Selective Labeling Strategies Research Articles

Discovery of novel inhibitors of ribosome biogenesis by innovative high throughput screening strategies.

Over the past two decades, ribosome biogenesis has emerged as an attractive target for cancer treatment. In this study, two high-throughput screens were used to identify ribosome biogenesis inhibitors. Our primary screen made use of the HaloTag selective labeling strategy to identify compounds that decreased the abundance of newly synthesized ribosomes in A375 malignant melanoma cells. This screen identified 5786 hit compounds. A subset of those initial hit compounds were tested using a secondary screen that directly measured pre-ribosomal RNA (pre-rRNA) abundance as a reporter of rRNA synthesis rate, using quantitative RT-PCR. From the secondary screen, we identified two structurally related compounds that are potent inhibitors of rRNA synthesis. These two compounds, Ribosome Biogenesis Inhibitors 1 and 2 (RBI1 and RBI2), induce a substantial decrease in the viability of A375 cells, comparable to the previously published ribosome biogenesis inhibitor CX-5461. Anchorage-independent cell growth assays further confirmed that RBI2 inhibits cell growth and proliferation. Thus, the RBI compounds have promising properties for further development as potential cancer chemotherapeutics.

Selective isotope labeling strategy and computational interpretation of spectra for protein NMR analyses

Signal assignment is a mandatory step for the site-specific analyses of proteins by NMR. In challenging cases, such as low solubility target proteins or in-cell NMR, amino-acid selective stable isotope labeling facilitates the main-chain assignment, since it can determine the amino-acid type of each signal, thus providing complementary information to that obtained by standard three-dimensional triple resonance experiments. Inspired by information science methodologies, we developed a combinatorial selective labeling strategy, named stable isotope encoding (SiCode), to enable amino-acid typing even from noisy NMR spectra, with small numbers of labeled samples. The high noise-resistance of SiCode is achieved by both optimization of the isotope labeling pattern according to the information distance between amino acids and model-based computational retrieval of the amino-acid information from spectra, using replica exchange Monte Carlo computation. We demonstrate amino-acid typing with simulated low signal-to-noise-ratio spectra, with sample concentrations as low as micromolar order. Since the main-chain signal assignment is often a bottleneck process for various amide-signal-based NMR analyses of challenging target proteins, SiCode will facilitate and accelerate such analyses.

SAM‐II Riboswitch Samples at least Two Conformations in Solution in the Absence of Ligand: Implications for Recognition

AbstractConformational equilibria are increasingly recognized as pivotal for biological function. Traditional structural analyses provide a static image of conformers in solution that sometimes present conflicting views. From 13C and 1H chemical exchange saturation transfer experiments, in concert with ligation and selective labeling strategies, we show that in the absence of metabolite, a Mg2+ (0–0.5 mm)‐bound apo SAM‐II riboswitch RNA exists in a minor (≈10 %) partially closed state that rapidly exchanges with a predominantly (≈90 %) open form with a lifetime of ≈32 ms. The base and sugar (H6,C6, H1′,C1′) chemical shifts of C43 for the dominant conformer are similar to those of a free CMP, but those of the minor apo species are comparable to shifts of CMPs in helical RNA regions. Our results suggest that these transient, low populated states stabilized by Mg2+ will likely enhance rapid ligand recognition and, we anticipate, will play potentially ubiquitous roles in RNA signaling.

Isobaric cross-sequence labeling of peptides by using site-selective N-terminus dimethylation

Highly site-selective dimethyl labeling of N-terminus of peptides has been obtained by adjusting the acidic strength of the reaction solution. And this selective labeling strategy combined with the use of different isotope formaldehyde reagents has been successfully used in the proteome quantification by using the isobaric peptide cross-sequence labeling method, which would play increasingly important roles in the clinical diagnosis, especially in the discovery of biomarkers for diseases.

Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.

Proteins are often made in more than one form, with alternate versions sometimes residing in different cellular compartments than the primary species. The mammalian prion protein (PrP), a cell surface GPI-anchored protein, is a particularly noteworthy example for which minor cytosolic and transmembrane forms have been implicated in disease pathogenesis. To study these minor species, we used a selective labeling strategy in which spatially restricted expression of a biotinylating enzyme was combined with asymmetric engineering of the cognate acceptor sequence into PrP. Using this method, we could show that even wild-type PrP generates small amounts of the (Ctm)PrP transmembrane form. Selective detection of (Ctm)PrP allowed us to reveal its N-terminal processing, long half-life, residence in both intracellular and cell surface locations, and eventual degradation in the lysosome. Surprisingly, some human disease-causing mutants in PrP selectively stabilized (Ctm)PrP, revealing a previously unanticipated mechanism of (Ctm)PrP up-regulation that may contribute to disease. Thus, spatiotemporal tagging has uncovered novel aspects of normal and mutant PrP metabolism and should be readily applicable to the analysis of minor topologic isoforms of other proteins.

Site‐specific Fluorescent Labeling of Poly‐histidine Sequences Using a Metal‐chelating Cysteine

Coupling genetically encoded target sequences with specific and selective labeling strategies has made it possible to utilize fluorescence spectroscopy in complex mixtures to investigate the structure, function, and dynamics of proteins. Thus, there is a growing need for a repertoire of such labeling approaches to deploy based on a given application and to utilize in combination with one another by orthogonal reactivity. We have developed a simple approach to synthesize a fluorescent probe that binds to a poly-histidine sequence. The amino group of cysteine was converted into nitrilotriacetate to create a metal-chelating cysteine molecule, Cys-nitrilotriacetate. Two Cys-nitrilotriacetate molecules were then cross-linked using dibromobimane to generate a fluorophore capable of binding a His-tag on a protein, NTA(2)-BM. NTA(2)-BM is a potential fluorophore for selective tagging of proteins in vivo.

Site-selective labeling strategies for screening by NMR.

NMR based screening has become an important tool in the pharmaceutical industry. Methods that provide information on the location of small molecule binding sites on the surface of a drug target (e. g. SAR-by-NMR and related techniques) are of particular interest. In order to extend the applicability of such techniques to drug targets of higher molecular weight, selective labeling strategies may be employed. Dual-amino acid selective labeling and site directed non-native amino acid replacement (SNAAR) allow for the selective detection of NMR resonances of a specific amino acid residue. This results in significantly reduced spectral complexity, which not only enables application to higher molecular weight systems, but also eliminates the need for sequential resonance assignment in order to identify the binding site. Regio-selective (or segmental) labeling of an entire protein domain of a multi domain protein may also be achieved. Labeling only a selected part of a multi domain protein (e. g. a catalytic or ligand binding domain) is an attractive way to simplify the spectral interpretation without disturbing the system under study.

Characterization of pheophytin ground states in Rhodobacter sphaeroides R26 photosynthetic reaction centers from multispin pheophytin enrichment and 2-D 13C MAS NMR dipolar correlation spectroscopy.

The electronic ground states of pheophytin cofactors potentially involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have been investigated through a characterization of the electron densities at individual atomic positions of pheophytin a from 13C chemical shift data. A new experimental approach involving multispin 13C labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26 were reconstituted with uniformly 13C biosynthetically labeled (plant) Pheo a in the two pheophytin binding sites. From the multispin labeled samples 1-D and 2-D solid-state 13C magic angle spinning NMR spectra could be obtained and used to characterize the pheophytin a ground state in the Rb. sphaeroides R26 RCs, i.e., without a necessity for time-consuming selective labeling strategies involving organic synthesis. From the 2-D solid state 13C-13C correlation spectra collected with spinning speeds of 8 and 10 kHz, with mixing times of 1 and 0.8 ms, many 13C resonances of the [U-13C]Pheo a molecules reconstituted in the RCs could be assigned in a single set of experiments. Parts of the pheophytins interacting with the protein, at the level of 13C shifts modified by binding, could be identified. Small reconstitution shifts are detected for the 17(2) side chain of ring IV. In contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective electrostatic interaction with Glu L104 on the active branch. The protonation states appear the same, and the NMR suggests a strong overall similarity between the ground states of the two Pheo a, which is of interest in view of the asymmetry of the electron transfer.

Identification of a Cysteine Residue Essential for Activity of Protein Farnesyltransferase: Cys299 IS EXPOSED ONLY UPON REMOVAL OF ZINC FROM THE ENZYME

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zinc-depleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM. These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [3H]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys299, that is in the beta subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified. While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys299 in the beta subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.