Design and Evaluation of a Peptidyl Fluorescent Chemosensor for Divalent Zinc

The rapid analysis of trace metal cations for environmental and biomedical applications is particularly demanding since it requires the specific recognition of a particular element in the presence of numerous closely related species. While remarkable progress has been achieved for inorganic analytes, such as Ca^(2+), there is still a significant need for the genesis of new fluorosensors. In light of the selectivity and avidity with which proteins bind divalent metal cations, we have chosen to use a polypeptide architecture as the framework for metal ion recognition.In addition, our ability to manipulate synthetic polypeptides allows the coordinated integration of fluorescent reporters for signal transduction within a metal-binding peptidyl construct. Herein we report the design, synthesis, and evaluation of a selective fluorescent chemosensor, sensitive to nanomolar concentrations of Zn^(2+)

The Renaissance of Bacillosamine and Its Derivatives: Pathway Characterization and Implications in Pathogenicity

Prokaryote-specific sugars, including N,N′-diacetylbacillosamine (diNAcBac) and pseudaminic acid, have experienced a renaissance in the past decade because of their discovery in glycans related to microbial pathogenicity. DiNAcBac is found at the reducing end of oligosaccharides of N- and O-linked bacterial protein glycosylation pathways of Gram-negative pathogens, including Campylobacter jejuni and Neisseria gonorrhoeae. Further derivatization of diNAcBac results in the nonulosonic acid known as legionaminic acid, which was first characterized in the O-antigen of the lipopolysaccharide (LPS) in Legionella pneumophila. Pseudaminic acid, an isomer of legionaminic acid, is also important in pathogenic bacteria such as Helicobacter pylori because of its occurrence in O-linked glycosylation of flagellin proteins, which plays an important role in flagellar assembly and motility. Here, we present recent advances in the characterization of the biosynthetic pathways leading to these highly modified sugars and investigation of the roles that each plays in bacterial fitness and pathogenicity.National Institutes of Health (U.S.) (NIH BiotechnologyTraining Program T32-GM08334)National Institutes of Health (U.S.) (NIH Grant GM097241

Monitoring protein interactions and dynamics with solvatochromic fluorophores

Solvatochromic fluorophores possess emission properties that are sensitive to the nature of the local microenvironment. These dyes have been exploited in applications ranging from the study of protein structural dynamics to the detection of protein-binding interactions. Although the solvatochromic indole fluorophore of tryptophan has been utilized extensively for in vitro studies to advance our understanding of basic protein biochemistry, the emergence of new extrinsic synthetic dyes with improved properties, in conjunction with recent developments in site-selective methods to incorporate these chemical tools into proteins, now open the way for studies in more complex systems. Herein, we discuss recent technological advancements and their application in the design of powerful reporters, which serve critical roles in modern cell biology and assay development.National Institutes of Health (U.S.) (NIH Cell Migration Consortium (GM064346))National Science Foundation (U.S.) (CHE-0414243)National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Grant T32 GM08334)European Commission (Marie Curie Postdoctoral Fellowship Program

Development of a fluorogenic sensor for activated Cdc42

Cdc42, a member of the Rho GTPase family, is a fundamental regulator of the actin cytoskeleton during cell migration. To generate a sensor for Cdc42 activation, we employed a multi-pronged approach, utilizing cysteine labeling and expressed protein ligation, to incorporate the environment sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) into the GTPase binding domain of the WASP protein. These constructs bind only the active, GTP-bound conformation of Cdc42 to produce a fluorescence signal. Studies with a panel of five sensor analogs revealed a derivative that exhibits a 32-fold increase in fluorescence intensity in the presence of activated Cdc42 compared to incubation with the inactive GDP-bound form of the protein. We demonstrate that this sensor can be exploited to monitor Cdc42 nucleotide exchange and GTPase activity in a continuous, fluorescence assay.National Institutes of Health (U.S.) (Cell Migration Consortium GM064346)National Institute of General Medical Sciences (U.S.) (Biotechnology Training Grant T32-GM08334

A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes

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 enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays. Current PGT assays include radioactivity-based methods, which rely on liquid-liquid or solid-liquid extractions, multienzyme systems linked to lactate dehydrogenase and NAD+ generation, and HPLC-based approaches, all of which may suffer from low sensitivity and low throughput. Herein, we present the validation of a new luminescence-based assay (UMP-Glo) for measuring activities of PGT enzymes. This assay measures UMP, the by-product of PGT reactions, in a sensitive and quantitative manner by measuring the luminescence output in a discontinuous coupled assay system. The assay is rapid and robust in nature, and also compatible with microtiter plate formats. Activity and kinetic parameters of PglC, a PGT from Campylobacter jejuni, were quickly established using this assay. The efficacy of the assay was further corroborated using two different PGTs; PglC from Helicobacter pullorum and WecA from Thermatoga maritima.National Institutes of Health (U.S.) (GM-039334

Derivatives of 8-Hydroxy-2-methylquinoline Are Powerful Prototypes for Zinc Sensors in Biological Systems

The recent emphasis on understanding the myriad roles of zinc in both normal and diseased cells and tissues has placed an ever increasing demand on methods for sensitive and selective methods for real-time monitoring of free Zn^(2+) in complex biological samples. Chelation-enhanced fluorescent sensors for zinc, based on fluorophores such as quinoline, dansyl, fluorescein, and anthracene, have been reported. While each of these agents has unique advantages, there remain issues with sensitivity, selectivity, and specificity that may be addressable with an alternate chromophore that is readily amenable to synthetic manipulation. Herein we report the systematic chemical modification of the 8-hydroxy-2-methylquinoline (Oxn) unit as a building block for the development of new sensors employing chelation-enhanced fluorescence. In particular, improvements in quantum yield from 0.004 to 0.70 and stepwise blue shifts in fluorescence emission wavelengths (to a total of over 70 nm) are reported

Design and Evaluation of a Peptidyl Fluorescent Chemosensor for Divalent Zinc

The rapid analysis of trace metal cations for environmental and biomedical applications is particularly demanding since it requires the specific recognition of a particular element in the presence of numerous closely related species. While remarkable progress has been achieved for inorganic analytes, such as Ca^(2+), there is still a significant need for the genesis of new fluorosensors. In light of the selectivity and avidity with which proteins bind divalent metal cations, we have chosen to use a polypeptide architecture as the framework for metal ion recognition.In addition, our ability to manipulate synthetic polypeptides allows the coordinated integration of fluorescent reporters for signal transduction within a metal-binding peptidyl construct. Herein we report the design, synthesis, and evaluation of a selective fluorescent chemosensor, sensitive to nanomolar concentrations of Zn^(2+)

Peptide platforms for metal ion sensing

Naturally occurring motifs have been redesigned to product fluorescent peptidyl-chemosensors that sensitively and selectively recognize Cu(II) or Fe(III). The modular nature of peptide architecture allows preparation and evaluation of potential sensors on solid supports

Two-Photon Fluorescence Spectroscopy and Imaging of 4-Dimethylaminonaphthalimide Peptide and Protein Conjugates

We report detailed photophysical studies on the two-photon fluorescence processes of the solvatochromic fluorophore 4-DMN as a conjugate of the calmodulin (CaM) and the associated CaM-binding peptide M13. Strong two-photon fluorescence enhancement has been observed which is associated with calcium binding. It is found that the two-photon absorption cross-section is strongly dependent on the local environment surrounding the 4-DMN fluorophore in the CaM conjugates, providing sensitivity between sites of fluorophore attachment. Utilizing time-resolved measurements, the emission dynamics of 4-DMN under various environmental (solvent) conditions are analyzed. In addition, anisotropy measurements reveal that the 4-DMN–S38C–CaM system has restricted rotation in the calcium-bound calmodulin. To establish the utility for cellular imaging, two-photon fluorescence microscopy studies were also carried out with the 4-DMN-modified M13 peptide in cells. Together, these studies provide strong evidence that 4-DMN is a useful probe in two-photon imaging, with advantageous properties for cellular experiments.German Science Foundation (SO 1100/1-1