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Biomedical subjects

Barbara Imperiali

Publications and source records attributed to Barbara Imperiali.

At least 37 records · Page 2Linked to original sources
PubMed2005

Chemical approaches for investigating phosphorylation in signal transduction networks.

The power and scope of chemical synthesis offer considerable opportunities to broaden the lexicon of chemical tools that can be implemented for the study of complex biological systems. To investigate individual signaling proteins and pathways, chemical tools provide a powerful complement to existing genetic, chemical genetic and immunologic methods. In particular, understanding phosphorylation-mediated signaling in real time yields important information about the regulation of cellular function and insights into the origin of disease. Recent advances in the development of photolabile caged analogs of bioactive species and fluorescence-based sensors of protein kinase activities are useful for investigating protein phosphorylation and the roles of phosphoproteins. Photolabile caged analogs allow spatial and temporal control over the release of a compound, while fluorescence-based sensors allow the real-time visualization of kinase activity. Here, we discuss recent advances that have increased the specificity and availability of these tools.

Animals↗
PubMed2004

Heterologous expression and biophysical characterization of soluble oligosaccharyl transferase subunits.

Oligosaccharyl transferase (OT) catalyzes the first committed step in N-linked protein glycosylation, a co-translational process that occurs in the lumen of the endoplasmic reticulum. The yeast Saccharomyces cerevisiae enzyme complex comprises nine integral membrane proteins, five of which are essential for catalysis. Due to the challenges with purifying the active enzyme complex for detailed biophysical studies, a systematic study to express, isolate, and characterize the soluble domains of three of the largest subunits in the complex (Nlt1p, Wbp1p, and Swp1p) is reported. The proteins are expressed using the lytic baculovirus expression system and the new constructs are well behaved, monomeric in solution, and glycosylated. Two of the proteins interact with each other as seen by gel filtration and circular dichroism. This study provides a framework to study the roles of these three essential subunits of the eukaryotic OT complex.

Baculoviridae↗
PubMed2004

X-ray structure analysis of a designed oligomeric miniprotein reveals a discrete quaternary architecture.

The x-ray crystal structure of an oligomeric miniprotein has been determined to a 1.2-A resolution by means of multiwavelength anomalous diffraction phasing with selenomethionine analogs that retain the biophysical characteristics of the native peptide. Peptide 1, comprising alpha and beta secondary structure elements with only 21 aa per monomer, associates as a discrete tetramer. The peptide adopts a previously uncharacterized quaternary structure in which alpha and beta components interact to form a tightly packed and well defined hydrophobic core. The structure provides insight into the origins of the unusual thermal stability of the oligomer. The miniprotein shares many characteristics of larger proteins, including cooperative folding, lack of 1-anilino-8-naphthalene sulfonate binding, and limited deuterium exchange, and possesses a buried surface area typical of native proteins.

Amino Acid Sequence↗
PubMed2004

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.

14-3-3 Proteins↗
PubMed2004

Effects of glycosylation on peptide conformation: a synergistic experimental and computational study.

Asparagine-linked glycosylation, the co-translational covalent attachment of carbohydrates to asparagine side chains, has a major effect on the folding, stability, and function of many proteins. The carbohydrate composition in mature glycoproteins is heterogeneous due to modification of the initial oligosaccharide by glycosidases and glycosyltransferases during the glycoprotein passage through the endoplasmic reticulum and Golgi apparatus. Despite the diversity of carbohydrate structures, the core beta-D-(GlcNAc)(2) remains conserved in all N-linked glycoproteins. Previously, results from our laboratory showed that the molecular composition of the core disaccharide has a critical and unique conformational effect on the peptide backbone. Herein, we employ a synergistic experimental and computational approach to study the effect of the stereochemistry of the carbohydrate--peptide linkage on glycopeptide structure. A glycopeptide derived from a hemagglutinin protein fragment was synthesized, with the carbohydrate attached to the peptide with an alpha-linked stereochemistry. Computational and biophysical analyses reveal that the conformations of the peptide and alpha- and beta-linked glycopeptides are uniquely influenced by the attached saccharide. The value of computational approaches for probing the influence of attached saccharides on polypeptide conformation is highlighted.

Biophysical Phenomena↗
PubMed2003

Versatile fluorescence probes of protein kinase activity.

We introduce a versatile fluorescent peptide reporter of protein kinase activity. The probe can be modified to target a desired kinase by changing the kinase recognition motif in the peptide sequence. The reporter motif contains the Sox amino acid, which generates a fluorescence signal when bound to Mg2+ present in the reaction mixture. The phosphorylated peptide exhibits a much greater affinity for Mg2+ than its unphosphorylated analogue and, thus, a greater fluorescence intensity. Product formation during phosphorylation by the kinase is easily followed by the increase in fluorescence intensity over time. These probes exhibit a 3-5-fold increase in fluorescence intensity upon phosphorylation, the magnitude of which depends on the substrate. Peptides containing the reporter functionality are phosphorylated on serine by Protein Kinase C and cAMP-dependent protein kinase and are shown to be good substrates for these enzymes. The principle of this design extends to peptides phosphorylated on threonine and tyrosine.

Fluorescent Dyes↗
PubMed2003

Protein alignment by a coexpressed lanthanide-binding tag for the measurement of residual dipolar couplings.

A protein fusion construct of human ubiquitin with an N-terminal lanthanide binding tag (LBT) enables observation of long-range orientational restraints in solution NMR from residual dipolar couplings (RDCs) due to paramagnetic alignment of the protein. The paramagnetic lanthanide ions Tb3+, Dy3+, and Tm3+ are shown to bind to the LBT and induce different alignment tensors, in agreement with theory. RDCs, measured relative to the diamagnetic Lu3+, range from -7.6 to 5.5 Hz for Tb3+ and -6.6 to 6.1 Hz for Dy3+, while an opposite alignment tensor is observed for Tm3+ (4.5 to -2.9 Hz) at 800 MHz. Experimental RDCs are in excellent agreement with those predicted on the basis of the X-ray structure of the protein.

Humans↗
PubMed2003

Modular and tunable chemosensor scaffold for divalent zinc.

A modular peptide scaffold has been developed for fluorescent sensing of divalent zinc. The signaling component of the chemosensor is the chelation-sensitive fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline, which is prepared as the protected amino acid derivative Fmoc-Sox-OH and integrated into peptide sequences. Nineteen synthetic peptides incorporating the signaling element exhibit a range of affinities for Zn(2+) through variation of the type and number of Zn(2+) ligands, ligand arrangement and the beta-turn sequence that acts as a preorganization element between the ligands. The stoichiometry of the peptide-Zn(2+) complexes is evaluated by several criteria. The fluorescence response of these peptides to pH and various important metal ions is reported. Eleven of these sequences form only 1:1 complexes with Zn(2+) and their affinities range from 10 nM to nearly 1 microM. When used in concert, these sensors can provide Zn(2+) concentration information in a valuable range.

Amino Acids↗
PubMed2003

Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations.

The development of chemical probes for the investigation of the complex phosphorylation signaling cascades that regulate biological events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biologically active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the enviromentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process. The binding of the peptide to 14-3-3 places the fluorescent reporter unit into a hydrophobic pocket, which changes the fluorescent maximum emission intensity and wavelength. At the same time, the newly developed photolabile 1-(2-nitrophenyl)ethyl-caged phosphoserine allows control of the release of the biologically active ligand through unmasking of the key phosphoserine functionality upon UV irradiation.

14-3-3 Proteins↗
PubMed2003

Caged phospho-amino acid building blocks for solid-phase peptide synthesis.

Three 1-(2-nitrophenyl)ethyl-caged phospho-amino acids have been synthesized for use in standard N(alpha)-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis (SPPS). The most common naturally occurring phospho-amino acids, serine, threonine, and tyrosine, were prepared as protected caged building blocks by modification with a unique phosphitylating reagent. In previous work, caged phospho-peptides were made using an interassembly approach (Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperiali, B. Org. Lett. 2002, 4, 2865-2868). However, this technique is limited to creating peptides without oxidation sensitive residues C-terminal to the amino acid to be modified and the methodology involves synthetic manipulations on the solid phase that may limit the utilization of the methodology. Herein we report the facile synthesis of N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-serine 1, N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-threonine 2, and N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-tyrosine 3. These building blocks allow the synthesis of any caged phospho-peptide sequence using standard Fmoc-based SPPS procedures.

Amino Acids↗
PubMed2003

Photolytic control of peptide self-assembly.

Herein, we present a methodology that allows for the temporal control of fibrillization of amyloidogenic peptides. This general approach implements a photolabile linker that connects the amyloidogenic peptide to a fibril-inhibitory unit, in this case, a pentamer of amino acids modified with the solubilizing N,N-dimethylethylenediamine (DMDA) units. Upon photolysis, the linker can be dissociated to afford the intact and native amyloidogenic peptide. This methodology should be of value in a variety of studies where spatial and temporal control of supramolecular association processes is desired.

Amino Acid Sequence↗
PubMed2003

The interplay of glycosylation and disulfide formation influences fibrillization in a prion protein fragment.

It is now accepted that the structural transition from cellular prion protein (PrPC) to proteinase K-resistant prion protein scrapie (PrPSc) is the major event leading to transmissible spongiform encephalopathies. Although the mechanism of this transition remains elusive, glycosylation has been proposed to impede the PrPC to PrPSc conversion. To address the role of glycosylation, we have prepared glycosylated and unglycosylated peptides derived from the 175-195 fragment of the human prion protein. Comparison of the structure, aggregation kinetics, fibril formation capabilities, and redox susceptibility of Cys-179 has shown that the N-linked glycan (at Asn-181) significantly reduces the rate of fibrillization by promoting intermolecular disulfide formation via Cys-179. Further-more, the aggressive fibrillization of a C179S mutant of this fragment highlights the significant role of disulfide stability in retarding the rate of fibril formation. The implications of these studies are discussed in the context of fibril formation in the intact prion protein.

Amino Acid Sequence↗
PubMed2003

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 Tb(3+) 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.

Amino Acid Sequence↗
PubMed2003

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 Tb(3+) 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, K(D)) 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 Tb(3+) ions.Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.

Amino Acid Sequence↗
PubMed2003

Peptides to peptidomimetics: towards the design and synthesis of bioavailable inhibitors of oligosaccharyl transferase.

Oligosaccharyl transferase (OT) is the enzyme responsible for asparagine-linked glycosylation in the lumen of the endoplasmic reticulum, which is a subcellular compartment within eukaryotic cells. Inhibition of this enzyme within a cellular environment would provide a valuable investigative tool for glycobiology. Due to the limitations of peptides, none of the existing peptide-based inhibitors of OT demonstrate activity in cell-based enzyme assays. We report herein the design, synthesis and preliminary biological characterization of a family of peptidomimetics that inhibit OT with Ki values in the nanomolar range. The hexapeptide Bz-Dab-Ala-Thr-Val-Thr-Nph-NH2 (Ki = 69 nM) was used as the prototype for the design of bioavailable inhibitors. Several aminobenzoic acid spacer groups were evaluated as potential isosteres of the Val-Thr dipeptide unit and the peptidomimetic incorporating 3-aminobenzoic acid proved to inhibit OT with similar potency to the parent compound (Ki = 84 nM). Further modifications explored the effects of size, hydrophobicity and conformational rigidity on enzyme affinity. This study yielded a family of potent non-peptidic inhibitors that are viable candidates for the in vivo inhibition of OT.

Asparagine↗