About

Barbara Imperiali is the Class of 1922 Professor of Chemistry and Biology at MIT. Her research group employs a multidisciplinary approach involving synthesis, state-of-the-art spectroscopy, molecular modeling, enzymology, and molecular biology to address fundamental problems at the interface of chemistry and biology. A primary focus of her work is on protein structure, function, and design, with particular emphasis on understanding enzyme-catalyzed protein glycosylation, especially N-linked glycosylation. Her group investigates the enzymatic processes involved in glycosylation, including the assembly of glycosyl donors and the action of oligosaccharyl transferase (OTase), aiming to develop inhibitors to probe glycosylation roles in pathogenic bacteria and to analyze active OTases from prokaryotic sources. Additionally, her research involves designing and synthesizing chemical tools such as fluorescent and luminescent probes to study complex biological systems, with a focus on protein kinases and signal transduction pathways. These efforts include developing probes for monitoring protein phosphorylation, protein-protein interactions, and cellular activities related to cell migration and cell cycle control. Her work ultimately aims to create chemical probes that elucidate the spatial and temporal dynamics of proteins in cellular pathways, contributing to a deeper understanding of biological processes at the molecular level.

Research topics

• Biochemistry
• Computational biology
• Genetics
• Chemistry
• Biology
• Crystallography
• Nanotechnology
• Optics
• Biophysics

Selected publications

• Detergent Exchange from Lipid Nanoparticles into Detergent Micelles Unlocks a Tool for Biochemical and Kinetic Characterization of Membrane Proteins

  Biochemistry · 2026-03-26

  articleCorresponding

  Bacterial membrane proteins make up ∼ 30% of the prokaryotic genome and play key roles in infection and virulence. Membrane protein chemistry has advanced in recent years, including purification strategies that mimic nativelike lipid environments, such as lipid nanoparticles, amphipols, and nanodiscs. The use of styrene maleic acid copolymers (SMALPs) to form a lipid nanoparticle has become increasingly common in membrane protein purification, especially for proteins which are not amenable to detergent extraction from the cellular membrane fraction. Yet, for some biochemical and biophysical methods, it is preferable to use detergent-solubilized protein. Here we show a general exchange screening method to transfer membrane proteins from lipid nanoparticles to detergent micelles while retaining protein fold, homogeneity, and function. Conditions were first optimized for copolymer dispersion and recovery into detergents, and analytical methods were employed to assess activity and quality of detergent-solubilized proteins. Thirteen protein targets were purified in copolymer based on a 16-polymer screen. This selection was followed by an eight-detergent screen in the presence of calcium and magnesium ions for optimal dissolution of the nanoparticle, producing detergent-stabilized protein. In all membrane proteins assessed, homogeneity and folding were retained from the initial purification in lipid nanoparticles through a detergent-exchange protocol. For membrane enzymes that have proven to be experimentally intractable when detergent solubilized, we were able to observe catalytic activity using the detergent-exchanged material. The use of this protocol to purify membrane proteins provides greater versatility for biochemical and kinetic characterization than was previously accessible.

  PublisherDOI

• Selection of nanobodies against liponanoparticle‐embedded membrane proteins by yeast‐surface display

  Protein Science · 2025-09-13 · 3 citations

  articleOpen accessCorresponding

  Abstract Single‐domain antibodies, known as nanobodies (Nbs), are widely used in structural biology, therapeutics, and as molecular probes in biology and biotechnology. Nbs towards soluble proteins are routinely developed via alpaca immunization or directed evolution in yeast cell‐surface display. However, for membrane proteins, the targets are generally detergent‐solubilized, and there remains a need for Nb development methods against membrane proteins in a native‐like membrane environment. To address this need, we present a protocol for Nb selection via extraction of membrane proteins into amphiphilic polymers such as those based on styrene‐maleic acid (SMA) to produce purified membrane proteins in stable liponanoparticles. Proof of generality is demonstrated by applying the pipeline to membrane‐resident enzymes of differing fold, oligomerization state, and membrane topology (reentrant membrane helix, transmembrane, membrane‐associated). Following screening for optimal stabilization into liponanoparticles, Nbs were selected against four target proteins from glycoconjugate biosynthesis pathways by yeast surface display. The selected Nbs showed high affinity and selectivity towards their target proteins with K D (apparent) values ranging from 15 to 200 nM, depending on the Nb–protein conjugate. In accordance with their tight binding, various Nb–protein complexes were found to be stable to size‐exclusion chromatography purification. The Nbs were also amenable to sortase‐mediated ligation, enabling their conversion into molecular probes for the target membrane protein. The ability to select for such high‐affinity Nb against membrane proteins in liponanoparticles based on SMA will facilitate their widespread application in cell biology and biomedical applications.

  PublisherOA PDFDOI

• Abstract 2343 Functional Characterization and Inhibitor Screening of the Monotopic Phosphoglycosyl Transferase Superfamily Using a Novel Protein-Purification-Free Approach for Antibiotic Drug Discovery

  Journal of Biological Chemistry · 2025-05-01

  articleOpen accessSenior author

  The rise of antibiotic resistance has intensified the need for new bacterial targets for therapeutic intervention. Monotopic phosphoglycosyl transferases (monoPGTs), a class of exclusively prokaryotic membrane proteins that remain poorly understood, represent promising targets for pharmacological intervention against human pathogens, including Campylobacter jejuni and Streptococcus pneumoniae. MonoPGTs play a key role in glycoconjugate synthesis, which is crucial for bacterial pathogenesis. These enzymes initiate glycan biosynthesis by transferring a sugar-phosphate from a soluble nucleotide diphosphate sugar to a polyprenol phosphate acceptor in the membrane.

  PublisherOA PDFDOI

• Detergent exchange from lipid nanoparticles into detergent micelles unlocks a tool for biochemical and kinetic characterization of membrane proteins

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-12-25

  articleOpen access

  Bacterial membrane proteins make up ~30% of the prokaryotic genome and play key roles in infection and virulence. Membrane protein chemistry has advanced in recent years, including purification strategies that mimic "native-like" lipid environments, such as lipid nanoparticles, amphipols, and nanodiscs. The use of styrene maleic acid co-polymers to form a lipid nanoparticle has become increasingly common in membrane protein purification, especially for proteins which are not amenable to detergent extraction from the cellular membrane fraction. Yet, for some biochemical and biophysical methods it is preferable to use detergent-solubilized protein. Here we show a general exchange method to transfer membrane proteins from lipid nanoparticles to detergent micelles while retaining protein fold, homogeneity and function. Conditions were first optimized for co-polymer dispersion and recovery into detergents, and analytical methods employed to assess activity and quality of detergent-solubilized proteins. Twelve protein targets were purified in co-polymer based on a 16-polymer screen. This selection was followed by an eight-detergent screen in the presence of calcium ions for optimal dissolution of the nanoparticle, producing detergent-stabilized protein. In all membrane proteins assessed, homogeneity and folding were retained from the initial purification in lipid nanoparticles through the detergent-exchange protocol. For membrane enzymes that have proven to be experimentally intractable once detergent solubilized, we were able to observe catalytic activity using the detergent-exchanged material. The use of this protocol to purify membrane proteins provides great versatility for biochemical and kinetic characterization than was previously accessible.

  PublisherDOI

• Selection of nanobodies against liponanoparticle-embedded membrane proteins by yeast surface display

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-05-23 · 2 citations

  preprintOpen access

  Abstract Single-domain antibodies, known as nanobodies (Nbs), are widely used in structural biology, therapeutics, and as molecular probes in biology and biotechnology. Nbs towards soluble proteins are routinely developed via alpaca immunization or directed evolution in yeast cell-surface display. However, for membrane proteins, the targets are generally detergent-solubilized, and there remains a need for Nb development methods against membrane proteins in a native-like membrane environment. To address this need, we present a protocol for Nb selection via extraction of membrane proteins into amphiphilic polymers such as styrene-maleic acid to produce purified membrane proteins in stable liponanoparticles. Proof of generality is demonstrated by applying the pipeline to four membrane-resident enzymes of differing fold, oligomerization state, and membrane topology (reentrant membrane helix, transmembrane, membrane-associated). Following screening for optimal stabilization into liponanoparticles, Nbs were selected against four target proteins from glycoconjugate biosynthesis pathways. The selected Nbs showed high affinity and selectivity towards their target proteins with K D apparent values ranging from 15 nM to 200 nM, depending on the Nb-protein conjugate. In accordance with their tight binding, various Nb-protein complexes were found to be stable to size-exclusion chromatography purification. The Nbs were also amenable to sortase-mediated ligation, enabling their conversion into molecular probes for the target membrane protein. The ability to select for such high-affinity Nb against membrane proteins in SMALP will facilitate their widespread application in cell biology and biomedical applications.

  PublisherOA PDFDOI

• Correlating membrane‐protein dynamics with function: Integrating bioinformatics, molecular dynamics, and single‐molecule <scp>FRET</scp>

  Protein Science · 2025-10-23 · 1 citations

  articleOpen accessSenior authorCorresponding

  We present a strategy that deploys structural bioinformatics, molecular simulation, and single-molecule Förster Resonance Energy Transfer (FRET) microscopy for observing the ligand-dependent conformational dynamics of integral membrane proteins in situ. We focus on representative members of the small monotopic phosphoglycosyl transferase (SmPGT) superfamily, which catalyze the transfer of a phosphosugar from a soluble nucleotide-sugar donor to a membrane-embedded polyprenol phosphate acceptor in the initiating step of glycoconjugate biosynthesis in prokaryotes. Substrate-specific structural features were identified across the superfamily and correlated with ligand-dependent conformational dynamics in all-atom simulations. To experimentally validate the role of this motion in ligand binding, we developed a platform to monitor intramolecular protein dynamics in a native-like lipid environment. The presented approach incorporates selective cysteine protein labeling and non-canonical amino acid mutagenesis with bicyclononyne-tetrazine click chemistry to assemble dual-labeled variants of PglC, the initiating enzyme of the N-linked protein glycosylation pathway from the Campylobacter genus. The modified proteins are solubilized in styrene-maleic acid liponanoparticles (SMALPs), which provide a model membrane environment. The conformational changes of PglC upon inhibitor binding correlate with inhibitor potency. The single-molecule FRET-SMALP strategy can be adapted to investigate protein dynamics across the superfamily of SmPGTs with different substrate selectivity, where structure prediction and molecular dynamics support significant conformational changes upon ligand binding.

  PublisherOA PDFDOI

• Tools for investigating host-microbe crosstalk using glycan analysis probes inspired by human lectins

  Glycobiology · 2025-05-27 · 1 citations

  articleOpen accessSenior author

  Human lectins are critical carbohydrate-binding proteins that recognize diverse glycoconjugates from microorganisms and can play a key role in host-microbe interactions. Despite their importance in immune recognition and microbe binding, the specific glycan ligands and functions of many human lectins remain poorly understood. Using previous proof-of-concept studies on selected lectins as the foundation for this work, we present ten additional glycan analysis probes (GAPs) from a diverse set of human soluble lectins, offering robust tools to investigate glycan-mediated interactions. We describe a protein engineering platform that enables scalable production of GAPs that maintain native-like conformations and oligomerization states, equipped with functional reporter tags for targeted glycan profiling. We demonstrate that the soluble GAP reagents can be used in various applications, including glycan array analysis, mucin-binding assays, tissue staining, and microbe binding in complex populations. These capabilities make GAPs valuable for dissecting interactions relevant to understanding host responses to microbes. The tools can also be used to probe differential microbial and mammalian glycan interactions, which are crucial for understanding the interactions of lectins in a physiological environment where both glycan types exist. GAPs have potential as diagnostic and prognostic tools for detecting glycan alterations in chronic diseases, microbial dysbiosis, and immune-related conditions.

  PublisherOA PDFDOI

• Abstract 2424 Defining structure and function of GT-A fold enzymes in bacterial glycan assembly

  Journal of Biological Chemistry · 2025-05-01

  articleOpen access

  Bacterial glycoconjugates mediate vital bacterial pathogen– host interactions and are also essential for bacterial survival. The machinery involved in bacterial glycoconjugate biosynthesis comprises an emerging set of antibiotic targets. The glycosyltransferase PglI attaches a branching glycan to the undecaprenyl phosphate-linked glycopolymer substrate. The branching position of the sugar varies widely amongst the different species of Campylobacter and the mode of action and structural determinants of PglI specificity are not known.

  PublisherOA PDFDOI

• Correlating membrane-protein dynamics with function: Integrating bioinformatics, molecular dynamics, and single-molecule FRET

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-05

  preprintOpen accessSenior authorCorresponding

  Abstract We present a strategy that deploys structural bioinformatics, molecular simulation, and single-molecule FRET microscopy for observing the ligand-dependent conformational dynamics of integral membrane proteins in situ. We focus on representative members of the small monotopic phosphoglycosyl transferase (SmPGT) superfamily, which catalyze transfer of a phosphosugar from a soluble nucleotide-sugar donor to a membrane-embedded polyprenol phosphate acceptor in the initiating step of glycoconjugate biosynthesis in prokaryotes. Substrate-specific structural features were identified across the superfamily and correlated with ligand-dependent conformational dynamics in all-atom simulations. To experimentally validate the role of this motion in ligand binding, we developed a platform to monitor intra-molecular protein dynamics in a native-like lipid environment. The presented approach incorporates selective cysteine protein labeling and non-canonical amino acid mutagenesis with bicyclononyne-tetrazine click chemistry to assemble dual-labeled variants of PglC, the initiating enzyme of the N-linked protein glycosylation pathway from Campylobacter jejuni. The modified proteins are then solubilized into styrene maleic acid liponanoparticles (SMALPs) to maintain an in situ membrane environment. The conformational changes of PglC upon inhibitor binding are diagnostic of inhibitor potency. The single-molecule FRET-SMALP strategy can be adapted to investigate protein dynamics across the superfamily of SmPGTs with different substrate selectivity where structure prediction and molecular dynamics support significant conformational changes upon ligand binding. Broader Impact Membrane protein structure-function relationships are critical for understanding fundamental biological processes and for the development of small-molecule drug treatments. Bacterial glycoconjugate biosynthesis pathways are a promising target for strain-specific antibiotics and exemplify the challenges of characterizing biomolecular systems that depend on highly specific protein, lipid, and carbohydrate chemistries. We integrate molecular simulation, structural bioinformatics, and single-molecule FRET to elucidate details of small-molecule binding to the PGT superfamily.

  PublisherOA PDFDOI

• Proteome-Wide Bioinformatic Annotation and Functional Validation of the Monotopic Phosphoglycosyl Transferase Superfamily

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-07-11 · 2 citations

  preprintOpen accessSenior authorCorresponding

  Abstract 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 enzymes high-value targets for biochemical investigation. In particular, the small monotopic PGT family is exclusively bacterial and represents the minimal functional unit of the monotopic PGT superfamily. Here, we combine a sequence similarity network (SSN) analysis with a generalizable, luminescence-based activity assay to probe the substrate specificity of this family of monoPGTs in a bacterial cell-membrane fraction. This strategy allows us to identify specificity on a far more significant scale than previously achievable and correlate preferred substrate specificities with predicted structural differences within the conserved monoPGT fold. Finally, we present the proof-of-concept for a small-scale inhibitor screen (eight nucleoside analogs) with four monoPGTs of diverse substrate specificity, thus building a foundation for future inhibitor discovery initiatives. Significance Uncovering the function and specificity of enzymes responsible for glycoconjugate biosynthesis traditionally requires a multi-faceted and individually curated approach. This is especially true for bacterial glycoconjugates due to greater monosaccharide diversity and a paucity of established structural information. Here we leverage bioinformatic and in-vitro tools to predict and validate substrate specificity for a unique, exclusively bacterial family of enzymes responsible for the first step in many of these glycan assembly pathways. We further show that this platform is suitable for enhanced functional annotation and inhibitor testing, paving the way for the development of urgently needed antibiotics.

  PublisherOA PDFDOI

Recent grants

• Development of multifunctional probes for profiling microbial glycans

  NIH · $1.4M · 2018–2022

• NIH Grant R01GM097241

  NIH · $1.8M · 2019

• NIH Grant R29GM039334

  NIH · $489k · 1993

• NIH Grant R01GM058716

  NIH · $486k · 2003

• Lanthanide Binding Tags: New Chemical Tools for Proteomics

  NSF · $1.6M · 2003–2008

Frequent coauthors

• Karen N. Allen

  53 shared

• Greg J. Dodge

  Massachusetts Institute of Technology

  33 shared

• Mark M. Chen

  Massachusetts Institute of Technology

  31 shared

• Harald Schwalbe

  Goethe University Frankfurt

  27 shared

• Christopher W. Reid

  Bryant University

  25 shared

• Jacek Stupak

  National Research Council Canada

  25 shared

• Langdon J. Martin

  Warren Wilson College

  22 shared

• Alyssa J. Anderson

  Massachusetts Institute of Technology

  22 shared

Education

• PhD, Chemistry

  Massachusetts Institute of Technology

  1983

• BSc (Hon. First class) Medicinal Chemistry, Chemistry

  University College London

  1979