Overview
Research in the Imperiali group is concerned with diverse aspects of protein structure, function and design. One area of investigation focuses on understanding the molecular details of the enzyme-catalyzed process of asparagine-linked protein glycosylation. A second program focuses on the development of new chemical and biochemical tools that can be implemented for the investigation of complex biochemical processes. Our approaches to these research areas at the interface between chemistry and biology are highly interdisciplinary and integrate organic synthesis, state-of-the-art spectroscopic analysis, enzymology, protein biochemistry and molecular biology.

 

Research Summary
In one area of research we are investigating the central biological process of enzyme catalyzed protein glycosylation. This objective presents a significant challenge because the substrates for these transformations are densely functionalized proteins, and the opportunities for competing processes are innumerable. The investigation of these complex processes has demanded highly interdisciplinary approach. The transformation that we have studied in the most depth is asparagine-linked glycosylation. N-linked glycosylation, involves the enzyme-catalyzed modification of an asparagine side chain in a nascent polypeptide with a triantennary tetradecasaccharide (GlcNAc₂Man₉Glc₃) moiety. This first committed step in the biosynthesis of N-linked glycoproteins is catalyzed by oligosaccharyl transferase (OT), a heteromeric membrane-associated enzyme complex, found in the lumen of the endoplasmic reticulum (ER) of eukaryotic cells. Glycosyl transfer to the nitrogen of the carboxamide side chain of an asparagine occurs through the intermediacy of a dolichol-linked pyrophosphate donor. The primary peptide sequence requirements for the glycosylation process include a minimum -Asn-Xaa-Ser/Thr- tripeptide recognition motif where Xaa. Thus, while OT exhibits rather simple substrate requirements, the enzyme catalyzes an unusual and specific reaction wherein the nucleophilicity of the asparagine side chain must be greatly enhanced in order to form a covalent linkage with the oligosaccharide. After transfer of the initial tetradecasaccharide, subsequent diversification of the primary glycoprotein conjugates arises from enzyme-catalyzed processing steps that occur in both the ER and Golgi apparatus. This eukaryotic process is essential for the structure and function of numerous proteins. We have developed synthetic probes and biophysical methods to gain insight on the mechanism and biochemical consequences of N-linked glycosylation. For example, we are currently investigating the conformational implications of N-linked glycosylation in several experimental systems including the toxin binding loop in the nicotinic acetylcholine receptor (AChR) family, glycoconjugates representing key turn-forming sequences in the viral hemagglutinin protein, and the glycosylated region of the soluble prion precursor protein [PrP(121-231)]. In addition, a major effort is being devoted to the cloning and heterologous expression of the oligosaccharyl transferase subunits. A significant long-term goal in these studies is the reconstitution of the catalytically active oligosaccharyl transferase complex. The knowledge of the essential subunits in the enzyme will allow for the manipulation of the protein function through site-directed mutagenesis and semi-synthetic methods. Furthermore, increased supplies of the enzyme will enable structural characterization of the protein and the development of new mechanistic insight.

A second area of research focuses on the development of chemical tools to probe complex biochemical processes. For example, we have used chemical and biochemical parallel synthesis strategies for the discovery of lanthanide binding coexpression tags, which may be used as multi-tasking tools for structural and functional proteomics. The lanthanide binding tags (LBTs) comprise short peptide sequences (fewer than 20 amino acids) that are optimized to bind trivalent lanthanide (Ln³⁺) ions. Because the tags are built from encoded amino acids, LBTs can be introduced as co-expression tags at the DNA level to create fusion proteins. Post-expression addition of a particular Ln ion provides the “function” of the LBT. The LBT sequence imparts on the fusion protein a built-in and site-specific fluorophore that can be used for monitoring protein expression and purification, and for assaying protein/protein and protein/ligand interactions. Lanthanides also provide excellent X-ray scattering power and powerful paramagnetic effects, therefore the tags can be implemented in numerous applications in structural and functional proteomics.

New strategies including the preparation of synthetic and semi-synthetic protein probes for interrogating the specific function of proteins involved in directed cell migration and cell cycle control are also being developed. Due to the essential signaling roles played by intracellular kinase-mediated phosphorylation in key cellular processes, the focus of these studies is on protein kinases as key protein targets. In particular, we have developed peptide and protein probes that will enable us to define the spatial and temporal characteristics of protein kinases and phosphoprotein mediators in complex cellular pathways. These probes include both environment-sensitive fluorophores for interrogating phosphorylation-dependent protein-protein interactions and caged phosphoamino acids for examining phosphorylation-mediated cellular functions with spatial and temporal control.

 

Selected Publications
In situ Photoactivation of a Caged Phosphotyrosine Peptide Derived from FAK Temporarily Halts Lamellar Extension of Single Migrating Tumor Cells David Humphrey, D.; Rajfur, Z.; Vazquez, M. E.; Scheswohl, D.; Schaller, M. D.; Jacobson, K.; Imperiali, B. J. Biol. Chem. 2005, 280, 22091-22101.

Photophysics and Biological Applications of the Environment-Sensitive Fluorophore 6-N,N-Dimethylamino-2,3-Naphthalimide Vázquez,  M. E.; Canosa,  J. B. B.; Imperiali, B. J. Am. Chem. Soc. 2005, 127, 1300-1306.

A Multiplexed Homogenous Fluorescence-Based Assay for Protein Kinase Activity in Cell Lysates, Shults, M. D.; Janes, K. A.; Lauffenburger, D. A.; Imperiali, B. Nature Methods, 2005, 2, 277-284.

Caged Phosphoproteins, Rothman, D. M.; Petersson, E. J.; Vázquez, M. E.; Brandt, G. S.; Dougherty, D. A.; Imperiali, B. J. Am. Chem. Soc. 2005, 127, 846-847

Structural Origin of the High Affinity of a Chemically Evolved Lanthanide Binding Peptide, Nitz, M.; Sherawat, M.; Franz, K. J.; Peisach, E.; Allen, K. N.; Imperiali, B. Angew. Chem. Int. Ed. 2004, 43, 3682-3685.

Caged Phosphopeptides Reveal a Temporal Role for 14-3-3 in G1 Arrest and S-Phase Checkpoint Function, Nguyen, A.; Rothman, D. M.; Stehn, J.; Imperiali, B.; Yaffe, M. B.  Nature Biotechnology, 2004, 22, 993-1000.

The Interplay of Glycosylation and Disulfide Formation Influences Fibrillization in a Prion Protein Fragment, Bosques, C. J.; Imperiali, B. Proc. Natl. Acad. Sci. USA 2003, 100, 7593-7598.

Oligosaccharyl Transferase: Gatekeeper to the Secretory Pathway, Dempski, R. E. Jr.; Imperiali, B. Curr. Op. Chem. Biol. 2002, 6, 844-850.

Search PubMed for Imperiali lab publications

Click image to view larger, plus legend.