Specific Protein Sites Research Articles

Dual-Color Fluorescent Labeling of G Protein-Coupled Receptors

Single-molecule FRET (smFRET) has become a powerful tool for biophysical and biological research. Currently, progress is limited by the absence of general methods to incorporate two different fluorophores at specific sites in proteins. Thus it would be of great value to develop a labeling scheme that enables two different fluorophores to be conjugated to one receptor (protein) in a well-defined manner. To prepare the dual-color labeled receptor two requirements need to be satisfied: 1) the generation of two different reactive handles at specific positions in the receptor and 2) the appropriate bioorthogonal labeling chemistries to distinguish between them. Here we show that the combination of cysteine/maleimide reaction and azido/alkyne cycloaddition (‘click’ chemistry) meets these conditions. Maleimide chemistry specifically targets at the thiol group of cysteine, which can be easily generated or removed by mutagenesis. In contrast, the click chemistry requires the azido or alkynyl functionality, which are not naturally present in proteins. We utilized the amber codon suppression technique to incorporate unnatural amino acids carrying these reactive groups. The first fluorophore is then attached using a maleimide handle and the second by a click reaction. In a series of proof-of-concept experiments utilizing the wellcharacterized visual photoreceptor rhodopsin, we have successfully demonstrated site-specific double labeling of a GPCR. Moreover, we have optimized the maleimide labeling protocol for rhodopsin and are conducting a comparative study on the copper-catalyzed click chemistry and copper-free click chemistry. We will discuss the results in terms of background level, material cost, and labeling stoichiometry. We envision a series of interesting applications for the dual-color labeled receptors, e.g., to probe the conformational change involved in GPCR activation. We propose to generalize this approach to other proteins, thereby permiting study of GPCR signalosome.

The Physical Motive of Early Tau Oligomer Formation and Aggregation into Fibrils is Seen through Site-Specific Tracking of Hydration Dynamics

The early onset and increase in severity of Alzheimer disease symptoms are often associated with accumulation and toxic effects of the early tau oligomers, rather than highly structured fibrils. These oligomers recently became an important therapeutic target for Alzheimer treatment, yet their structural and physical characteristics are purely understood due to challenges associated with capturing and studying these transient and dynamic species that form under physiological conditions. While the essential role of biological water in mediating the protein aggregation and toxicity of oligomeric intermediates is undeniable, experimental demonstration has been lagging behind. We have recently developed and utilized a novel ultra-sensitive technique, termed Overhauser Enhanced Dynamic Nuclear Polarization (OE-DNP), for temporal tracking of the biological water dynamics at specific protein sites. This methodology has enabled us to determine the role of solvation water on the surface of tau protein as it undergoes transformations into early oligomers and further formation of amyloid fibrils. We find tau oligomers accumulate and dominate the early stages of in-vitro aggregation before appearance of elongated fibrils. Our data shows that these early intermediates are partially structured, but mostly disordered, where the core of the oligomers is filled with slow water. We find that further aggregation proceeds through structural rearrangements of tau as the early oligomers mature into the structured oligomeric blocks, that anneal in a linear manner to form elongated amyloid fibrils. We also observe that the slow water, homogeneously spanning the core of the early oligomers, shifts its signature toward the more polar and charged amino-acid regions of tau as mature fibrils are formed. Thus the loosely bound water plays an immediate role in hydrating the early oligomeric globule of tau and further self-assembly.

O2‐01‐07: Baseline cerebrospinal fluid markers in healthy middle‐aged adults predict brain health at three‐year follow‐up

reflect specific cleavage sites in proteins and, due to their smaller size, will pass the BBB. Importantly, these smaller protein fragments include more information than their intact counterparts, namely that the proteins are degraded by specific enzymes, a cardinal feature of the brain damage in AD. Methods: Tau was cleaved with BACE1, ADAM10 and caspase 3, and fragments were identified by Mass spectrometry. 10 different fragments of tau was selected for monoclonal antibody development, and one assay measuring tau degradation mediated by the protease ADAM10 using an ELISA assay (NB191) was developed. 33 paired CSF and serum samples from patients with severe ADwere investigated. Brains fromwild-type (wt) and Tg4510 transgenic mice known to develop some aspects of Alzheimer’s disease were extracted. Results: The NB191 antibody recognized the cleavage site generated by ADAM10 with an IC50 of 12.5ng/mL, but not an elongated sequence with one more amino acid (see figure). Technically robust and measurable levels of the tau neoepitope in human Alzheimer’s disease patient sera and CSF samples with averages of 21ng/ml and 2ng/ mL respectively were obtained. Finally, we compared the levels of NB191 in CSF to the levels in serum and found a correlation of R 1⁄4 0.45, borderline significant. Brain extracts from Tg4510 mice had 10-fold elevated levels of the tau neoepitope when compared to wildtype controls (p <0.001). Conclusions: In summary, we have developed a serological tau neoepitope biomarker assay that, as it crosses the BBB, may be useful for assessment of neurodegeneration in Alzheimer’s disease patients.

Protein-Induced Excited-State Dynamics of Protochlorophyllide

The light-driven NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. POR's unique requirement for light to become catalytically active makes the enzyme an attractive model to study the dynamics of enzymatic reactions in real time. Here, we use picosecond time-resolved fluorescence and femtosecond pump-probe spectroscopy to examine the influence of the protein environment on the excited-state dynamics of the substrate, protochlorophyllide (PChlide), in the enzyme/substrate (PChlide/POR) and pseudoternary complex including the nucleotide cofactor NADP(+) (PChlide/NADP(+)/ POR). In comparison with the excited-state processes of unbound PChlide, the lifetime of the thermally equilibrated S(1) excited state is lengthened from 3.4 to 4.4 and 5.4 ns in the PChlide/POR and PChlide/NADP(+)/POR complex, whereas the nonradiative rates are decreased by ∼30 and 40%, respectively. This effect is most likely due to the reduced probability of nonradiative decay into the triplet excited state, thus keeping the risk of photosensitized side reactions in the enzyme low. Further, the initial reaction path involves the formation of an intramolecular charge-transfer state (S(ICT)) as an intermediate product. From a strong blue shift in the excited-state absorption, it is concluded that the S(ICT) state is stabilized by local interactions with specific protein sites in the catalytic pocket. The possible relevance of this result for the catalytic reaction in the enzyme POR is discussed.

Photocleavage of avidin by a new pyrenyl probe

In this study, a new small-molecule-based reagent was designed to recognize and bind to specific site in protein. A new pyrenyl probe, d-biotinyl-1(1-pyrene)methylamide (Py–biotin) was designed and synthesized by coupling of d-biotin to 1(1-pyrene)methylamine hydrochloride. Binding studies and site-specific photocleavage of avidin by Py–biotin were demonstrated. Binding of Py–biotin to avidin was studied using absorbance and fluorescence spectroscopic techniques. Red shifts of the absorption peak positions of the pyrenyl chromophore followed by hyperchromism were observed upon binding to avidin. The photocleavage of avidin was achieved when a mixture of the protein, Py–biotin, and an electron acceptor, cobalt(III) hexammine trichloride (CoHA), was irradiated at 342 nm. No reaction occurred in the absence of the probe, CoHA, or light. N-terminal sequencing of the peptide fragments indicated a cleavage site of avidin between Thr 77 and Val 78. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

A Ruthenium Antimetastasis Agent Forms Specific Histone Protein Adducts in the Nucleosome Core

His-tones are not ruth-less: A ruthenium anticancer agent, RAPTA-C, was found to associate with chromatin in cancer cells and to form stable, well-defined adducts in the nucleosome core at specific histone protein sites (see picture). This illuminates the macromolecular binding properties of this antimetastasis agent and indicates potential for developing metallodrugs for site-selective targeting of chromatin.

Structural Basis for Alcohol Modulation of a Ligand-Gated Ion Channel Homolog

The molecular mechanisms underlying direct effects of ethanol and other n-alcohols on the nervous system, including intoxication and anesthesia, are poorly understood. Although physiological and behavioral evidence implicates ligand-gated ion channels in the action of alcohols and other general anesthetics, a lack of high-resolution structural information for these receptors has limited structural characterization of alcohol modulation. Recent structural and functional studies of the prokaryotic ligand-gated ion channel homolog GLIC show it to be a valuable model for structure, function, and modulation of these receptors. We are investigating the effects of alcohols on GLIC, and identifying specific protein sites underlying alcohol modulation. Our results demonstrate that GLIC exhibits differential modulation by short- and long-chain n-alcohols, similar to homologous cys-loop receptors. Mutations at putative alcohol binding sites modify alcohol modulation, while labeling with MTS reagents mimics alcohol effects. Our work supports a role for specific residues in the GLIC transmembrane domain in mediating alcohol binding and modulation.

Multiple Sequence Signals Direct Recognition and Degradation of Protein Substrates by the AAA+ Protease HslUV

Proteolysis is important for protein quality control and for the proper regulation of many intracellular processes in prokaryotes and eukaryotes. Discerning substrates from other cellular proteins is a key aspect of proteolytic function. The Escherichia coli HslUV protease is a member of a major family of ATP-dependent AAA+ degradation machines. HslU hexamers recognize and unfold native protein substrates and then translocate the polypeptide into the degradation chamber of the HslV peptidase. Although a wealth of structural information is available for this system, relatively little is known about mechanisms of substrate recognition. Here, we demonstrate that mutations in the unstructured N-terminal and C-terminal sequences of two model substrates alter HslUV recognition and degradation kinetics, including changes in V max. By introducing N- or C-terminal sequences that serve as recognition sites for specific peptide-binding proteins, we show that blocking either terminus of the substrate interferes with HslUV degradation, with synergistic effects when both termini are obstructed. These results support a model in which one terminus of the substrate is tethered to the protease and the other terminus is engaged by the translocation/unfolding machinery in the HslU pore. Thus, degradation appears to consist of discrete steps, which involve the interaction of different terminal sequence signals in the substrate with different receptor sites in the HslUV protease.

Nanometer-Scale Distance Measurements in Proteins Using Gd3+ Spin Labeling

Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd(3+) spin labeling for nanometer-range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and tau(C)14, labeled at strategically selected sites with either two nitroxides or two Gd(3+) spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd(3+) tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (approximately 9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd(3+)-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd(3+)-Gd(3+) distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein tau(C)14, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd(3+)-Gd(3+) distance distribution with a maximum at 3.4 nm. The Gd(3+)-Gd(3+) distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd(3+) labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection.

Exchange of the Coronavirus Replicase Polyprotein Cleavage Sites Alters Protease Specificity and Processing

Coronavirus nonstructural proteins 1 to 3 are processed by one or two papain-like proteases (PLP1 and PLP2) at specific cleavage sites (CS1 to -3). Murine hepatitis virus (MHV) PLP2 and orthologs recognize and cleave at a position following a p4-Leu-X-Gly-Gly-p1 tetrapeptide, but it is unknown whether these residues are sufficient to result in processing by PLP2 at sites normally cleaved by PLP1. We demonstrate that exchange of CS1 and/or CS2 with the CS3 p4-p1 amino acids in engineered MHV mutants switches specificity from PLP1 to PLP2 at CS2, but not at CS1, and results in altered protein processing and virus replication. Thus, the p4-p1 residues are necessary for PLP2 processing but require a specific protein or cleavage site context for optimal PLP recognition and cleavage.

Identification of Specific Lipid-binding Sites in Integral Membrane Proteins

Protein-lipid interactions are increasingly recognized as central to the structure and function of membrane proteins. However, with the exception of simplified models, specific protein-lipid interactions are particularly difficult to highlight experimentally. Here, we used molecular dynamics simulations to identify a specific protein-lipid interaction in lactose permease, a prototypical model for transmembrane proteins. The interactions can be correlated with the functional dependence of the protein to specific lipid species. The technique is simple and widely applicable to other membrane proteins, and a variety of lipid matrices can be used.

Profiling Carbonylated Proteins in Human Plasma

This study reports the first proteomic-based identification and characterization of oxidized proteins in human plasma. The study was conducted by isolating carbonylated proteins from the plasma of male subjects (age 32-36) with avidin affinity chromatography subsequent to biotinylation of carbonyl groups with biotin hydrazide and sodium cyanoborohydride reduction of the resulting Schiff's bases. Avidin selected proteins were digested with trypsin, and the peptide fragments were separated by C18 reversed phase chromatography and identified and characterized by both electrospray ionization and matrix assisted laser desorption ionization mass spectrometry. Approximately 0.2% of the total protein in plasma was selected with this method. Sixty-five high, medium, and low abundance proteins were identified, the majority appearing in all subjects. An interesting feature of the oxidized proteins isolated was that in addition to carbonylation they often bore other types of oxidative modification. Twenty-four oxidative modifications were mapped in 14 proteins. Fifteen carbonylation sites carried on 7 proteins were detected. Methionine oxidation was the most frequent single type of oxidative modification followed by tryptophan oxidation. Apolipoprotein B-100 had 20 oxidative modifications, the largest number for any protein observed in this study. Among the organs contributing oxidized proteins to plasma, kidney, liver, and soft tissues were the most frequent donors. One of the more important outcomes of this work was that mass spectral analysis allowed differentiation between different biological mechanisms of oxidation in individual proteins. For the first time, oxidation products arising from direct ROS oxidation of amino acid side chains in proteins, formation of advanced glycation endproducts (AGEs) adducts, and formation of adducts with lipid peroxidation products were simultaneously recognized and assigned to specific sites in proteins.

Photophysics and photochemistry of dyes bound to human serum albumin are determined by the dye localization

The photophysics and photochemistry of rose bengal (RB) and methylene blue (MB) bound to human serum albumin (HSA) have been investigated under a variety of experimental conditions. Distribution of the dyes between the external solvent and the protein has been estimated by physical separation and fluorescence measurements. The main localization of protein-bound dye molecules was estimated by the intrinsic fluorescence quenching, displacement of fluorescent probes bound to specific protein sites, and by docking modelling. All the data indicate that, at low occupation numbers, RB binds strongly to the HSA site I, while MB localizes predominantly in the protein binding site II. This different localization explains the observed differences in the dyes' photochemical behaviour. In particular, the environment provided by site I is less polar and considerably less accessible to oxygen. The localization of RB in site I also leads to an efficient quenching of the intrinsic protein fluorescence (ascribed to the nearby Trp residue) and the generation of intra-protein singlet oxygen, whose behaviour is different to that observed in the external solvent or when it is generated by bound MB.

Interdomain signalling in the blue-light sensing and GTP-binding protein YtvA: A mutagenesis study uncovering the importance of specific protein sites

YtvA from Bacillus subtilis is a blue-light responsive, flavin-binding photoreceptor, built of a light-sensing LOV domain (aa 25-126) and an NTP (nucleoside triphosphate)-binding STAS domain (aa 147-261). The STAS domain is supposed to be the effector part of the protein or a secondary switch. Both domains are connected by a linker polypeptide. The active form of YtvA is generated upon light excitation, causing the formation of a covalent bond between a cysteine residue (Cys62) in the LOV domain and the position 4a of the flavin chromophore. This photoadduct formation within the LOV domain results in a conformational change of the NTP-binding cavity, evidencing intra-protein signal transmission. We have previously shown that Glu105, localized on the beta-scaffold of the LOV-core, is involved in this process. Here, we extend this work by the identification of further residues that upon mutation supress or strongly impair signal transmission by interfering with the communication between the two domains. These comprise L106 and D109 on the LOV domain; K130 and K134 on the linker region; D193, L194 and G196 within the DLSG GTP-binding motif (switch region) and N201 on the STAS domain. Furthermore in the mutated S195A and D193A proteins, GTP affinity is diminished. Other mutations investigated have little or no effect on signal transmission and GTP-binding affinity: R63K that was found to accelerate the thermal recovery of the parent state ca. ten-fold; K128A, Q129A and Y132A within the linker region, and S183A and S212A on the STAS domain. The results show a key role of the LOV domain beta-scaffold and of positively charged residues within the linker for intra-protein signal transmission. Furthermore they evidence the conformational switch function of a structurally conserved strand-loop-helix region (bearing the DLSG GTP-binding motif and N201) within the STAS domain that constitutes a novel GTP-binding fold.

Cell-free production of Gaussia princeps luciferase – antibody fragment bioconjugates for ex vivo detection of tumor cells

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc–scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide–alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA–tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc–scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Ternary Protein Adsorption onto Brushes: Strong versus Weak

Attractive interactions between proteins and polyethylene glycol (PEG) give rise to ternary adsorption within PEG brushes. Experimental evidence suggests two ternary adsorption modes: (i) weak, due to nonspecific weak attraction between PEG monomers and the surface of the protein, as exemplified by serum albumin and (ii) strong, due to strong binding of PEG segments to specific protein sites as it occurs for PEG antibodies, which can involve the terminal adsorption of free chain ends or backbone adsorption due to binding to interior chain segments. Ternary adsorption affects the capacity of brushes to repress protein adsorption. The strong adsorption of antibodies can trigger an immune response that may affect the biocompatibility of the surface. Theoretical adsorption isotherms and protein concentration profiles of the three cases are compared for "parabolic" brushes, allowing for the grafting density, 1/Sigma, and degree of polymerization of the PEG chains, N, as well as the volume and surface area of the proteins. The amount of adsorbed protein per unit area, Gamma, exhibits a mode-specific maximum in all three cases. For backbone and weak adsorption, Gamma approximately N, whereas for terminal adsorption, Gamma approximately N0. In every case, the concentration profile of adsorbed proteins, ctern(z), exhibits a maximum at zmax>0 that shifts outward as Sigma decreases; zmax=0 occurs only for weak and backbone adsorption at a high Sigma value.

Epigenetics and Neonatology

Although an individual's cells all have the same DNA, each cell type has a different pattern of active (expressed) and inactive genes. Such patterns are established or programmed during development by epigenetic modifications involving small molecules that covalently bind to specific sites in DNA or DNA-packaging proteins. The modifications regulate gene activity without changing the DNA sequence. Diet, lifestyle factors, and exposure to toxins or other adverse factors as well as random epigenetic mutations can result in de novo epigenetic modifications that may influence the course of development. The perinatal period is a time of rapid physiologic change during which some epigenetic reprogramming is likely to occur. Further, adverse events at this time may lead to epigenetic changes that have implications for future health and well-being. Evidence suggests that epigenetics plays a part in mediating effects of the perinatal environment and that such epigenetic changes may be reversed later in life. This has major implications for understanding of the cause of preterm birth as well as the consequences of prematurity.

The Chemical Biology of Protein Phosphorylation

The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain.

Site-specific dynamic nuclear polarization of hydration water as a generally applicable approach to monitor protein aggregation

We present a generally applicable approach for monitoring protein aggregation by detecting changes in surface hydration water dynamics and the changes in solvent accessibility of specific protein sites, as protein aggregation proceeds in solution state. This is made possible through the Overhauser dynamic nuclear polarization (DNP) of water interacting with stable nitroxide spin labels tethered to specific proteins sites. This effect is highly localized due to the magnetic dipolar nature of the electron-proton spin interaction, with >80% of their interaction occurring within 5 A between the unpaired electron of the spin label and the proton of water. We showcase our tool on the aggregation of tau proteins, whose fibrillization is linked to neurodegenerative disease pathologies known as taupathies. We demonstrate that the DNP approach to monitor local changes in hydration dynamics with residue specificity and local contrast can distinguish specific and neat protein-protein packing leading to fibers from non-specific protein agglomeration or precipitation. The ability to monitor tau assembly with local, residue-specific, resolution, under ambient conditions and in solution state will help unravel the mechanism and structural characteristics of the gradual process of tau aggregation into amyloid fibers, which remains unclear to this day.

Characterisation of Cisplatin Binding Sites in Human Serum Proteins Using Hyphenated Multidimensional Liquid Chromatography and ESI Tandem Mass Spectrometry

Cisplatin binding sites in human serum proteins have been characterised by using combined multidimensional liquid chromatography and ESI tandem mass spectrometry (MudPIT). Following incubation periods of 3 h for cisplatin-blood serum mixtures and subsequent trypsin digestion, MS-MS spectra were recorded for individual peptides that had been separated by SCX and RP liquid chromatography. Matching of the MS-MS spectra to theoretical sequences that were generated for human proteins in the SWISS-PROT database led to the identification of specific binding sites in human serum albumin (HSA), serotransferrin (Trfe) and other abundant serum proteins (A2mg, A1at, Apoa1, Apoa2). The cisplatin coordination sites in HSA and Trfe were confirmed by independent MudPIT studies on cisplatin reaction mixtures with the individual proteins. A total of five specific binding sites were identified for HSA, including the cysteine residue C34, two methionine sites (M329, M548) and the tyrosine and aspartate O-donor sites Y150 (or Y148) and D375 (or E376). Methionine-256 was established as a cisplatin coordination site for Trfe in addition to the O-donor sites E265, Y314, E385 and T457. Inspection of the protein structures indicates that the preferred residues belong either to peripheral alpha helices or to flexible loops within the protein-binding pockets. O-donor residues dominate as cisplatin binding sites for other abundant serum proteins.