Barbara Imperiali

Barbara Imperiali
Massachusetts Institute of Technology | MIT · Department of Biology

BSc, PhD

About

317
Publications
31,044
Reads
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14,659
Citations
Additional affiliations
January 2011 - present
MIT
Position
  • Professor (Full)
Description
  • Teaching General Biology and Chemical Biology
January 2005 - present
MIT
Position
  • Professor (Full)
July 1999 - July 2017
Massachusetts Institute of Technology
Position
  • Professor (Full)

Publications

Publications (317)
Article
Full-text available
Phosphoglycosyl transferases (PGTs) are membrane proteins that initiate glycoconjugate biosynthesis by transferring a phospho-sugar moiety from a soluble nucleoside diphosphate sugar to a membrane-embedded polyprenol phosphate acceptor. The centrality of PGTs in complex glycan assembly and the current lack of functional information make these enzym...
Article
Full-text available
Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for...
Article
Full-text available
In selected Campylobacter species, the biosynthesis of N‐linked glycoconjugates via the pgl pathway is essential for pathogenicity and survival. However, most of the membrane‐associated GT‐B fold glycosyltransferases responsible for diversifying glycans in this pathway have not been structurally characterized which hinders the understanding of the...
Preprint
Full-text available
Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for...
Preprint
Full-text available
Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for...
Preprint
Full-text available
Complex bacterial glycoconjugates are essential for bacterial survival, and drive interactions between pathogens and symbionts, and their human hosts. Glycoconjugate biosynthesis is initiated at the membrane interface by phosphoglycosyl transferases (PGTs), which catalyze the transfer of a phosphosugar from a soluble uridine diphospho-sugar (UDP-su...
Article
Full-text available
The Bacillus subtilis extracellular biofilm matrix includes an exopolysaccharide (EPS) that is critical for the architecture and function of the community. To date, our understanding of the biosynthetic machinery and the molecular composition of the EPS of B. subtilis remains unclear and incomplete. This report presents synergistic biochemical and...
Article
Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked...
Article
Nucleoside analogs show useful bioactive properties. A versatile solid-phase synthesis that readily enables the diversification of thymine-containing nucleoside analogs is presented. The utility of the approach is demonstrated with the...
Preprint
Full-text available
Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for...
Poster
Cross-linking mass spectrometry (XL-MS) has grown dramatically into a routinely utilized strategy for characterizing protein higher-order structure and mapping protein-protein interaction networks on a proteome-wide scale. However, the XL-MS analysis of membrane proteins is still a significant challenge due to their hydrophobic properties as well a...
Article
Full-text available
The oral microbiome is critical to human health and disease, yet the role that host salivary proteins play in maintaining oral health is unclear. A highly expressed gene in human salivary glands encodes the lectin zymogen granule protein 16 homolog B (ZG16B). Despite the abundance of this protein, its interaction partners in the oral microbiome are...
Article
Full-text available
Monotopic phosphoglycosyl transferases (monoPGTs) are an expansive superfamily of enzymes that catalyze the first membrane‐committed step in the biosynthesis of bacterial glycoconjugates. MonoPGTs show a strong preference for their cognate nucleotide diphospho‐sugar (NDP‐sugar) substrates. However, despite extensive characterization of the monoPGT...
Preprint
Full-text available
Complex glycans serve important functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked...
Article
Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of...
Preprint
Full-text available
The Bacillus subtilis extracellular biofilm matrix includes an exopolysaccharide that is critical for the architecture and function of the community. To date, our understanding of the biosynthetic machinery and the molecular composition of the exopolysaccharide of B. subtilis remains unclear and incomplete. This report presents synergistic biochemi...
Article
Glycan-binding proteins (GBPs) are widely used reagents for basic research and clinical applications. These reagents allow for the identification and manipulation of glycan determinants without specialized equipment or time-consuming experimental methods. Existing GBPs, mainly antibodies and lectins, are limited, and discovery or creation of reagen...
Article
Monotopic phosphoglycosyl transferase enzymes (monoPGTs) initiate the assembly of prokaryotic glycoconjugates essential for bacterial survival and proliferation. MonoPGTs belong to an expansive superfamily with a diverse and richly annotated sequence space; however, the biochemical roles of most monoPGTs in glycoconjugate biosynthesis pathways rema...
Article
Phosphoglycosyl transferases (PGTs) play a pivotal role at the inception of complex glycoconjugate biosynthesis pathways across all domains of life. PGTs promote the first membrane-committed step in the en bloc biosynthetic strategy by catalyzing the transfer of a phospho-sugar from a nucleoside diphospho-sugar to a membrane-resident polyprenol pho...
Article
Significance Glycoconjugates and glycopolymers are involved in critical and varied biological functions across domains of life. Many of these complex molecules are biosynthesized via multistep membrane-associated pathways initiated through the action of phosphoglycosyl transferases (PGTs) and propagated by the collective action of glycosyl transfer...
Article
The influences of glycans impact all biological processes, disease states, and pathogenic interactions. Glycan-binding proteins (GBPs), such as lectins, are decisive tools for interrogating glycan structure and function because of their ease of use and ability to selectively bind defined carbohydrate epitopes and glycosidic linkages. GBP reagents a...
Article
Bacterial virulence and viability are strongly dependent on extracellular glycoconjugates including lipopolysaccharide (LPS), capsular polysaccharide (CPS), wall teichoic acid (WTA) and glycoproteins. While these glycoconjugates are diverse in structure and function, they share a common biosynthetic logic. For example, in Gram‐negative bacteria the...
Article
In humans, a collection of diverse proteins known as lectins are responsible for binding to glycan epitopes. Recognition of extracellular glycan epitopes represents a key facet of the innate immune response in eukaryotes. These epitopes can be used to distinguish cell types, self‐from non‐self, and perhaps even pathogenic from commensal bacteria. U...
Article
An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the N-linked protein glycosylation pathway in Campylobacter jejuni pathogenicity. The gut-immune co-culture (GIC) was developed to model important aspects of the human...
Article
We report the application of lanthanide-binding tags (LBTs) for two- and three-dimensional X-ray imaging of individual proteins in cells with a sub-15 nm beam. The method combines encoded LBTs, which are tags of minimal size (ca 15-20 amino acids) affording high-affinity lanthanide ion binding, and X-ray Fluorescence Microscopy (XFM). This approach...
Article
Full-text available
High‐throughput small‐molecule screening in drug discovery processes commonly rely on fluorescence‐based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false‐negative rates and background signals, or might invol...
Article
Biological membranes are complex barriers in which membrane proteins and thousands of lipidic species participate in structural and functional interactions. Developing a strategic approach that allows uniform labeling of membrane proteins while maintaining a lipidic environment that retains functional interactions is highly desirable for in vitro f...
Article
Long-chain polyprenol phosphates feature in membrane-associated glycoconjugate biosynthesis pathways across domains of life. These unique amphiphilic molecules are best known as substrates of polytopic membrane proteins, including polyprenol-phosphate phosphoglycosyl and glycosyl transferases, and as components of more complex substrates. The linea...
Preprint
An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the Nlinked protein glycosylation (Pgl) pathway in Campylobacter jejuni pathogenicity. The gutimmune co-culture (GIC) was developed to model important aspects of the hu...
Article
Peripheral and integral membrane proteins feature in stepwise assembly of complex glycans and glycoconjugates. Catalysis on membrane-bound substrates features challenges with substrate solubility and active-site accessibility. However, advantages in enzyme and substrate orientation and control of lateral membrane diffusion provide order to the mult...
Article
Monotopic membrane proteins, classified by topology, are proteins that embed into a single face of the membrane. These proteins are generally underrepresented in the Protein Data Bank (PDB), but the past decade of research has revealed new examples that allow the description of generalizable features. This Opinion article summarizes shared characte...
Article
Full-text available
Monotopic membrane proteins integrate into the lipid bilayer via reentrant hydrophobic domains that enter and exit on a single face of the membrane. Whereas many membrane-spanning proteins have been structurally characterized and transmembrane topologies can be predicted computationally, relatively little is known about the determinants of membrane...
Data
Corresponding key residues in C. jejuni and C. concisus PglC
Data
Results of covariance analyses performed on sequences in Table 1 using the GREMLIN web-server (http://gremlin.bakerlab.org/). Interactions between residues of the hydrophobic and globular domains are highlighted in red (>90% probability) and orange (80–90% probability).
Data
Primers used for cloning and mutagenesis of PglC and LpxM variants
Article
The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity...
Article
Introduction: Protein kinases are a diverse group of 518 enzymes whose dysregulation lies at the center of many diseases across therapeutic areas and especially oncology. Currently, 30% of all drug development efforts are focused on protein kinases. Although many drugs are approved or in clinical trials, these are predominantly ATP-competitive inhi...
Article
Full-text available
Polyprenol phosphate phosphoglycosyl transferases (PGTs) catalyze the first membrane-committed step in assembly of essential glycoconjugates. Currently there is no structure-function information to describe how monotopic PGTs coordinate the reaction between membrane-embedded and soluble substrates. We describe the structure and mode of membrane ass...
Article
The human enteropathogen Campylobacter jejuni, like many bacteria, employs siderophores such as enterobactin for cellular uptake of ferric iron. This transport process has been shown to be essential for virulence and presents an attractive opportunity for further study of the permissiveness of this pathway to small-molecule intervention and as insp...
Article
Phosphoglycosyl transferases (PGTs) initiate the biosynthesis of both essential and virulence-associated bacterial glycoconjugates including lipopolysaccharide, peptidoglycan and glycoproteins. PGTs catalyze the transfer of a phosphosugar moiety from a nucleoside diphosphate sugar to a polyprenol phosphate, to form a membrane-bound polyprenol dipho...
Chapter
This method describes the chemoenzymatic synthesis of several nucleotide sugars, which are essential substrates in the biosynthesis of prokaryotic N- and O-linked glycoproteins. Protein glycosylation is now known to be widespread in prokaryotes and proceeds via sequential action of several enzymes, utilizing both common and modified prokaryote-spec...
Article
Full-text available
Encodable lanthanide binding tags (LBTs) have become an attractive tool in modern structural biology as they can be expressed as fusion proteins of targets of choice. Previously, we have demonstrated the feasibility of inserting encodable LBTs into loop positions of interleukin-1β (Barthelmes et al. in J Am Chem Soc 133:808-819, 2011). Here, we inv...
Article
Introduction: Protein kinases are a diverse group of over 500 enzymes whose dysregulation lies at the center of many human diseases, spanning all therapeutic areas. Oncology is the most active area, where 30% of all drug development efforts are focused on protein kinases. Although 30 drugs have been approved by the FDA, and another 120 are in clini...
Article
Full-text available
Significance Escalating antibiotic resistance has stimulated interest in understanding the mechanisms of phosphoglycosyl transferases (PGTs) that initiate glycoconjugate biosynthesis and are implicated in bacterial survival and pathogenicity. This study provides compelling evidence that the action of PglC, a prototypic dual domain PGT, proceeds thr...
Article
Full-text available
Scientific Reports 6 : Article number: 39430; 10.1038/srep39430 published online: 23 December 2016 ; updated: 22 March 2017 . The original version of this Article contained typographical errors in the Abstract.
Article
Full-text available
The glycoproteins of selected microbial pathogens often include highly modified carbohydrates such as 2,4-diacetamidobacillosamine (diNAcBac). These glycoconjugates are involved in host cell interactions and may be associated with the virulence of medically-significant Gram-negative bacteria. In light of genetic studies demonstrating the attenuated...
Article
Full-text available
In autoimmune diseases, there have been proposals that exogenous “molecular triggers”, i.e., specific this should be ‘non-self antigens’ accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of the multiple sclerosis (MS) remains unclear. However, epidemiologic data sugges...
Article
Full-text available
Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these en...
Article
Full-text available
Site selectivity of protein N-linked glycosylation is dependent on many factors, including accessibility of the modification site, amino acids that make up the glycosylation consensus sequence and cellular localization of target proteins. Previous studies have shown that the bacterial oligosaccharyltransferase, PglB, of Campylobacter jejuni favors...
Article
Quantifying protein location and concentration is critical for understanding function in situ. SuCESsFul biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can in principle detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical scr...
Article
Campylobacter jejuni is a major cause of gastroenteritis, often resulting in death of the young, elderly and immunocompromised. Campylobacteritis also may result in complications leading to autoimmune and neurological disorders such as Guillain‐Barre syndrome. In this organism, a consistent structure‐function relationship has emerged in recent year...
Article
Integral membrane proteins play essential roles in all living systems; however, major technical hurdles challenge analyses of this class of proteins. Biophysical approaches that provide structural information to complement and leverage experimentally-determined and computationally-predicted structures are urgently needed. Herein we present the appl...
Article
Phosphoglycosyltransferases (PGTs) represent "gatekeeper" enzymes in complex glycan assembly pathways by catalyzing transfer of a phosphosugar from an activated nucleotide diphosphosugar to a membrane-resident polyprenol phosphate. The unique structures of selected nucleoside antibiotics, such as tunicamycin and mureidomycin A, which are known to i...
Article
Phosphoglycosyltransferases (PGTs) catalyze the transfer of a C1'-phosphosugar from a soluble sugar nucleotide diphosphate to a polyprenol-phosphate. These enzymes act at the membrane interface, forming the first membrane-associated intermediates in the biosynthesis of cell-surface glycans and glycoconjugates including glycoproteins, glycolipids an...
Article
The cell surfaces of bacteria are replete with diverse glycoconjugates that play pivotal roles in determining how bacteria interact with the environment and the hosts that they colonize. Studies to advance our understanding of these interactions rely on the availability of chemically defined glycoconjugates in a form that can be selectively modifie...
Article
We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1β (IL1β) and measured (1)H, (15)N-pseudocontact shifts (PCSs) by NM...
Article
Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic post-grafting approach is presented. Sortase-mediated li...
Article
Full-text available
In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans cha...
Article
Full-text available
The display of cell-surface glycolipids and glycoproteins is essential for the motility, adhesion and colonization of pathogenic bacteria such as Campylobacter jejuni. Recently, the cell-surface display of C. jejuni glycoconjugates has been the focus of considerable attention, however, our understanding of the roles that glycosylation plays in bact...
Article
PglB, a monomeric membrane-bound protein, is the oligosacchryltransferase (OTase) of the N-linked glycosylation system from the gram-negative bacterium Campylobacter jejuni (C. jejuni). PglB is a homologue of the Stt3p subunit of the multimeric eukaryotic OTase and represents a simplified model of this highly conserved enzymatic process. OTases tra...
Article
Consider the lanthanide metals, comprising lanthanum through lutetium. Lanthanides form stable cations with a +3 charge, and these ions exhibit a variety of useful physical properties (long-lifetime luminescence, paramagnetism, anomalous X-ray scattering) that are amenable to studies of biomolecules. The absence of lanthanide ions in living systems...
Article
A major challenge in the in vitro organogenesis field is to create microscale tissues with intercalated vascular networks - similar challenges exist in virtually every tissue type as blood and lymph networks must productively coexist while remaining physically separated by the intervening tissue cells. We address the important challenge of how to c...
Article
Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation-sensitive amino acid, termed CSox, to afford kinase-selective biosensors capable of reporting on enzymatic act...
Article
Reagentless biosensors rely on the interaction of a binding partner and its target to generate a change in fluorescent signal using an environment sensitive fluorophore or Förster Resonance Energy Transfer. Binding affinity can exert a significant influence on both the equilibrium and the dynamic response characteristics of such a biosensor. We her...
Article
Accurate and quantitative methods for measuring the dynamic fluctuations of protein kinase activities are critically needed as diagnostic tools and for the evaluation of kinase-targeted inhibitors, which represent a major therapeutic development area in the treatment of cancer and other diseases. In particular, rapid and economical methods that uti...