Research

Chromatin

chromatin-intro-illustration-400x459The eukaryotic genome is organized as a nucleoprotein complex called chromatin. The repeating unit of chromatin is the nucleosome, containing two copies of each core histone (H2A, H2B, H3, and H4) and typically 147 bp of DNA. Histones carry a plethora of post-translational modifications (PTMs or marks), which are dynamically installed and removed by specific enzymes (“writers” and “erasers”), forming a covalent language that is often referred to as the “histone code“. Patterns of these modifications are interpreted by “reader” proteins, regulating the function of chromatin and thereby controlling cell homeostasis, differentiation, and development.

In the Muir lab, we use synthetic designer chromatin as well as chemical, biophysical, and biological methods to investigate the molecular basis of the histone code ranging from the nucleosome to the cellular level.

 

Inteins

cis-splicing-intein-320x597Inteins, found in a variety of unicellular organisms, are intervening polypeptide sequences that are able to catalyze their own excision from flanking protein regions (i.e. exteins) and ligate them together.  While the biological function of inteins remains an area of investigation, this class of proteins has found widespread use in the fields of chemical and cell biology.  Our lab recognizes the unique intein splicing reaction as platform for development of chemical biological tools.  At a fundamental level, we aim to characterize the precise biochemical requirements for intein splicing to identify characteristics that enable the engineering of inteins with enhanced properties and thus the development of improved and completely new intein-based technologies.

 

 pHis

chasing-pHis

Protein phosphorylation is one of the most common and extensively studied posttranslational modifications (PTMs). Phosphorylation and dephosphorylation of proteins control protein function, and their misregulation has been linked to numerous human diseases. Accordingly, protein kinases and phosphatases have emerged as important drug targets.

Not unlike Ser, Thr, and Tyr residues, histidine can also be phosphorylated at its imidazole nitrogens. The role of protein histidine phosphorylation, first discovered over 50 years ago, is best understood in two-component signaling pathways in bacteria and lower eukaryotes, and we are just beginning to appreciate its roles in higher eukaryotes. In recent years, the PTM has been implicated in several important processes, including G-protein signaling, ion conduction, metabolism, and chromatin biology. Still, much detail remains to be elucidated about the prevalence and mechanistic role of the modification.

Studying the functional role of pHis, either in vitro or in vivo, has proven extremely difficult, largely due to the chemical instability of the modification. As a consequence, the lack of adequate chemical and biochemical tools to study histidine phosphorylation has been the major roadblock in the comprehensive understanding of pHis functions in biological systems.

In the Muir group, we develop and utilize novel research tools to study histidine phosphorylation by synergistically combining our expertise in synthetic chemistry, protein biochemistry, and molecular biology. We have recently developed the first ever pHis-specific antibody, which enabled the selective detection and identification of pHis proteins from complex biological samples. Currently we are utilizing this antibody, along with other tools, to investigate the detailed functional roles played by pHis in signaling, metabolism, and epigenetics.

 

AGR

agr-illustration-640x360

Bacteria gain significant evolutionary advantage from having individual cells working synergistically.  This intra-species collaboration often entails coordination of the physiological behavior of individual cells according to the population density, a process termed quorum sensing (QS). Staphylococcus aureus, a notorious human pathogen, develops virulence after achieving growth due to the activation of its quorum sensing. Interfering with the quorum sensing has been shown effective in controlling the infection of S. aureus, which attracts certain pharmaceutical interests to the study of the mechanism of its action.

Molecules that play central roles in S. aureus quorum sensing are encoded in a chromosomal locus named agr, which contains two back-to-back operons, P2 and P3. The transcript of the P3 operon, RNAIII, is the major downstream effector of agr, which regulates the production of a series of toxins and surface protein. The P2 operon encodes four proteins responsible to the production or recognition of a peptidic pheromone, AIP (for auto-inducing peptide). AgrD is the precursor of AIP, whose maturation and secretion involves the membrane-bound peptidase AgrB. AgrC and AgrA forms into a two-component system that regulate the transcription level of both P2 and P3 operons in response to the extracellular level of AIP. The entire pathway closes into a positive-feedback loop and is therefore named the agr auto-induction circuit. Four distinct groups in terms of quorum-sensing specificity has been identified for S. aureus: each group produces distinct AgrB and AgrC proteins as well as one distinct AIP. Remarkably, AIPs act as agonists of the quorum sensing within a group but, in most occasions, as antagonists between groups.

Since the 1990s, the Muir Lab has participated in the identification of the structures of S. aureus AIPs and studied extensively the structure-activity relationship for AIPs using chemically synthesized analogs. More recently, we have studied the mechanism by which AIP activates the quorum-sensing signaling, using a combination of synthetic chemistry and bacterial genetic approach. Now, we are aiming to reconstitute all biochemical events involved in the agr circuit in vitro using purified recombinant proteins. This undertaking will provide a platform for the anti-staphylococcal drug design and quantitative biophysical/biochemical studies in the future.