Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids

An efficient method for the heteroconjugation of biomolecules carrying free amino groups was reported previously, where mixed polyfluorophenyl diesters of dicarboxylic acids with varied aliphatic chain length were shown to be efficient reagents for the conjugation of a variety of model biomolecules. The concept was based on the differential reactivity of the esters towards amines. The concept has now been further optimized, and a 2,6‐difluorophenyl‐pentafluorophenyl diester combination has been demonstrated to be the most efficient, both with respect to selectivity and to reaction rate. A pentafluorophenyl ester reacts faster with an amino group and requires a weaker base than a 2,6‐difluorophenyl ester that requires a stronger base and longer reaction time. With the use of this combination of esters, we obtained considerably shortened reaction times compared with those reported previously, yet still retaining the desired selectivity in heteroconjugation. The increased reactivity of the bifunctional reagent allowed the construction of sophisticated peptide heteroconjugates from peptides, carbohydrates and proteins, showing a wide scope of applicability in the field of assembling functional bioconjugates. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Protein heteroconjugation by the peroxidase-catalyzed tyrosine coupling reaction

Combining different proteins can integrate the functions of each protein to produce novel protein conjugates with wider ranges of applications. We have previously introduced a peptide containing tyrosine residues (Y-tag) at the C-terminus of Escherichia coli alkaline phosphatase (BAP). The tyrosine residues in the Y-tag were efficiently recognized by horseradish peroxidase (HRP) and were site-specifically cross-linked with each other to yield BAP homoconjugates. In this study, the HRP-catalyzed tyrosine coupling reaction was used for protein heteroconjugation. Streptavidin (SA) was selected as the conjugation partner for BAP. The Y-tag (GGGGY) was genetically introduced to the C-terminus of SA. Prior to heteroconjugation, the reactivity of the Y-tagged SA was examined. The Y-tagged SA cross-linked to form an SA homoconjugate upon HRP treatment, whereas wild-type SA remained essentially intact. In the heteroconjugation reaction of BAP and SA, the Y-tagged BAP and SA were efficiently cross-linked with each other upon HRP treatment. The functions of the BAP-SA conjugates were evaluated by measuring the BAP enzymatic activity on a biotin-coated plate. The BAP-SA conjugate tethered to the plate showed BAP enzymatic activity, indicating that both BAP and SA retained their functions following heteroconjugation. The BAP-SA conjugate prepared from both Y-tagged BAP and SA showed the highest enzymatic activity on the biotin-coated plates. This result illustrates the advantage of the protein conjugation reaction in which multiple numbers of proteins can be conjugated at the same time.     

Molecular beacons: fluorogenic probes for living cell study

Molecular beacons are a new class of fluorescent probes that can report the presence of specific nucleic acids with high sensitivity and excellent specificity. In addition to their current wide applications in monitoring the progress of polymerase chain reactions, their unique properties make them promising probes for the detection and visualization of target biomolecules in living cells. This article is focused on our recent research in exploring the potential of using molecular beacon for living-cell studies in three important areas: the monitoring of mRNA in living cells, the development of ultrasmall DNA/RNA biosensors, and the novel approach of combining molecular beacon's signal transduction mechanism with aptamer's specificity for real-time protein detection. These applications demonstrate molecular beacon's unique properties in bioanalysis and bioassay development.     

Functions of toll-like receptors: lessons from KO mice

The innate immune response is a first-line defense system in which individual Toll-like receptors (TLRs) recognize distinct pathogen-associated molecular patterns (PAMPs) and exert subsequent immune responses against a variety of pathogens. TLRs are composed of an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic domain that is homologous to that of the IL-IR family. Upon stimulation, TLR recruits a cytoplasmic adaptor molecule MyD88, then IL-IR-associated kinase (IRAK), and finally induces activation of NF-kappaB and MAP kinases. However, the responses to TLR ligands differ, indicating the diversity of TLR signaling pathways. Besides MyD88, several novel adaptor molecules have recently been identified. Differential utilization of these adaptor molecules may provide the specificity in the TLR signaling.     

Side-by-Side Comparison of Commonly Used Biomolecules That Differ in Size and Affinity on Tumor Uptake and Internalization

The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization.     

Molecular beacons

This opinion covers the field of molecular beacons (MBs), in which nucleic acids are molecularly engineered to have unique functions for the investigation of biomolecules. Molecular beacons have been used in a variety of formats, and this review discusses four: first, in vitro RNA and DNA monitoring; second, biosensors and biochips based on MBs; third, real-time monitoring of genes and gene expression in living systems; and finally, the next generation of molecular beacons that will be highly useful for studies with proteins, molecular beacon aptamers. These unique applications have shown that MBs holds great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.     

Chiroptical spectroscopy and metabolomics for blood‐based sensing of pancreatic cancer

To enable the early diagnosis of pancreatic cancer, the search for and definition of reliable biomarkers remain a subject of great interest, with the specificity and sensitivity of the currently used biomarkers being below the required values. We tested a novel diagnostic approach for pancreatic cancer based on the specific molecular signature of blood plasma components. To acquire more detailed structural information, structure‐sensitive chiroptical methods (electronic circular dichroism and Raman optical activity) were supplemented by conventional Raman and infrared spectroscopies. The obtained spectra were subsequently processed by linear discriminant analysis yielding high values of specificity and sensitivity. In addition, to monitor not only large biomolecules as potential biomarkers but also those of low molecular weight, we conducted an analysis of blood plasma samples by using metabolomics. The achieved results suggest a panel of promising biomarkers for a reliable detection of pancreatic cancer.     

Structural Basis for the Interaction of the Adaptor Protein Grb14 with Activated Ras

Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region, and a C-terminal Src-homology-2 (SH2) domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V). The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM) and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.     

Detailed structural insights into the p97-Npl4-Ufd1 interface

The AAA ATPase, p97, achieves its versatility through binding to a wide range of cofactor proteins that adapt it to different cellular functions. The heterodimer UN (comprising Ufd1 and Npl4) is an adaptor complex that recruits p97 for numerous tasks, many of which involve the ubiquitin pathway. Insights into the structural specificity of p97 for its UN adaptor are currently negligible. Here, we present the solution structure of the Npl4 "ubiquitin-like" domain (UBD), which adopts a beta-grasp fold with a 3(10) helical insert. Moreover we performed a chemical shift perturbation analysis of its binding surface with the p97 N domain. We assigned the backbone amides of the p97 N domain and probed both its reciprocal binding surface with Npl4 UBD and its interaction with the p97-binding region of Ufd1. NMR data recorded on a 400-kDa full-length UN-hexamer p97 complex reveals an identical mode of interaction. We calculated a structural model for the p97 N-Npl4 UBD complex, and a comparison with the p97-p47 adaptor complex reveals subtle differences in p97 adaptor recognition and specificity.     

Hydrazinonicotinic acid (HYNIC) - Coordination chemistry and applications in radiopharmaceutical chemistry

HYNIC (hydrazinonicotinamide) is an efficient bifunctional chelator for Tc-99m used for labelling biomolecules for molecular imaging. Developments and enhancements to improve its efficacy and versatility, including applications beyond Tc-99m labelling, include designs to allow site specificity, availability of amino acid building blocks, improved protecting groups, and a varied choice of co-ligands. In this review, these enhancements are summarised, along with an assessment of the opportunities afforded and problems posed by the use of HYNIC, a discussion of its coordination mode, and the prospects for improving its use and overcoming some of the limitations. There is now an opportunity to exploit the excellent labelling kinetics associated with the tricine-HYNIC system with better co-ligand design to enable both efficient production of labelled proteins and peptides and better specific activity and in vivo properties. In summary, HYNIC represents a well-established way to exploit the highly reactive hydrazine group, to generate bioconjugate chemistry with a degree of bioorthogonality offering the possibility for highly efficient and site specific modification of biomolecules for imaging.     

Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA

In Escherichia coli, protein degradation is performed by several proteolytic machines, including ClpAP. Generally, the substrate specificity of these machines is determined by chaperone components, such as ClpA. In some cases, however, the specificity is modified by adaptor proteins, such as ClpS. Here we report the 2.5 A resolution crystal structure of ClpS in complex with the N-terminal domain of ClpA. Using mutagenesis, we demonstrate that two contact residues (Glu79 and Lys 84) are essential not only for ClpAS complex formation but also for ClpAPS-mediated substrate degradation. The corresponding residues are absent in the chaperone ClpB, providing a structural rationale for the unique specificity shown by ClpS despite the high overall similarity between ClpA and ClpB. To determine the location of ClpS within the ClpA hexamer, we modeled the N-terminal domain of ClpA onto a structurally defined, homologous AAA+ protein. From this model, we proposed a molecular mechanism to explain the ClpS-mediated switch in ClpA substrate specificity.     

Control of a tyrosyl radical mediated protein cross-linking reaction by electrostatic interaction

Herein, we demonstrate the control of protein heteroconjugation via a tyrosyl coupling reaction by using electrostatic interaction. Aspartic acid and arginine were introduced to a tyrosine containing peptide tag (Y-tag) to provide electrostatic charge. Designed negatively or positively charged Y-tags were tethered to the C-terminus of Escherichia coli alkaline phosphatase (BAP) and streptavidin (SA), and these model proteins were subjected to horseradish peroxidase (HRP) treatment. The negatively charged Y-tags showed low reactivity due to repulsive interactions between the Y-tags with the negatively charged BAP and SA. In contrast, the positively charged Y-tags showed high reactivity, indicating that the electrostatic interaction between Y-tags and proteins significantly affects the tyrosyl radical mediated protein cross-linking. From the heteroconjugation reaction of BAP and SA, the SA with the positively charged Y-tags exhibited favorable cross-linking toward negatively charged BAP, and the BAP-SA conjugates prepared from BAP with GY-tag (GGGGY) and SA with RYR-tag (RRYRR) had the best performance on a biotin-coated microplate. Encompassing the reactive tyrosine residue with arginine residues reduced the reactivity against HRP, enabling the modulation of cross-linking reaction rates with BAP-GY. Thus, by introducing a proper electrostatic interaction to Y-tags, it is possible to kinetically control the heteroconjugation behavior of proteins, thereby maximizing the functions of protein heteroconjugates.     

TiO2 nanowire FET device: encapsulation of biomolecules by electro polymerized pyrrole propylic acid

Silane-based methods have become the standards for the conjugation of biomolecules, especially for the preparation of one-dimensional nanomaterial biosensors. However, the specific binding of those target molecules might raise problems with regard to the sensing and non-sensing regions, which may contaminate the sensing devices and decrease their sensitivity. This paper attempts to explore the encapsulation of biomolecules on a one-dimensional nanomaterial field effect transistor (FET) biosensor using polypyrrole propylic acid (PPa). Specifically, the encapsulation of biomolecules via the electropolymerization of pyrrole propylic acid (Pa), a self-made low-conductivity polymer, on TiO(2)-nanowire (NW)-based FETs is presented. The energy dispersive spectrum (EDS) was obtained and electrical analysis was conducted to investigate PPa entrapping anti-rabbit IgG (PPa/1°Ab) on a composite film. The specificity, selectivity and sensitivity of the sensor were analyzed in order to determine the immunoreaction of PPa/1°Ab immobilized NW biosensors. Our results show that PPa/1°Ab achieved high specificity immobilization on NWs under the EDS analysis. Furthermore, the TiO(2)-NW FET immunosensor developed in this work successfully achieved specificity, selectivity and sensitivity detection for the target protein rabbit IgG at the nano-gram level. The combination of PPa material and the electropolymerization method may provide an alternative method to immobilize biomolecules on a specific surface, such as NWs.     

Oligonucleotide-coated metallic nanoparticles as a flexible platform for molecular imaging agents

Targeted metallic nanoparticles have shown promise as contrast agents for molecular imaging. To obtain molecular specificity, the nanoparticle surface must be appropriately functionalized with probe molecules that will bind to biomarkers of interest. The aim of this study was to develop and characterize a flexible approach to generate molecular imaging agents based on gold nanoparticles conjugated to a diverse range of probe molecules. We present two complementary oligonucleotide-based approaches to develop gold nanoparticle contrast agents which can be functionalized with a variety of biomolecules ranging from small molecules, to peptides, to antibodies. The size, biocompatibility, and protein concentration per nanoparticle are characterized for the two oligonucleotide-based approaches; the results are compared to contrast agents prepared using adsorption of proteins on gold nanoparticles by electrostatic interaction. Contrast agents prepared from oligonucleotide-functionalized nanoparticles are significantly smaller in size and more stable than contrast agents prepared by adsorption of proteins on gold nanoparticles. We demonstrate the flexibility of the oligonucleotide-based approach by preparing contrast agents conjugated to folate, EGF peptide, and anti-EGFR antibodies. Reflectance images of cancer cell lines labeled with functionalized contrast agents show significantly increased image contrast which is specific for the target biomarker. To demonstrate the modularity of this new bioconjugation approach, we use it to conjugate both fluorophore and anti-EGFR antibodies to metal nanoparticles, yielding a contrast agent which can be probed with multiple imaging modalities. This novel bioconjugation approach can be used to prepare contrast agents targeted with biomolecules that span a diverse range of sizes; at the same time, the bioconjugation method can be adapted to develop multimodal contrast agents for molecular imaging without changing the coating design or material.     

Substrate specificity of lymphoid-specific tyrosine phosphatase (Lyp) and identification of Src kinase-associated protein of 55 kDa homolog (SKAP-HOM) as a Lyp substrate

A missense single-nucleotide polymorphism in the gene encoding the lymphoid-specific tyrosine phosphatase (Lyp) has been identified as a causal factor in a wide spectrum of autoimmune diseases. Interestingly, the autoimmune-predisposing variant of Lyp appears to represent a gain-of-function mutation, implicating Lyp as an attractive target for the development of effective strategies for the treatment of many autoimmune disorders. Unfortunately, the precise biological functions of Lyp in signaling cascades and cellular physiology are poorly understood. Identification and characterization of Lyp substrates will help define the chain of molecular events coupling Lyp dysfunction to diseases. In the current study, we identified consensus sequence motifs for Lyp substrate recognition using an "inverse alanine scanning" combinatorial library approach. The intrinsic sequence specificity data led to the discovery and characterization of SKAP-HOM, a cytosolic adaptor protein required for proper activation of the immune system, as a bona fide Lyp substrate. To determine the molecular basis for Lyp substrate recognition, we solved crystal structures of Lyp in complex with the consensus peptide as well as the phosphopeptide derived from SKAP-HOM. Together with the biochemical data, the structures define the molecular determinants for Lyp substrate specificity and provide a solid foundation upon which novel therapeutics targeting Lyp can be developed for multiple autoimmune diseases.     

Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors

AAA+ proteases are frequently regulated by adaptors that modulate spatial and temporal control of protein turnover. Caulobacter crescentus is an alpha-proteobacterium which requires protein degradation by the AAA+ ClpXP protease for cell-cycle progression, and contains an adaptor (SspBalpha) that binds ssrA-tagged proteins and targets them to ClpXP. Here we determine the tag-binding specificity and crystal structure of SspBalpha. Despite poor sequence homology, the overall SspBalpha fold resembles orthologs from other bacteria. However, several structural features are specific to the SspBalpha subfamily, including the dimerization interface, binding surfaces optimized for ssrA-tag delivery, and residues in the tag-binding groove that act as selectivity gatekeepers for substrate recognition. Mutagenesis of these residues broadens specificity, creating a promiscuous adaptor that recognizes an expanded substrate repertoire. These results highlight general features of adaptor-mediated substrate recognition and shed light on design principles that underlie adaptor function.     

Dynamic control of signaling by modular adaptor proteins

Adaptor proteins are composed exclusively of domains and motifs that mediate molecular interactions, and can thereby link signaling proteins such as activated cell-surface receptors to downstream effectors. Recent data supports the notion that adaptors are not simply coupling devices that hard-wire successive components of signaling pathways. Rather, they display highly dynamic properties that direct the flow of information through signaling networks. The binding activity of adaptors can be regulated by conformational reorganization, and by the cooperative association of domains within the same adaptor. Furthermore, an individual adaptor can deliver different outputs by utilizing distinct combinations of binding partners. Adaptors can also control the oligomerization of receptor signaling complexes, and the subcellular location and duration of signaling events, and act as coincidence detectors to enhance specificity in cellular responses.     

Molecular crowding effects on structure and stability of DNA

Living cells contain a variety of biomolecules including nucleic acids, proteins, polysaccharides, and metabolites as well as other soluble and insoluble components. These biomolecules occupy a significant fraction (20-40%) of the cellular volume. The total concentration of biomolecules reaches 400gL(-1), leading to a crowded intracellular environment referred to as molecular crowding. Therefore, an understanding of the effects of molecular crowding conditions on biomolecules is important to broad research fields such as biochemical, medical, and pharmaceutical sciences. In this review, we describe molecular conditions in the cytoplasm and nucleus, which are totally different from in vitro conditions, and then show the biochemical and biophysical consequences of molecular crowding. Finally, we discuss the effect of molecular crowding on the structure, stability, and function of nucleic acids and the significance of molecular crowding in biotechnology and nanotechnology.     

Adaptor Ligation-Based Polymerase Chain Reaction-Mediated Walking

An improved method of adaptor ligation PCR was developed for isolation of unknown sequences flanking a known DNA sequence. It was determined that the specificity of the adaptor ligation-based walking technique could be significantly enhanced by using uniquely blocked adaptors along with removal of unligated genomic DNA by exonuclease III digestion. This technique was utilized to isolate three novel promoter regions from three differentZea mays(maize) peroxidase genes. Sequences encoding a putative maize 6-phosphogluconate dehydrogenase gene were also isolated and confirmed by sequence analysis. The described improvements could be applied to other existing adaptor ligation-based PCR walking techniques.     

Characterizing rare-event property distributions via replicate molecular dynamics simulations of proteins

As computational resources increase, molecular dynamics simulations of biomolecules are becoming an increasingly informative complement to experimental studies. In particular, it has now become feasible to use multiple initial molecular configurations to generate an ensemble of replicate production-run simulations that allows for more complete characterization of rare events such as ligand-receptor unbinding. However, there are currently no explicit guidelines for selecting an ensemble of initial configurations for replicate simulations. Here, we use clustering analysis and steered molecular dynamics simulations to demonstrate that the configurational changes accessible in molecular dynamics simulations of biomolecules do not necessarily correlate with observed rare-event properties. This informs selection of a representative set of initial configurations. We also employ statistical analysis to identify the minimum number of replicate simulations required to sufficiently sample a given biomolecular property distribution. Together, these results suggest a general procedure for generating an ensemble of replicate simulations that will maximize accurate characterization of rare-event property distributions in biomolecules.