Site-specific labeling of proteins with small molecules in live cells

The principal bottleneck for the utilization of small-molecule probes in live cells is the shortage of methodologies for targeting them with very high specificity to biological molecules or compartments of interest. Recently developed methods for labeling proteins with small-molecule probes in cells employ special protein or peptide handles that recruit small-molecule ligands, harness enzymes to catalyze small-molecule conjugation or hijack the cell's protein translation machinery.     

Development of a small peptide tag for covalent labeling of proteins

A 21-mer peptide that can be used to covalently introduce synthetic molecules into proteins has been developed. Phage-displayed peptide libraries were subjected to reaction-based selection with 1,3-diketones. The peptide was further evolved by addition of a randomized region and reselection for improved binding. The resulting 21-mer peptide had a reactive amino group that formed an enaminone with 1,3-diketone and was used as a tag for labeling of maltose binding protein. Using this peptide tag and 1,3-diketone derivatives, a variety of molecules such as reporter probes and functionalities may be covalently introduced into proteins of interest.     

Axonal transport of proteins. A new view using in vivo covalent labeling

The injection of [2,3-3H]N-succinimidyl propionate ([3H]N-SP) into the rat sciatic nerve was used to covalently label both intra- and extra- axonal proteins. While extra-axonal proteins (e.g., myelin proteins) remained in the injection site, the intra-axonal proteins were transported in both the anterograde and retrograde directions. The mobile labeled proteins appeared to move by normal axonal transport processes because: (a) autoradiographic studies showed that they were localized exclusively within the axon at considerable distances from the injection site, (b) specific and identifiable proteins (by SDS gel electrophoresis) moved at expected rates in the anterograde direction, and (c) an entirely different profile of proteins moved in the anterograde vs. retrograde direction. This novel experimental approach to axonal transport, which is independent of de novo protein synthesis, provided a unique view of slow anterograde transport, and particularly of retrograde transport of endogenous proteins. A large quantity of a 68,000 mol wt proteins, moving at approximately 3-6 mm/day, dominated the retograde transport profile. [3H]N-SP, therefore, represents a new and unique "vital stain" which may find many applications in cell biology.     

Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro

The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro.     

A method for the covalent capture and screening of diverse small molecules in a microarray format

This protocol describes a robust method for the covalent capture of small molecules with diverse reactive functional groups in microarray format, and outlines a procedure for probing small-molecule microarrays (SMMs) with proteins of interest. A vapor-catalyzed, isocyanate-mediated surface immobilization scheme is used to attach bioactive small molecules, natural products and small molecules derived from diversity-oriented synthesis pathways. Additionally, an optimized methodology for screening SMMs with purified proteins and cellular lysates is described. Finally, a suggested model for data analysis that is compatible with commercially available software is provided. These procedures enable a platform capability for discovering novel interactions with potential applications to immunoglobulin profiling, comparative analysis of cellular states and ligand discovery. With the appropriate materials and experimental setup, the printing of SMMs can be completed in 14 hours over 3 days. Screening and data analysis requires 2 days. A detailed timeline is provided.     

Site-specific covalent labeling of proteins inside live cells using small molecule probes

The study of dynamic movement and interactions of proteins inside living cells in real time is critical for a better understanding of cellular mechanisms and functions in molecular detail. Genetically encoded fusions to fluorescent protein(s) (FP) have been widely used for this purpose [Annu. Rev. Biochem. 1998, 67, 509-544]. To obviate some of the drawbacks associated with the use of FPs [Curr. Opin. Biotechnol. 2005, 16, 1-6; Nat. Methods2006, 3, 591-596], we report a small molecule-based approach that exploits the unique reactivity between the cysteine residue at the N-terminus of a target protein and cell-permeable, thioester-based small molecule probes resulting in site-specific, covalent tagging of proteins. This approach has been demonstrated by the in vivo labeling of proteins in both bacterial and mammalian systems thereby making it potentially useful for future bioimaging applications.     

A method for efficient isotopic labeling of recombinant proteins

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for ¹³C labeling of the C-terminal domain of angiopoietin-2, ¹⁵N labeling of ubiquitin and for ²H/¹³C/¹⁵N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.     

The use of diaminodithiol for labeling small molecules with technetium-99m

To investigate the labeling of small molecules with ^99mTc by the bifunctional chelate approach, we have synthesized both a fatty acid and an estrone derivative containing a chelator of the N₂S₂ type. In the case of the fatty acid, this was a diaminodithiol (DADT) while for the estrone, a diaminodisulfide (DADS) was attached. The estrone derivative (5-(2-methylene estrone 3-methyl ether)-3,3,10,10-tetramethyl-1, 2-dithia-5,8-diazacyclodecane hydrochloride, DADS-E) was prepared by alkylation of DADS while the fatty acid derivative (N-(11-undecanoic acid)-N,N′-bis(2-methyl-2-mercaptopropyl) ethylenediamine hydrochloride, DADT-FA) was synthesized by alkylation of DADS followed by reduction. DADS-E was labeled in ethanol at elevated temperatures while DADT-FA was labeled at room temperature, both by stannous reduction. Paper chromatography showed both to be labeled and reverse-phase HPLC showed multiple peaks for both. Serum stability studies were performed by incubation at 37 °C with aliquots removed at 1 min and 1 day for analysis by size-exclusion HPLC. Initially, little pertechnetate or binding to serum proteins was observed whereas after 1 day the majority of activity in both cases was protein bound with 20 and 38% pertechnetate appearing for DADT-FA and DADS-E respectively. In conclusion, small biologically active molecules may be labeled with ^99mTc through an attached diaminodithiol or diaminodisulfide group.     

A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies

Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state or time-resolved fluorescence resonance energy transfer (FRET) detection of the kinetics of protein folding require relatively large quantities (approximately 10-100 mg) of site-specific doubly labeled protein samples. Engineered cysteine residues are common targets for labeling of proteins. The challenge here is to develop methods for selective modification of one of two reactive sulfhydryl groups in a protein molecule. A general systematic procedure for selective labeling of each of two cysteine residues in a protein molecule was developed, using Escherichia coli adenylate kinase (AKe) as a model protein. Potential sites for insertion of cysteine residues were selected by examination of the crystal structure of the protein. A series of single-cysteine mutants was prepared, and the rates of the reaction of each engineered cysteine residue with a reference reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] were determined. Two-cysteine mutants were prepared by selection of pairs of sites for which the ratio of this reaction rate constant was high (>80). The conditions for the selective labeling reaction were optimized. In a first cycle of labeling, the more reactive cysteine residue was labeled with a fluorescent probe (donor). The second probe was attached to the less reactive site under unfolding conditions in the second cycle of labeling. The doubly and singly labeled mutants retained full enzymatic activity and the capacity for a reversible folding-unfolding transition. High yields (70-90%) of the preparation of the pure, site-specific doubly labeled AK mutant were obtained. The procedure described herein is a general outline of procedures, which can meet the double challenge of both site specificity and large-scale preparation of doubly labeled proteins.     

A method for covalent insertion of mercury into the cysteine disulfide bridges of proteins

A method is described by which atomic mercury can be taken up by thiol groups and inserted into the disulfide bridges of proteins which can be reversibly reduced and denatured. The method utilizes tandem columns of Sephadex G-10 and Biogel P2. Protein samples are separated from reducing and denaturing agent on the Sephadex column and then react with mercury, which is bound to the Biogel P2 column. Of eight proteins tested, all took up mercury using this method. The amount of mercury incorporated by this method differed from that found using other methods and was closer to the stoichiometry of the disulfide bridges of the protein than these methods.     

A novel method for selective isotope labeling of bacterially expressed proteins

Summary A novel method for isotope labeling in selected amino acids is presented for use with the T7 RNA polymerase system. The protocol is illustrated with the DNA-binding domain from the E2 protein of bovine papillomavirus, BPV-1. On addition of rifampicin, protein expression occurs exclusively from the gene controlled by the T7 promoter. Since the bacteria are now dedicated to the production of E2 protein, labeling with specific amino acids is efficiently performed. For example, 10 mg/l of ¹⁵N-labeled phenylalanine is shown to be sufficient for incorporation of the label, without scrambling, and without the use of an auxotrophic strain.     

An improved method for the covalent coupling of proteins to liposomes

A method is described for the preparation of liposomes carrying covalently attached protein at the outer surface. It is based upon the reaction between proteins, thiolated with the heterobifunctional reagent succinimidyl-S-acetylthioacetate (SATA), and liposomal maleimido-4-(p-phenylbutyryl)phosphatidyl-ethanolamine. Advantages of the procedure are that it is not restricted to proteins carrying native SH-groups and that it is substantially more convenient and less time-consuming than previously published methods. Applications for these liposomes are to be found in the fields of liposome targeting and the production of monoclonal antibodies.     

A method for the determination of diffusion coefficients for small molecules in aqueous solution

A method is described for determining the diffusion coefficients of small solutes in limited volumes (approximately equal to 4-9 ml) of fluid. Diffusion is measured in a three-chamber diffusion cell across a central unstirred compartment. Compartments are separated by nitrocellulose membranes. The instantaneous concentration gradient and the instantaneous flux of solute into the dilute end compartment are derived from changes in the concentration of solute in the two stirred end compartments through time. The diffusion coefficient is calculated from the slope of the least-squares regression line relating the magnitude of the instantaneous solute flux to that of the instantaneous concentration gradient. The apparatus is calibrated with a solute of known diffusivity (KCl). Diffusion coefficients thus determined in water at 25 degrees C for CaCl2 (7.54 X 10(-6) cm2.s-1), Na2-ATP (7.01 X 10(-6) cm2.s-1), 2-deoxyglucose (5.31 X 10(-6) cm2.s-1), and D-Na-lactate (5.62 X 10(-6) cm2.s-1) differed by an average of 3.7% from literature values. The method described results in accurate estimates of diffusion coefficients by a simple and relatively rapid procedure.     

Synthesis and characterization of bifunctional probes for the specific labeling of fusion proteins

Labeling proteins with synthetic probes is important for studying and characterizing protein function. We have recently introduced a general method for the specific in vivo and in vitro labeling of fusion proteins that is based on the reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine derivatives. Here we report two complementary routes for the synthesis of O6-benzylguanine derivatives, which allow for the labeling of AGT fusion proteins with bifunctional synthetic probes and demonstrate the specific labeling of AGT fusion proteins with these probes. These molecules should become useful tools for various applications in functional proteomics.     

Functional solubilization of aggregation-prone HIV envelope proteins by covalent fusion with chaperone modules

The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus (HTLV) mediate cell attachment and membrane fusion. For HIV-1, the precursor protein gp160 is cleaved proteolytically into two fragments, the surface-associated receptor binding subunit gp120 and the membrane spanning subunit gp41, which is involved in membrane fusion during virus entry. Soluble and immunoreactive variants of gp41 are essential for the reliable diagnosis of HIV-1 infections. Hitherto, gp41 was solubilized by adding detergents, or in acidic or alkaline solvents. We find that covalent fusions with SlyD or FkpA, two homodimeric Escherichia coli chaperones with peptidyl-prolyl isomerase activity, solubilize retroviral envelope proteins without compromising their immunological reactivity. gp41 from HIV-1, gp36 from HIV-2 and gp21 from HTLV could be expressed in large amounts in the Escherichia coli cytosol when fused with one or two subunits of SlyD or FkpA. The fusion proteins could be easily isolated and refolded, and showed high solubility and immunoreactivity, thus providing sensitive and reliable tools for diagnostic applications. Covalent fusions with SlyD or FkpA might be valuable generic tools for the solubilization and activation of aggregation-prone proteins.     

A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells.

A method to obtain uniformly isotopically labeled (15N and 15N/13C) protein from mammalian cells is described. The method involves preparation of isotopically labeled media consisting of amino acids isolated from bacterial and algal extracts supplemented with cysteine and enzymatically synthesized glutamine. The approach is demonstrated by producing 15N-labeled and 15N/13C-labeled urokinase from Sp2/0 cells and successfully growing Chinese hamster ovary (CHO) cells on the labeled media. Thus, using the procedures described, isotopically labeled proteins that have been expressed in mammalian cells can be prepared, allowing them to be studied by heteronuclear multidimensional NMR techniques.