Site-specific fluorescence labelling of recombinant polyomavirus-like particles

For the development of gene therapy protocols based on polyomavirus-like particles, we describe a method for fluorescence labelling of virions in order to study virus-cell interactions preceding gene delivery. Site-specific fluorescence labelling of polyomavirus-like particles is achieved via a single cysteine residue and maleimide conjugates of fluorescence dyes (fluorescein, Texas Red). Polyomavirus-like particles can be assembled in vitro from recombinant capsomers produced in E. coli. Since the assembly process is independent of disulfide bond formation, all cysteine residues of the wild-type protein were replaced by serines, and a new unique cysteine residue was introduced for the attachment of the fluorescence marker.     

The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residues in ficin

Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and alpha-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined.     

On the hypothetical protein F154 of the TTV1 virus from Thermoproteus tenax. Part III: Immunological identification of the protein with anti-peptide antibodies.

To assess the expression of an hypothetical protein F154 encoded in an open reading frame of the DNA sequence of the virus TTV1 identified in the archaebacterium Thermoproteus tenax, two synthetic peptides related to characteristic repeats of the primary structure and corresponding to the sequence portions [Ser81] (73-84) and (79-101) were selectively grafted at their N-termini to the surface-exposed single cysteine residue of iso-1-cytochrome c via the maleimide thiol principle. Antisera were raised in rabbits against the two conjugates and the antipeptide-(79-101) immunoselected antibodies were found to crossreact with a strong protein band in Western blots of cell lysates of TTV1-infected bacteria, thus confirming expression of the protein F154 in Thermoproteus tenax.     

N-(1-pyrene)maleimide: a fluorescent cross-linking reagent.

N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.     

Evidence of ball-and-chain transport of ferric enterobactin through FepA

The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane beta-barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy- and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the beta-barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal beta-barrel domain confirmed the feasibility of this mechanism.     

Preparation of oligonucleotide-peptide conjugates.

A procedure for preparing oligonucleotide-peptide conjugates is presented. It is based on appending a maleimide group to the oligonucleotide for selective coupling to the thiol side chain of a cysteine residue in the peptide. A convenient chromatographic purification procedure, based on Fmoc-on/Fmoc-off, is described.     

Spin-labeling proton NMR study on aromatic amino acid residues in the guanine nucleotide binding site of human c-Ha-ras(1-171) protein

A truncated human c-Ha-ras gene product, ras(1-171) protein, was prepared and chemically modified with maleimide spin-label (MSL). By trypsin digestion of the MSL-labeled ras(1-171) protein, MSL-labeled peptide fragments were isolated and sequenced. The cysteine residue in position 118 of the protein, but not the other cysteine residues, Cys-51 or Cys-80, was found to be specifically labeled by MSL. The ESR spectrum of the MSL-labeled ras(1-171) protein indicates that the MSL group attached to Cys-118 is strongly immobilized. Proton NMR spectra at 400-MHz were measured for this MSL-labeled ras(1-171) protein and also for a control sample of a labeled ras(1-171) protein whose MSL was reduced by sodium ascorbate. In the difference spectra for these two proteins, resonances of protons in the vicinity of the MSL group attached to Cys-118 of the ras(1-171) protein were observed. Thus, the MSL group was found to be in the vicinity of the protein-bound GDP. A phenylalanine residue and two histidine residues, which were characterized by 2D HOHAHA and DQF-COSY spectra, were also found to be in the vicinity of MSL. NOE and pH titration analyses indicate that this phenylalanine residue is close to the bound GDP and one of the two histidine residues. By carboxypeptidase digestion, the two histidine residues near MSL were identified as His-27 and His-94.(ABSTRACT TRUNCATED AT 250 WORDS)     

The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.     

"Cystope tagging" for labeling and detection of recombinant protein expression

A labeling and detection method, based on the addition of a single cysteine residue at the C terminus of a recombinant protein and the subsequent sulfhydryl-specific Michael addition to the double bond of maleimide and its derivatives, was developed. The method was named "cystope tagging." Sorbit dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase family of proteins that contains three inherent cysteines, was used as a model recombinant protein. By labeling with fluorescein-maleimide, it was demonstrated that only the single accessory cysteine is accessible under nonreducing conditions. After the addition of beta-mercaptoethanol, the inherent cysteines of SDH were also detectable by coupling to fluorescein-maleimide. The data were obtained using Autodisplay, an efficient surface expression system in Escherichia coli, but the method presented in this article represents a rather general solution for analyzing the expression of recombinant proteins, irrespective of the expression system used. The authors conclude that cystope tagging is an interesting alternative to other tagging methods applied in recombinant protein techniques.     

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.     

Cysteine 288: an essential hyperreactive thiol of cytosolic phosphoenolpyruvate carboxykinase (GTP)

Phosphoenolpyruvate carboxykinase from the cytosol of rat liver has 13 cysteines, at least one of which is known to be very reactive and essential for catalytic activity (Carlson, G. M., Colombo, G., and Lardy, H. A. (1978) Biochemistry 17, 5329-5338). In order to identify the essential cysteine, this enzyme was modified with the fluorescent sulfhydryl reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Incubation of phosphoenolpyruvate carboxykinase with a 10% molar excess of this maleimide at 0 degrees C results in the rapid and nearly complete loss of catalytic activity. Under these conditions, 1 mol of the maleimide is incorporated per mol inactivated enzyme. The substrate GDP provides almost complete protection against inactivation and modification, while phosphoenolpyruvate protects against the rate, but not the extent, of modification. The pH dependence of the rate of enzyme inactivation suggests that the modified residue has a pK alpha of approximately 7.0. Purification and sequencing of the labeled peptide identifies the hyperreactive essential cysteine as Cys-288. This cysteine lies between two putative phosphoryl-binding domains and within a hydrophobic sequence.     

Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli.

AcrA/B in Escherichia coli is a multicomponent system responsible for intrinsic resistance to a wide range of toxic compounds, and probably cooperates with the outer membrane protein TolC. In this study, acrAB genes were cloned from the E. coli W3104 chromosome. To determine the topology of the inner membrane component AcrB, we employed a chemical labeling approach to analyse mutants of AcrB in which a single cysteine residue had been introduced. The cysteine-free AcrB mutant, in which the two intrinsic Cys residues were replaced by Ala, retained full drug resistance. We constructed 33 cysteine mutants in which a single cysteine was introduced into each putative hydrophilic loop region of the cysteine-free AcrB. The binding of [(14)C]N-ethylmaleimide (NEM) to the Cys residue and the competition of NEM binding with the binding of a membrane-impermeant maleimide, 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), in intact cells were investigated. The results revealed that the N- and C-terminals are localized on the cytoplasmic surface of the membrane and the two large loops are localized on the periplasmic surface of the membrane. The results supported the 12-membrane-spanning structure of AcrB. Three of the four short periplasmic loop regions were covered by the two large periplasmic loop domains and were not exposed to the water phase until one of the two large periplasmic loops was removed.     

Studies on immunoassays of peptide factors. V. Synthesis of cholecystokinin-58-[1-11]/iso-1-cytochrome c conjugate

In previous studies on model compounds we have found that the maleimide function is sufficiently stable under the conditions of peptide synthesis to allow its incorporation at preselected positions of a peptide chain in earlier steps of the synthetic route. Taking advantage of this observation the N-terminal undecapeptide of canine cholecystokinin-58 containing at its N-terminus the maleimide group became accessible in high yields as chromatographically homogenous and analytically well characterized compound. Via the incorporated anchor group the undecapeptide was linked selectively at its N-terminus to the cysteine residue 107 of iso-1-cytochrome c to yield a well characterized conjugate of 1:1 stoichiometry for immunization experiments.     

Photocontrol of the GTPase activity of the small G protein K-Ras by using an azobenzene derivative

The small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switch mechanisms utilizing conformational changes in nucleotide-binding motifs have been well studied at the molecular level. Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis and trans forms upon exposure to ultraviolet and visible light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the nucleotide-binding motif of Ras to regulate the GTPase activity by photoirradiation. We prepared four Ras mutants (G12C, Y32C, I36C, and Y64C) that have a single reactive cysteine residue in the nucleotide-binding motif. PAM was stoichiometrically incorporated into the cysteine residue of the mutants. The PAM-modified mutants exhibited reversible alterations in GTPase activity, nucleotide exchange rate, and interaction between guanine nucleotide exchange factor and Ras, accompanied by photoisomerization upon exposure to ultraviolet and visible light irradiation. The results suggest that incorporation of photochromic molecules into its nucleotide-binding motif enables photoreversible control of the function of the small G protein Ras.     

Comparative study on the active sites of ficin and papain by the spin labeling method

Ficin was alkylated with a series of haloacetamide spin labels with various distances between the spin probes and reactive groups. From the relation of these distances to the tau c values of the labels incorporated into protein, it was estimated that the depth of the active site hole of ficin is ca. 8 A. The results are somewhat different from those reported previously for papain (S. Nakayama et al. (1981) Biochem. Biophys. Res. Commun. 98, 471-475). Examination of the pH dependence of the ESR spectra for ficin and papain alkylated with an iodoacetamide or a maleimide spin label suggested that these enzymes have an amino acid residue of pKa 4 (probably a histidine residue) around the active site cysteine and that the active site conformations change at around pH 5.     

Assignment of catalytically essential cysteine residues in aspartase by selective chemical modification with N-(7-dimethylamino-4-methylcoumarynyl)maleimide

N-(7-Dimethylamino-4-methylcoumarynyl)maleimide (DACM), a fluorescent reagent for sulfhydryl groups, was employed to determine the functionally essential cysteine residues in aspartase from Escherichia coli. Analysis of the tryptic peptides containing DACM-labeled residues by reverse phase HPLC revealed that Cys-140 and Cys-430 were selectively modified, among 11 residues whose loci were recently determined by a DNA sequencing study (Takagi, J.S., et al. (1985) Nucl. Acids Res. 13, 2063-2074). When the modification was carried out in the presence of Mg2+ and L-aspartate, the enzyme activity remained unchanged and no cysteine residue was modified. This suggests that two cysteine residues are located at the L-aspartate binding site and that at least one of them is involved in the catalytic reaction.     

Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pK(a) values of the cysteines at the two splice junctions. The apparent pK(a) of the cysteine at the upstream splice junction, which initiates the N-S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pK(a) of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.     

Analysis of the membrane topology of transmembrane segments in the C-terminal hydrophobic domain of the yeast vacuolar ATPase subunit a (Vph1p) by chemical modification

The integral V(0) domain of the vacuolar (H(+))-ATPases (V-ATPases) provides the pathway by which protons are transported across the membrane. Subunit a is a 100-kDa integral subunit of V(0) that plays an essential role in proton translocation. To better define the membrane topology of subunit a, unique cysteine residues were introduced into a Cys-less form of the yeast subunit a (Vph1p) and the accessibility of these cysteine residues to modification by the membrane permeant reagent N-ethylmaleimide (NEM) and the membrane impermeant reagent polyethyleneglycol maleimide (PEG-mal) in the presence and absence of the protein denaturant SDS was assessed. Thirty Vph1p mutants containing unique cysteine residues were constructed and analyzed. Cysteines introduced between residues 670 and 710 and between 807 and 840 were modified by PEG-mal in the absence of SDS, indicating a cytoplasmic orientation. Cysteines introduced between residues 602 and 620 and between residues 744 and 761 were modified by NEM but not PEG-mal in the absence of SDS, suggesting a lumenal orientation. Finally, cysteines introduced at residues 638, 645, 648, 723, 726, 734, and at nine positions between residue 766 and 804 were modified by NEM and PEG-mal only in the presence of SDS, consistent with their presence within the membrane or at a protein-protein interface. The results support an eight transmembrane helix (TM) model of subunit a in which the C terminus is located on the cytoplasmic side of the membrane and provide information on the location of hydrophilic loops separating TM6, 7, and 8.     

Phosphoenolpyruvate carboxylase of Escherichia coli K-12. N- and C-terminal sequences and tentative assignment of the catalytically essential cysteine residue

The N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were determined to establish the primary structure deduced from the nucleotide sequence of the cloned gene for the enzyme (Fujita, N., Miwa, T., Ishijima, S., Izui, K., & Katsuki, H. (1984) J. Biochem. 95, 909-916). As predicted from the nucleotide sequence, two polypeptides were produced upon treatment with hydroxylamine, which specifically cleaves the Asn-Gly bond, and their amino acid compositions were also in accordance with those predicted. The tryptic peptides which contained cysteine residues labeled with a fluorescent reagent, N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide, were isolated by high-performance liquid chromatography and partially sequenced. All of them could be assigned on the deduced primary structure. The modified cysteine residues were Cys-157, Cys-385, Cys-458, Cys-568, Cys-665, and Cys-754. Furthermore, the essential cysteine residue which is presumably located at or near the active site was tentatively identified as Cys-568, since it was consistently protected against the modification by 2-phospholactate, a substrate analog.     

Role of the human and murine cyclin T proteins in regulating HIV-1 tat-activation.

Human cyclin T1 markedly stimulates tat-activation in rodent cells which are normally poorly responsive to the effects of Tat. This result suggests that there are likely to be critical differences in the murine and human cyclin T1 proteins. Here, we analyzed the role of the murine and human cyclin T1 proteins in addition to the human cyclin T2a and T2b proteins on regulating tat-activation. Only the human cyclin T1 protein efficiently formed a complex with Tat bound to TAR RNA. This difference in function was due to the presence of a cysteine residue in human cyclin T1 at position 261 rather than a tyrosine or asparagine residue which are found in the murine cyclin T1 protein and the human cyclin T2a and T2b proteins, respectively. A mouse cyclin T1 protein containing a substitution of tyrosine residue 261 with a cysteine residue, was able to interact with Tat and stimulate tat-transactivation in rodent cells. Likewise, substitution of a cysteine residue for an asparagine residue at position 260 of the cyclin T2a and T2b proteins also resulted in their ability to interact with Tat and stimulate tat-activation in rodent cells. The data indicate that a specific residue in the cyclin T proteins is required for their in vitro interaction with Tat and their ability to stimulate in vivo tat-activation.