Barbara Imperiali

Effect of N-linked glycosylation on glycopeptide and glycoprotein structure

Asparagine-linked glycosylation is an enzyme-catalyzed, co-translational protein modification reaction that has the capacity to influence either the protein folding process or the stability of the native glycoprotein conjugate. Advances in both glycoconjugate chemical synthesis and glycoprotein expression methods have increased the availability of these once elusive biopolymers. The application of spectroscopic methods to these proteins has begun to illuminate the various ways in which the saccharide affects the structure, function and stability of the proteins.

A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates

New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase–associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.

Modulation of protein structure and function by asparagine-linked glycosylation

In eukaryotic cells, many enzymes are devoted to the construction of the complex glycan structures that decorate secreted and cell-surface proteins. Recent studies have begun to elucidate the effects of asparagine-linked glycosylation on protein folding and on the structure and function of mature glycoproteins.

The Expanding Horizons of Asparagine-Linked Glycosylation

Asparagine-linked glycosylation involves the sequential assembly of an oligosaccharide onto a polyisoprenyl donor, followed by the en bloc transfer of the glycan to particular asparagine residues within acceptor proteins. These N-linked glycans play a critical role in a wide variety of biological processes, such as protein folding, cellular targeting and motility, and the immune response. In the past decade, research in the field of N-linked glycosylation has achieved major advances, including the discovery of new carbohydrate modifications, the biochemical characterization of the enzymes involved in glycan assembly, and the determination of the biological impact of these glycans on target proteins. It is now firmly established that this enzyme-catalyzed modification occurs in all three domains of life. However, despite similarities in the overall logic of N-linked glycoprotein biosynthesis among the three kingdoms, the structures of the appended glycans are markedly different and thus influence the functions of elaborated proteins in various ways. Though nearly all eukaryotes produce the same nascent tetradecasaccharide (Glc(3)Man(9)GlcNAc(2)), heterogeneity is introduced into this glycan structure after it is transferred to the protein through a complex series of glycosyl trimming and addition steps. In contrast, bacteria and archaea display diversity within their N-linked glycan structures through the use of unique monosaccharide building blocks during the assembly process. In this review, recent progress toward gaining a deeper biochemical understanding of this modification across all three kingdoms will be summarized. In addition, a brief overview of the role of N-linked glycosylation in viruses will also be presented.

A Powerful Combinatorial Screen to Identify High‐Affinity Terbium(III)‐Binding Peptides

Lanthanide‐binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb3+ ions and intense luminescence properties. This methodology utilizes solid‐phase split‐and‐pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two‐step screening procedure. The initial screen avoids the interference caused by on‐bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aqueous solution. Employment of this methodology on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140‐fold increase in affinity (57 nM dissociation constant, KD) over previously reported lanthanide‐binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2‐nM apparent dissociation constant for Tb3+ ions.

Lanthanide-Binding Tags as Versatile Protein Coexpression Probes

Comprehensive proteomic analyses require new methodologies to accelerate the correlation of gene sequence with protein function. Key tools for such efforts include biophysical probes that integrate into the covalent architecture of proteins. Lanthanide‐binding tags (LBTs) are expressible, multitasking fusion partners that are optimized to bind lanthanide ions and have several desirable attributes, which include long‐lived luminescence, excellent X‐ray scattering power for phase determination, and magnetic properties to facilitate NMR spectroscopic structure elucidation. Herein, we present peptide sequences with a 40‐fold higher affinity for Tb3+ ions and significantly brighter luminescence intensity compared with existing peptides. Incorporation of an LBT onto ubiquitin as a prototype fusion protein allows the use of powerful protein‐visualization techniques, which include rapid luminescence detection of LBT‐tagged proteins in SDS‐PAGE gels, as well as determination of protein concentrations in complex mixtures. The LBT strategy is a new alternative for expressing fluorescent fusion proteins by routine molecular biological techniques.

Engineering Encodable Lanthanide-Binding Tags into Loop Regions of Proteins

Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β (IL1β) and varied the length of the spacer between the LBT and the protein (denoted 1−3). Luminescence studies demonstrate that all nine constructs bind Tb3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing the 1H−15N heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modeling.

Structure determination of a Galectin‐3–carbohydrate complex using paramagnetism‐based NMR constraints

The determination of the location and conformation of a natural ligand bound to a protein receptor is often a first step in the rational design of molecules that can modulate receptor function. NMR observables, including NOEs, often provide the basis for these determinations. However, when ligands are carbohydrates, interactions mediated by extensive hydrogen‐bonding networks often reduce or eliminate NOEs between ligand and protein protons. In these cases, it is useful to look to other distance‐ and orientation‐dependent observables that can constrain the geometry of ligand–protein complexes. Here we illustrate the use of paramagnetism‐based NMR constraints, including pseudo‐contact shifts (PCS) and field‐induced residual dipolar couplings (RDCs). When a paramagnetic center can be attached to the protein, field‐induced RDCs and PCS reflect only bound‐state properties of the ligand, even when averages over small fractions of bound states and large fractions of free states are observed. The effects can also be observed over a long range, making it possible to attach a paramagnetic center to a remote part of the protein. The system studied here is a Galectin‐3–lactose complex. A lanthanide‐binding peptide showing minimal flexibility with respect to the protein was integrated into the C terminus of an expression construct for the Galectin‐3–carbohydrate‐binding domain. Dysprosium ion, which has a large magnetic susceptibility anisotropy, was complexed to the peptide, making it possible to observe both PCSs and field‐induced RDCs for the protein and the ligand. The structure determined from these constraints shows agreement with a crystal structure of a Galectin‐3–N‐acetyllactosamine complex.

Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function

Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.

Lanthanide-tagged proteins — an illuminating partnership

Lanthanide-tagged proteins are valuable for exploiting the unique properties of Ln ions for investigating protein structure, function, and dynamics. Introduction of the Ln into the target is accomplished via chemical modification with synthetic lanthanide-chelating prosthetic groups or by coexpression with peptide-based binding tags. Complexed Ln-tags offer a heavy-atom site for solving the phase problem in X-ray crystallography. In NMR, paramagnetic lanthanide ions induce residual dipolar couplings and pseudo-contact shifts that yield valuable distance constraints for structural analysis. Lanthanide luminescence-based techniques and Ln-tagged proteins are valuable for investigating the functions and dynamics of large proteins and protein complexes and have been applied in vivo. Overall, the reach of Ln-tagged proteins will increase our ability to understand cellular functions on the molecular level.