Rewiring kinase specificity with a synthetic adaptor protein

Signaling cascades are managed in time and space by interactions between and among proteins. These interactions are often aided by adaptor proteins, which guide enzyme-substrate pairs into proximity. Miniature proteins are a class of small, well-folded protein domains possessing engineered binding properties. Here we made use of two miniature proteins with complementary binding properties to create a synthetic adaptor protein that effectively redirects a ubiquitous signaling event: tyrosine phosphorylation. We report that miniature-protein-based adaptor 3 uses templated catalysis to redirect the Src family kinase Hck to phosphorylate hDM2, a negative regulator of the p53 tumor suppressor and a poor Hck substrate. Phosphorylation occurs with multiple turnover and at a single site targeted by c-Abl kinase in the cell.     

Modulation of the substrate specificity of purified human protein kinase C by its activators

The substrate specificity of purified human protein kinase C was modulated by 12-O-tetradecanoyl-4 beta-phorbol-13-acetate (TPA), dioleoylglycerol, arachidonic acid and lipid A when histone type III-S and myelin basic protein were used as phosphate acceptors. Each activator also showed a distinct pattern in the stimulation of phosphorylation of the kinase itself and of cytosolic placental proteins. The nature of the substrate and the presence of calcium and phospholipid determined the magnitude of the effect observed upon addition of all activators and also the dose dependency of kinase activation by TPA. The apparent Km value for phosphorylation of histone type III-S by the kinase activated by phorbol ester alone and with calcium was 20-30 fold higher than that observed for the enzyme activated by calcium and phospholipid. These observations indicate that the nature and extent of cellular response induced by the activation of C-kinase(s) may be determined by the type of cellular stimulus.     

Design of a functional protein for molecular recognition: specificity of ligand binding in a metal-assembled protein cavity probed by 19f NMR

A metal-assembled homotrimeric coiled coil based on the GCN4-p1 sequence has been designed that noncovalently binds hexafluorobenzene and other similar ligands in a hydrophobic cavity, created by making the core substitution Asn16Ala ([Fe(bpyGCN4-N16A)3]2+). The KD of binding of hexafluorobenzene with [Fe(bpyGCN4-N16A)3]2+ was observed to be 1.1(9) x 10(-4) M by diffusion NMR experiments. A control coiled coil with the core substitution Asn16Val ([Fe(bpyGCN4-N16V)3]2+) exhibited a significantly weaker association with hexafluorobenzene, providing evidence that even in the absence of structural data, benzene-like ligands bind in the cavity created by the Asn16Ala substitution. 19F NMR was employed to observe hexafluorobenzene binding and to monitor titrations with competing hydrophobic and polar ligands similar in size and shape to hexafluorobenzene. All hydrophobic ligands bound with greater affinity than the polar ligands in the hydrophobic core, although the cavity seems to be somewhat flexible in terms of the sizes of molecules it can accommodate. Thus 19F NMR has proved to be a useful spectral tool to probe molecular recognition in a hydrophobic cavity of a metal-assembled coiled coil.     

Prioritization of charge over geometry in transition state analogues of a dual specificity protein kinase

The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using (19)F NMR analysis of MEK6 in complex with tetrafluoroaluminate (AlF(4)(-)), ADP, and p38α MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF(4)(-) moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF(4)(-) complex involving the alternative acceptor residue in p38α (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF(4)(-) species for both acceptor residues, rather than the trigonal bipyramidal AlF(3)(0) previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion.     

Control of protein structure and function through surface recognition by tailored nanoparticle scaffolds

Thioalkyl and thioalkylated oligo(ethylene glycol) (OEG) ligands with chain-end functionality were used to fabricate water-soluble CdSe nanoparticle scaffolds. Surface recognition of chymotrypsin (ChT) was achieved using these functionalized nanoparticle scaffolds, with three levels of interaction demonstrated: no interaction (OEG terminated with hydroxyl group), inhibition with denaturation (carboxylate-terminated thioalkyl ligands), and inhibition with retention of structure (carboxylate-terminated OEG). The latter process was reversible upon an increase in ionic strength, with essentially complete restoration of enzymatic activity.     

Use of hydrophilic interaction chromatography for the study of tyrosine protein kinase specificity.

A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [32P]phosphorylated peptide, unreacted [gamma-32P]ATP, and 32P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase.     

Molecularly imprinted polymer grafted on polysaccharide microsphere surface by the sol-gel process for protein recognition

An interfacial organic–inorganic hybridization concept was applied to the preparation of a new spherical imprinted material for protein recognition. The functional biopolymer chitosan (CS), shaped as microsphere and high-density cross-linked, constituted of the polysaccharide core for surface imprinting. After the model template protein, bovine serum albumin, was covalently immobilized by forming imine bonds with the functional amine groups of CS, two kinds of organic siloxane (3-aminopropyltrimethoxysiloxane: APTMS, and tetraethoxysiloxane: TEOS) assembled and polymerized on the polysaccharide–protein surface via sol–gel process in aqueous solution at room temperature. After template removal, the protein-imprinted sol–gel surface exhibited a prevalent preference for the template protein in adsorption experiments, as compared with four contrastive proteins. Bioinformatics methods were also employed to investigate the imprinting process and the recognition effect. The influence of siloxane type, pH, siloxane/water ratio on template removal and recognition selectivity was assessed. Under optimized imprinting conditions, a large quantity of well-distributed pores was observed on the immobilized-template imprinted surface. The surface-imprinted adsorbent offered a fast kinetics for template re-adsorption and could be reused. Compared with the imprinted material prepared with free-template, material prepared with immobilized-template possessed higher adsorption capacity towards template protein. Easy preparation of the described imprinted material, high affinity and good reusability make this approach attractive and broadly applicable in biotechnology for down-stream processing and biosensor.     

Molecular recognition in partially folded states of a transporter protein: temperature-dependent specificity of bovine serum albumin

The specificity of molecular recognition of a transporter protein bovine serum albumin (BSA) in its different partially folded states has been studied. In order to avoid complications due to chemical denaturation, we have prepared thermally induced partially folded states of the protein. The partially folded states have been structurally characterized by circular dichroism and differential thermal analysis techniques. The change in the globular structure of the protein as a consequence of thermal unfolding has also been characterized by dynamic light scattering. Steady state, picosecond‐resolved fluorescence and polarization gated spectroscopies on the ligands (DCM, LDS 750) in the protein reveal the dynamics of the binding sites and the specificity of ligand binding of BSA. Picosecond resolved Förster resonance energy transfer studies on the donor DCM and acceptor LDS 750 confirm that the specificity of ligand binding in the binding site is maintained up to 70°C. At 75°C, the protein loses its specificity of recognition at the aforesaid site.     

Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing of arrays under different assay conditions. These new array processors provide researchers and assay developers with novel possibilities to construct highly specific DNA arrays even towards regions of DNA greatly varying in G?+?C content. These array processors are also a powerful development tool for building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit microarray-based loci selection prior to high-throughput sequencing.     

Increasing specificity of tandem mass spectrometry by laser‐induced dissociation

Mass spectrometry offers an arsenal of tools for diverse proteomic investigations. This perspective article reviews some of the recent developments in the field of coupling laser‐induced dissociation with mass spectrometry (LID‐MS). Strategies involving labelling with a chromophore to induce specific photo‐absorption properties are considered, with a focus on specific amino acid derivatization. Some of the opportunities and challenges of LID‐MS after targeted labelling for increasing specificity in complex sample analysis are discussed.     

Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening

Background  

The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified.     

Protein surface recognition by synthetic receptors based on a tetraphenylporphyrin scaffold

[structure: see text] Receptors based on a tetraphenylporphyrin scaffold bearing different charged and hydrophobic groups have been synthesized. The interactions of these with horse heart cytochrome c were studied by fluorescence spectroscopy. Receptor 4 was identified to be the strongest synthetic receptor (K(d) = 20 nM) for cytochrome c. The differences in affinity among the receptors reflected a dependence on the number of anionic and hydrophobic groups.     

Understanding the relative affinity and specificity of the pleckstrin homology domain of protein kinase B for inositol phosphates

Protein kinase B (PKB) is a serine/threonine kinase that plays a key role in the phosphoinositide 3-kinase (PI3K) pathway-one of the most frequently activated proliferation pathways in cancer. In this pathway, PKB is recruited to the plasma membrane by direct interaction of its pleckstrin homology (PH) domain with the inositol phosphate head-group of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] or phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)]. This recruitment is a critical stage in the activation of PKB, whose downstream effectors play important roles in cell survival, proliferation and growth. It is therefore of great interest to understand PKB's mode of binding, as well as its specificity and affinity for different phosphoinositides. We have used a total of 3 μs of molecular dynamics (MD) simulations to better understand the interactions of the PKB PH domain with the inositol phosphate head-groups of phosphoinositides involved in the PI3K pathway. Our computational models successfully mirror PKB's in vivo selectivity for 3-phosphorylated phosphoinositides. Furthermore, the models also help to rationalize unexpected in vitro data in which inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] binds with a relatively high affinity to the PKB PH domain, despite its parent lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] being known not to bind in vivo. With the support of computational simulations, we propose that when not bonded to a phosphatidate tail Ins(1,4,5)P(3) binds in an orientation in which its inositol ring is flipped with respect to the 3-phosphorylated inositol phosphate ligands and its parent lipid.     

Nanosized aptameric cavities imprinted on the surface of magnetic nanoparticles for high-throughput protein recognition

The authors introduce a new kind of surface artificial biomimetic receptor, referred to as aptameric imprinted polymer (AIP), for separation of biological macromolecules. Highly dispersed magnetic nanoparticles (MNPs) were coated with silica and then functionalized with methacrylate groups via silane chemistry. The aptamer was covalently immobilized on the surface of nanoparticles via a “thiol-ene” click reaction. Once the target analyte (bovine serum albumin; BSA) has bound to the aptamer, a polymer is created by 2-dimensional copolymerization of short-length poly(ethylene glycol) and (3-aminopropyl)triethoxysilane. Following removal of BSA from the polymer, the AIP-MNPs presented here can selectively capture BSA with a specific absorbance (κ) as high as 65. When using this AIP, the recovery of BSA from spiked real biological samples is >97%, and the adsorption capacity is as high as 146 mg g^?1. In our perception, this method has a wide scope in that it may be applied to the specific extraction of numerous other biomolecules.

Open image in new window

Graphical abstract Schematic presentation of the AIP (aptamer-imprinted polymer) introduced here. The surface of silica coated magnetic nanoparticles is modified with a polymer that is covalently modified with an aptamer against bovine serum albumin (BSA).

     

Molecular recognition forces between immunoglobulin G and a surface protein adhesin on living Staphylococcus aureus

We report AFM measurements of binding events between immunoglobulin G (IgG) and protein A (PA) on the surface of live Staphylococcus aureus bacteria. The experiments were carried out with IgG molecules tethered via CM-amylose linkers to thiol SAMs on gold-coated AFM tips. For comparison, a model system consisting of protein A molecules tethered to thiol SAMs on gold-coated silicon substrates was also investigated. Histograms of binding forces for the PA-IgG bond showed comparable rupture forces of 59 and 64 pN for the model system and live bacteria, respectively. We suggest that linker molecules with a length comparable to the AFM tip radius should make it possible to detect specific binding events on the surface of live bacteria with a lateral resolution of a few tens of nanometers. Furthermore, because S. aureus is an important human pathogen, especially methicillin-resistant strains (MRSA), it is possible that additional virulence factors beyond PA can be probed using this technique.     

Chiral protein scissors activated by light: recognition and protein photocleavage by a new pyrenyl probe

Strong chiral discrimination and site-selective photocleavage of two model proteins, lysozyme and bovine serum albumin (BSA), by new pyrenyl probes are reported here. The enantiomeric pyrenyl probes D-phenylalanine-1(1-pyrene)methylamide (PMA- D-Phe) and L-phenylalanine-1(1-pyrene)methylamide (PMA- l-Phe) were synthesized by coupling the carboxyl function of D-phenylalanine or L-phenylalanine with the amino group of 1(1-pyrene)methylamine. Binding affinities of the two enantiomers with the proteins were quantitated in absorption titrations. BSA indicated 10-fold selectivity for PMA- D-Phe, and the binding constants for the L- and D-enantiomers were 3.8 x 10(5) and 4.0 x 10(6) M(-1), respectively. Lysozyme, similarly, indicated a 6-fold preference for PMA- D-Phe with binding constants of 3.3 x 10 (5) and 2.0 x 10(6) M(-1) for the L- and D-isomers, respectively. Such strong chiral discrimination illustrates the key role of the chiral center of the probe (Phe) in the binding interactions. The enantiomers were tested to examine how the chiral discrimination for their binding influences reactivity toward protein photocleavage. Irradiation of the probe-protein complexes, at 342 nm in the presence of hexammine cobalt(III) chloride, resulted in the cleavage of the protein backbone. Photocleavage did not proceed in the dark or in the absence of the pyrenyl probes. Both enantiomers indicated low reactivity with BSA (<5% yield), while large photocleavage yields ( approximately 57%) have been noted with lysozyme. This lysozyme photocleavage yield is a significant improvement over previous reports. However, both enantiomers cleaved lysozyme at the same location between Trp108-Val109, despite the strong chiral selectivity for binding. H-atom abstraction from Trp 108, accessible from the active site cleft, could initiate the observed peptide bond cleavage.     

Proximity-induced catalysis by the protein kinase ERK2

Five hundred protein kinases phosphorylate 10 000 proteins in human cells. Frequently, more than one site in a protein is phosphorylated, and often by more than one protein kinase. The mechanistic basis underlying the overlapping specificity of the phospho-proteome is not well understood. We are interested in understanding why ERK2, a proline-directed protein kinase, phosphorylates only a fraction of the (S/T-P) sites found in the surface loops of proteins, at an appreciable rate. To address this fundamental question, we utilized a well-established protein substrate EtsDelta138, which comprises a globular ERK2-recognition domain (pnt domain) and an unstructured peptide-like N-terminal tail. This tail contains T38, the sole ERK2 phosphorylation site. We mutated the TP motif, which is recognized by the active site and found that mutagenesis of the T-38/P-39 motif to TD, TR, TA, TG, and TV has no effect on the stability of the ternary complex but does decrease kcat. We also investigated the effect of perturbing the binding between ERK2 and the pnt domain, which occurs outside the active site, to find that mutation of the pnt domain (F120A) leads to a 10-fold decrease in binding but the kcat remains the same. The data support a mechanism of proximity-mediated catalysis, where the docking of the pnt domain, outside the active site, increases the effective concentration of the TP motif near the active site. The data are consistent with the notion that the interaction between ERK2 and the pnt domain provides uniform binding energy and stabilizes each enzyme intermediate and transition state to an equal extent. While other steps on the reaction pathway contribute towards the specificity of the ERK2 reaction, a docking interaction provides the initial basis for substrate recognition. Those residues within the docked complex, which have the ability to access the active site with an appropriate geometry, can be phosphorylated at an efficient rate if followed by a proline or small hydrophobic amino acid.