biology.

Stability of Nucleosomes Containing Homogenously Ubiquitylated H2A and H2B Prepared Using Semisynthesis

Co-reporter:Beat Fierz ; Sinan Kilic ; Aaron R. Hieb ; Karolin Luger

Journal of the American Chemical Society 2012 Volume 134(Issue 48) pp:19548-19551

Publication Date(Web):November 19, 2012

DOI:10.1021/ja308908p

Post-translational modifications (PTMs) of histones are an essential feature in the dynamic regulation of chromatin. One of these modifications, ubiquitylation, has been speculated to directly influence the stability of the nucleosome, which represents the basic building block of chromatin. Here we report a strategy for the semisynthesis of site-specifically ubiquitylated histone H2A (uH2A). This branched protein was generated through a three-piece expressed protein ligation approach including a traceless ligation at valine. uH2A could be efficiently incorporated into nucleosomes, thereby opening the way to detailed biochemical and biophysical studies on the function of this PTM. Accordingly, we used uH2A, as well as a previously generated ubiquitylated H2B, in chaperone-coupled nucleosome stability assays to demonstrate that the direct effect of ubiquitylated histones on nucleosomal stability is in fact modest.

Streamlined Expressed Protein Ligation Using Split Inteins

Co-reporter:Miquel Vila-Perelló ; Zhihua Liu ; Neel H. Shah ; John A. Willis ; Juliana Idoyaga

Journal of the American Chemical Society 2012 Volume 135(Issue 1) pp:286-292

Publication Date(Web):December 24, 2012

DOI:10.1021/ja309126m

Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

Chasing Phosphohistidine, an Elusive Sibling in the Phosphoamino Acid Family

Co-reporter:Jung-Min Kee and Tom W. Muir

ACS Chemical Biology 2012 Volume 7(Issue 1) pp:44

Publication Date(Web):December 9, 2011

DOI:10.1021/cb200445w

This year (2012) marks the 50th anniversary of the discovery of protein histidine phosphorylation. Phosphorylation of histidine (pHis) is now widely recognized as being critical to signaling processes in prokaryotes and lower eukaryotes. However, the modification is also becoming more widely reported in mammalian cellular processes and implicated in certain human disease states such as cancer and inflammation. Nonetheless, much remains to be understood about the role and extent of the modification in mammalian cell biology. Studying the functional role of pHis in signaling, either in vitro or in vivo, has proven devilishly hard, largely due to the chemical instability of the modification. As a consequence, we are currently handicapped by a chronic lack of chemical and biochemical tools with which to study histidine phosphorylation. Here, we discuss the challenges associated with studying the chemical biology of pHis and review recent progress that offers some hope that long-awaited biochemical reagents for studying this elusive posttranslational modification (PTM) might soon be available.

From EPOthilone to EPO: A challenge for natural product synthesis

Co-reporter:Zachary Z. Brown

PNAS 2012 Volume 109 (Issue 19 ) pp:7196-7201

Publication Date(Web):2012-05-08

DOI:10.1073/pnas.1205667109

Split Inteins: Natures Protein Ligases

Co-reporter:Neel H. Shah

Israel Journal of Chemistry 2011 Volume 51( Issue 8-9) pp:854-861

Publication Date(Web):

DOI:10.1002/ijch.201100094

Abstract

Split inteins carry out a naturally occurring process known as protein trans-splicing, where two protein fragments bind to form a catalytically competent enzyme, then catalyze their own excision and the ligation of their flanking sequences. In the past thirteen years since their discovery, chemists and biologists have utilized split inteins in exogenous contexts for a number of biotechnological applications centered around the formation of native peptide bonds. While many protein trans-splicing technologies have emerged and flourished in recent years, several factors still limit their wide-spread practical use. Here, we discuss the development, applications, and limitations of split intein-based technologies and propose that further advancement in this field will require a more fundamental understanding of split intein structure and function.

Activation and Inhibition of the Receptor Histidine Kinase AgrC Occurs through Opposite Helical Transduction Motions

Co-reporter:Boyuan Wang, Aishan Zhao, Richard P. Novick, Tom W. Muir

Molecular Cell (20 March 2014) Volume 53(Issue 6) pp:929-940

Publication Date(Web):20 March 2014

DOI:10.1016/j.molcel.2014.02.029

•A quantitative study of the signal transduction in AgrC incorporated to nanodiscs•Rheostat-like activity control in response to twisting force on interdomain linkers•Linker rotation in opposite directions with agonist or inverse-agonist binding•AgrC’s low affinity to ATP is a probable reason of agr shutdown under energy stressStaphylococcus aureus virulence is regulated when secreted autoinducing peptides (AIPs) are recognized by a membrane-bound receptor histidine kinase (RHK), AgrC. Some AIPs are agonists of virulence gene expression, while others are antagonists. It is unclear how AIP binding regulates AgrC activity. Here, we reconstitute an AgrC family member, AgrC-I, using nanometer-scale lipid bilayer discs. We show that AgrC-I requires membranes rich in anionic lipids to function. The agonist, AIP-I, binds AgrC-I noncooperatively in a 2:2 stoichiometry, while an antagonist ligand, AIP-II, functions as an inverse agonist of the kinase activity. We also demonstrate the kinase and sensor domains in AgrC are connected by a helical linker whose conformational state exercises rheostat-like control over the kinase activity. Binding of agonist or inverse-agonist peptides results in twisting of the linker in different directions. These two observations provide a view of the molecular motions triggered by ligand binding in an intact membrane-bound RHK.Download high-res image (235KB)Download full-size image

Inteins: nature's gift to protein chemists

Co-reporter:Neel H. Shah and Tom W. Muir

Chemical Science (2010-Present) 2014 - vol. 5(Issue 2) pp:NaN461-461

Publication Date(Web):2013/12/10

DOI:10.1039/C3SC52951G

Inteins are auto-processing domains found in organisms from all domains of life. These proteins carry out a process known as protein splicing, which is a multi-step biochemical reaction comprised of both the cleavage and formation of peptide bonds. While the endogenous substrates of protein splicing are specific essential proteins found in intein-containing host organisms, inteins are also functional in exogenous contexts and can be used to chemically manipulate virtually any polypeptide backbone. Given this, protein chemists have exploited various facets of intein reactivity to modify proteins in myriad ways for both basic biological research as well as potential therapeutic applications. Here, we review the intein field, first focusing on the biological context and phylogenetic diversity of inteins, followed by a description of intein structure and biochemical function. Finally, we discuss prevalent intein-based technologies, focusing on their applications in chemical biology, followed by persistent caveats of intein chemistry and approaches to alleviate these shortcomings. The findings summarized herein describe two and a half decades of research, leading from a biochemical curiosity to the development of powerful protein engineering tools.