A competitive labeling method for the determination of the chemical properties of solitary functional groups in proteins.

The properties of the functional groups in a protein can be used as built-in-probes of the structure of the protein. We have developed a general procedure whereby the ionization constant and chemical reactivity of solitary functional groups in proteins may be determined. The method may be applied to the side chain of histidine, tyrosine, lysine, and cysteine, as well as to the amino terminus of the protein. The method, which is an extension of the competitive labeling technique using [3H]- and [14C]1-fluoro-2,4-dinitrobenzene (N2ph-F) in a double-labeling procedure, is rapid and sensitive. Advantage is taken of the fact that after acid hydrolysis of a dinitrophenylated protein, a derivative is obtained which must be derived from a unique position in the protein. The method has been applied to the solitary histidine residue of lysozyme, alpha-lytic protease, and Streptomyces griseus (S.G.) trypsin, as well as to the amino terminus of the latter protein. The following parameters were obtained for reaction with N2ph-F at 20 degrees C in 0.1 N KCl: the histidine of hen egg-white lysozyme, pKa of 6.4 and second-order velocity constant of 0.188 M-1 min-1; the histidine of alpha-lytic protease, pKa of 6.5 and second-order velocity constant of 0.0235 M-1 min-1; the histidine of S.G. trypsin, pKa of 6.5 and second-order velocity constant of 0.0328 M-1 min-1; the valyl amino terminus of S.G. trypsin, pKa of 8.1 and second-order velocity constant of 0.403 M-1 min-1. In addition, the results obtained provide clues as to the microenvironments of these functional groups, and indicate that the proteins studied undergo pH-dependent conformational changes which affect the microenvironment, and hence the chemical reactivity of these groups.     

Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins

The membrane-permeant fluorogenic biarsenicals FlAsH-EDT(2) and ReAsH-EDT(2) can be prepared in good yields by a straightforward two-step procedure from the inexpensive precursor dyes fluorescein and resorufin, respectively. Handling of toxic reagents such as arsenic trichloride is minimized so the synthesis can be carried out in a typical chemistry laboratory, usually taking about 2-3 d. A wide range of other biarsenical reagents and intermediates that also bind to tetracysteine-tagged (CysCysProGlyCysCys) proteins can be prepared similarly using this general procedure.     

Specific inhibition of sensitized protein tyrosine phosphatase 1B (PTP1B) with a biarsenical probe

Protein tyrosine phosphatase 1B (PTP1B) is a key regulator of the insulin-receptor and leptin-receptor signaling pathways, and it has therefore emerged as a critical antitype-II-diabetes and antiobesity drug target. Toward the goal of generating a covalent modulator of PTP1B activity that can be used for investigating its roles in cell signaling and disease progression, we report that the biarsenical probe FlAsH-EDT(2) can be used to inhibit PTP1B variants that contain cysteine point mutations in a key catalytic loop of the enzyme. The site-specific cysteine mutations have little effect on the catalytic activity of the enzyme in the absence of FlAsH-EDT(2). Upon addition of FlAsH-EDT(2), however, the activity of the engineered PTP1B is strongly inhibited, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. We show that the cysteine-rich PTP1B variants can be targeted with the biarsenical probe in either whole-cell lysates or intact cells. Together, our data provide an example of a biarsenical probe controlling the activity of a protein that does not contain the canonical tetra-cysteine biarsenical-labeling sequence CCXXCC. The targeting of "incomplete" cysteine-rich motifs could provide a general means for controlling protein activity by targeting biarsenical compounds to catalytically important loops in conserved protein domains.     

Use of Iodo-Gen and Iodine-125 to label the outer membrane proteins of whole cells of Neisseria gonorrhoeae

The outer membrane proteins of Neisseria gonorrhoeae are specifically labeled by use of 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodo-Gen) and ¹²⁵I under the conditions described in this report. Use of this procedure with whole cells of N. gonorrhoeae produces a clear labeling pattern which can be visualized by electrophoretic separation of the proteins, followed by autoradiography. Electrophoretograms reveal some 70 polypeptide bands, while autoradiograms reveal only 5 or 6 labeled bands. The labeled polypeptide bands correspond to isolated outer membrane proteins, the most intensely labeled of which is the principal outer membrane protein. The method described in this report is both specific and gentle, as well as rapid and convenient.     

Fluorescent labeling of cysteinyl residues to facilitate electrophoretic isolation of proteins suitable for amino-terminal sequence analysis

A protein labeling procedure which enables detection of subpicomole quantities of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gels is described. Proteins are rendered fluorescent by reduction of disulfide bonds with dithiothreitol followed by alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) or 5-iodoacetamido-fluorescein. Labeling is performed prior to electrophoresis, thus eliminating the need for staining with dyes and destaining after electrophoresis. As little as 375 fmol (25 ng) of prelabeled bovine serum albumin can be readily visualized after electrophoresis. Bands are still visible after electrophoretic transfer to nitrocellulose. Simultaneous labeling of proteins in complex mixtures is possible using this technique. This includes cysteine containing proteins of disrupted Newcastle disease virus. The magnitudes of the molecular weight increases which occur upon labeling reflect the cysteine contents of proteins. The mode of chemical modification for the prelabeling procedure was chosen because of its compatibility with analytical techniques, such as amino acid analysis, peptide mapping, or sequence analysis, which may be applied to the protein after electroelution from SDS-acrylamide gels. It replaces the need for reduction and carboxymethylation prior to these analytical procedures. Protein-sequence analysis of prelabeled bovine serum albumin, including samples electroeluted from SDS-acrylamide gels, has justified the choice of this method to facilitate isolation of proteins for sequence analysis. Equivalent sequence data were obtained with reduced bovine serum albumin S-alkylated with iodoacetic acid or 5-I-AEDANS.     

Determining the number of specific proteins in cellular compartments by quantitative microscopy

This protocol describes a method for determining both the average number and variance of proteins, in the few to tens of copies, in isolated cellular compartments such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number, but lack information on the variance, or they are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling of the cellular compartment with fluorescent primary-secondary antibody complexes, total internal reflection fluorescence microscopic imaging of the cellular compartment, digital image analysis and deconvolution of the fluorescence intensity data. A minimum of 2.5 d is required to complete the labeling, imaging and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes.     

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.     

Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry

A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.     

Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing

Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a ¹³C-labeled retinal cofactor and extensively ¹³C-¹⁵N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications.     

Thinking outside the crystal: complementary approaches for examining transporter conformational change

As the number of high-resolution structures of membrane proteins continues to rise, so has the necessity for techniques to link this structural information to protein function. In the case of transporters, function is achieved via coupling of conformational changes to substrate binding and release. Static structural data alone cannot convey information on these protein movements, but it can provide a high-resolution foundation on which to interpret lower resolution data obtained by complementary approaches. Here, we review selected biochemical and spectroscopic methods for assessing transporter conformational change. In addition to more traditional techniques, we present 1?F-NMR as an attractive method for characterizing conformational change in transporters of known structure. Using biosynthetic labeling, multiple, non-perturbing fluorine-labeled amino acids can be incorporated throughout a protein to serve as reporters of conformational change. Such flexibility in labeling allows characterization of movement in protein regions that may not be accessible via other methods.     

A combination of protein profiling and isotopomer analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometry reveals an active metabolism of the extracellular matrix of 3T3-L1 adipocytes

Differential gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a commonly used protein profiling method. However, observed changes can be explained in multiple ways, one of which is by the protein turnover rate. In order to easily and rapidly obtain information on both the identity and turnover of individual proteins, we applied a combination of protein labeling with L-(ring-2,3,4,5,6 2H5) phenylalanine and MALDI-TOF MS. While the spectrum reveals the identity of a protein, mass isotopomer analysis provides information about the rate of protein labeling as a measure of synthesis or turnover. Using this approach on mature 3T3-L1 adipocytes, we were able to discriminate between rapidly and slowly metabolised proteins. In our isolate, proteins of the cytoskeleton appeared to be slowly metabolised, whereas components of the extracellular matrix, in particular collagen type I alpha 1 (COL1A1) and collagen type I alpha 2 (COL1A2) showed rapid accumulation of newly synthesized proteins. Both proteins appeared to be metabolised in the same ratio as they are present in collagen fibers, i.e. 2:1 (COL1A1: COL1A2). In addition, functionally related proteins were also readily labeled. Taken together, we have shown that a combination of stable isotope labeling and protein profiling by gel electrophoresis and MALDI-TOF analysis can simultaneously provide information on the identity and relative metabolic rate of proteins in eukaryotic cells in a simple, nonhazardous and rapid-throughput way.     

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P?], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P? that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P? headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P? headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P?-binding proteins.     

An efficient on-column expressed protein ligation strategy: application to segmental triple labeling of human apolipoprotein E3

Expressed protein ligation (EPL) is an intein-based approach that has been used for protein engineering and biophysical studies of protein structures. One major problem of the EPL is the low yield of final ligation product, primarily due to the complex procedure of the EPL, preventing EPL from gaining popularity in the research community. Here we report an efficient on-column EPL strategy, which focuses on enhancing the expression level of the intein-fusion protein that generates thioester for the EPL. We applied this EPL strategy to human apolipoprotein E (apoE) and routinely obtained 25-30 mg segmental, triple-labeled apoE from 1-L cell culture. The approaches reported here are general approaches that are not specific for apoE, thus providing a general strategy for a highly efficient EPL. In addition, we also report an isotopic labeling scheme that double-labels one domain and keeps the other domain of apoE deuterated. Such an isotopic labeling scheme can only be achieved using the EPL strategy. Our data indicated that the segmental triple-labeled apoEs using this labeling scheme produced high-quality, simplified NMR spectra, facilitating NMR spectral assignment. For large proteins, such as apoE, perdeuterated protein samples have to be used to reduce the linewidth of NMR signals, causing a major problem for the NOE-based NMR method, since perdeuterated proteins lack protons for NOE measurement. The new labeling strategy solves this problem and provides (13)C/(15)N double-labeled, protonated protein domains, allowing for determination of high-resolution NMR structure of these large proteins.     

Radioiodination of proteins by reductive alkylation

The use of the aliphatic aldehyde, para-hydroxyphenylacetaldehyde as the reactive moiety in the radioiodination of proteins by reductive alkylation is described. The para-hydroxyphenyl group is radiolabeled with 125I, reacted through its aliphatic aldehyde group with primary amino groups on proteins to form a reversible Schiff base linkage which can then be stabilized with the mild reducing agent NaCNBH3. The introduction of the methylene group between the benzene ring and the aldehyde group increases its reactivity with protein amino groups permitting efficient labeling at low aldehyde concentrations. Using this method, radioiodinated proteins with high specific activity can be produced. The reductive alkylation procedure is advantageous in that the labeling conditions are mild, the reaction is specific for lysyl residues, and the modification of the epsilon-ammonium group of lysine results in ionizable secondary amino groups avoiding major changes in protein charge.     

Protein folding mechanisms studied by pulsed oxidative labeling and mass spectrometry

Deciphering the mechanisms of protein folding remains a considerable challenge. In this review we discuss the application of pulsed oxidative labeling for tracking protein structural changes in a time-resolved fashion. Exposure to a microsecond OH pulse at selected time points during folding induces the oxidation of solvent-accessible side chains, whereas buried residues are protected. Oxidative modifications can be detected by mass spectrometry. Folding is associated with dramatic accessibility changes, and therefore this method can provide detailed mechanistic insights. Solvent accessibility patterns are complementary to H/D exchange investigations, which report on the extent of hydrogen bonding. This review highlights the application of pulsed OH labeling to soluble proteins as well as membrane proteins.     

Site-specific labeling of proteins with cyclen-bound transition metal ions

A general procedure for site-specific and reversible labeling of proteins with transition metal ions is described. The method is based on the use of the novel ligand 1-(2-thioethyl)-1,4,7,10-tetraazacyclododecane (TETAC), which specifically and readily reacts with thiol groups. Synthesis of TETAC from 1,4,7,10-tetraazacyclododecane (cyclen) and ethylene disulfide yielded a mixture of products, including TETAC and its oxidized disulfide in 56.4% yield. The procedure for labeling proteins with TETAC is straightforward and led to separation of the TETAC-containing product mixture through gel-filtration chromatography. The resulting protein-TETAC adducts were shown to contain a single TETAC group which bound transition metal ions. Protein-TETACCu²⁺ had a UV-Vis spectrum similar to that of Cu²⁺(cyclen) while the protein-TETACCo²⁺ complex had a different spectrum to that of the cobalt-containing cyclen. This is because attachment to the protein prevented the Co²⁺-containing TETAC from dimerising and binding O₂, which the cobalt-containing cyclen is able to do. The proteins used to develop this labeling procedure were the DNase domain of colicin E9 and its inhibitor protein Im9. Unlike Im9, the DNase does not contain a cysteine residue but the Ser30Cys variant of the DNase was prepared by site-directed mutagenesis. Both Im9 and the Ser30Cys DNase were modified with TETAC and the modifications shown to be structurally and functionally benign through NMR spectroscopy of the modified Im9 and fluorescence spectroscopy binding assays in which DNase-Im9 complexes were formed. The simplicity of the method, and its general application to any protein through the introduction of cysteine by site-directed mutagenesis, suggests it will be of wide use in protein chemistry applications.     

A versatile procedure for the radioiodination of proteins and labeling reagents

For tracer or analytical studies it is often useful to label proteins by direct iodination or by reacting them with an iodinated reagent. A simple iodination technique with hydrogen peroxide is described for use with either carrier-free or low-specific-activity iodine. The method introduces less oxidative damage to proteins than any other procedure tested, yet the efficiency of labeling approaches that offered by the chloramine T or Iodogen methods. The method has been applied to the facile and inexpensive preparation of the iodinated Bolton-Hunter reagent. This peroxide iodination procedure should be particularly useful for labeling proteins or peptides for structural investigations or for immunoassays.     

Inverse labeling-mass spectrometry for the rapid identification of differentially expressed protein markers/targets

Comparative proteomic studies can lead to the identification of protein markers for disease diagnostics and protein targets for potential disease interventions. An inverse labeling strategy based on the principle of protein stable isotope labeling and mass spectrometric detection has been successfully applied to three general protein labeling methods. In contrast to the conventional single experiment approach, two labeling experiments are performed in which the initial labeling is reversed in the second experiment. Signals from differentially expressed proteins will distinguish themselves by exhibiting a characteristic pattern of isotope intensity profile reversal that will lead to the rapid identification of these proteins. Application of the inverse labeling method is demonstrated using model systems for protein chemical labeling, protein proteolytic labeling, and protein metabolic labeling. The methodology has clear advantages which are illustrated in the various studies. The inverse labeling strategy permits quick focus on signals from differentially expressed proteins (markers/targets) and eliminates ambiguities caused by the dynamic range of detection. In addition, the inverse labeling approach enables the unambiguous detection of covalent changes of proteins responding to a perturbation.     

Minimizing the overlap problem in protein NMR: a computational framework for precision amino acid labeling

MOTIVATION: Recent advances in cell-free protein expression systems allow specific labeling of proteins with amino acids containing stable isotopes ((15)N, (13) C and (2)H), an important feature for protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. Given this labeling ability, we present a mathematical optimization framework for designing a set of protein isotopomers, or labeling schedules, to reduce the congestion in the NMR spectra. The labeling schedules, which are derived by the optimization of a cost function, are tailored to a specific protein and NMR experiment. RESULTS: For 2D (15)N-(1)H HSQC experiments, we can produce an exact solution using a dynamic programming algorithm in under 2 h on a standard desktop machine. Applying the method to a standard benchmark protein, calmodulin, we are able to reduce the number of overlaps in the 500 MHz HSQC spectrum from 10 to 1 using four samples with a true cost function, and 10 to 4 if the cost function is derived from statistical estimates. On a set of 448 curated proteins from the BMRB database, we are able to reduce the relative percent congestion by 84.9% in their HSQC spectra using only four samples. Our method can be applied in a high-throughput manner on a proteomic scale using the server we developed. On a 100-node cluster, optimal schedules can be computed for every protein coded for in the human genome in less than a month. AVAILABILITY: A server for creating labeling schedules for (15)N-(1)H HSQC experiments as well as results for each of the individual 448 proteins used in the test set is available at http://nmr.proteomics.ics.uci.edu.     

Differential membrane proteomics using 18O-labeling to identify biomarkers for cholangiocarcinoma

Quantitative proteomic methodologies allow profiling of hundreds to thousands of proteins in a high-throughput fashion. This approach is increasingly applied to cancer biomarker discovery to identify proteins that are differentially regulated in cancers. Fractionation of protein samples based on enrichment of cellular subproteomes prior to mass spectrometric analysis can provide increased coverage of certain classes of molecules. We used a membrane protein enrichment strategy coupled with 18O labeling based quantitative proteomics to identify proteins that are highly expressed in cholangiocarcinomas. In addition to identifying several proteins previously known to be overexpressed in cholangiocarcinoma, we discovered a number of molecules that were previously not associated with cholangiocarcinoma. Using immunoblotting and immunohistochemical labeling of tissue microarrays, we validated Golgi membrane protein 1, Annexin IV and Epidermal growth factor receptor pathway substrate 8 (EPS8) as candidate biomarkers for cholangiocarcinomas. Golgi membrane protein 1 was observed to be overexpressed in 89% of cholangiocarcinoma cases analyzed by staining tissue microarrays. In light of recent reports showing that Golgi membrane protein 1 is detectable in serum, further investigation into validation of this protein has the potential to provide a biomarker for early detection of cholangiocarcinomas.