Estimation of intramolecular distance distributions in bovine pancreatic trypsin inhibitor by site-specific labeling and nonradiative excitation energy-transfer measurements

A series of four bovine pancreatic trypsin inhibitor (BPTI) derivatives, site specifically labeled by (2-methoxy-1-naphthyl)methyl (MNA) at the N-terminal amino group and by [7-(dimethylamino)-coumarin-4-yl]acetyl (DA-coum) at one of the four epsilon-amino groups, was prepared. The four derivatives, N alpha-MNA-Arg1-N epsilon-DA-coum-Lysn-BPTI [(1-n)BPTI] (n = 15, 26, 41, and 46), were purified by affinity chromatography and high-performance liquid chromatography (HPLC). The homogeneity of each derivative and its site of labeling were characterized by HPLC tryptic peptide mapping. Nonradiative energy transfer from MNA (donor) to DA-coum (acceptor) was measured by monitoring donor emission and acceptor excitation spectra. Transfer efficiencies between 45% and 85% were observed. The fluorescence decay of MNA in MNA-BPTI, a derivative labeled by a donor without an acceptor, is monoexponential, with a lifetime of 6.8 +/- 0.15 ns. The decay kinetics of MNA fluorescence measured for derivatives labeled both by donor and acceptor showed a small deviation from monoexponential decay with shorter average lifetimes. Analysis of the experimental decay curves yielded the detailed intramolecular distance distribution functions for each pair of labeled sites. The averages of the calculated distance distribution functions are close to the values expected from the known structure of BPTI in the crystalline state. The derivatives thus obtained are suitable for investigation of conformational transitions of the labeled protein and for monitoring localized changes such as those involved in the folding or unfolding transitions.     

Chemical modification of guanidinium groups of vasoactive intestinal peptide

Molecular characterization of receptors depends on the availability of ligand derivatives carrying a reactive group to covalently link the active sites. Two vasoactive intestinal peptide (VIP) derivatives, each labeled either at the two arginine residues 12 and 14 or singly in position 14, were prepared. In the first case, this was achieved by a selective chemical modification using azidophenylglyoxal. In the second, the amino acids of VIP, buried in the active site of the receptor, were protected and one arginine residue of bound VIP was successfully modified using azidophenylglyoxal. The two molecules were resolved by radioimmunocompetition and reversed phase high performance liquid chromatography. Identification of sites of labeling was achieved by tryptic peptide mapping and amino acid analysis. One derivative (Az-Bz-Arg14-VIP) retains a high binding affinity for the receptor and was found to be biologically active. The present method yields a derivative which is useful in structural analysis of the receptor.     

Conformational unfolding in the N-terminal region of ribonuclease A detected by nonradiative energy transfer

Unfolding in the N-terminal region of RNase A was studied by the nonradiative energy-transfer technique. RNase A was labeled with a nonfluorescent acceptor (2,4-dinitrophenyl) on the alpha-amino group and a fluorescent donor (ethylenediamine monoamide of 2-naphthoxyacetic acid) on a carboxyl group in the vicinity of residue 50 (75% at Glu-49 and 25% at Asp-53). The distribution of donor labeling sites does not affect the results of this study since they are close in both the sequence and the three-dimensional structure. The sites of labeling were determined by peptide mapping. The derivatives possessed full enzymatic activity and underwent reversible thermal transitions. However, there were some quantitative differences in the thermodynamic parameters. When the carboxyl groups were masked, there was a 5 degrees C lowering of the melting temperature at pH 2 and 4, and no significant change in delta H(Tm). Labeling of the alpha-amino group had no effect on the melting temperature or delta H(Tm) at pH 2 but did result in a dramatic decrease in delta H(Tm) of the unfolding reaction at pH 4. The melting temperature did not change appreciably at pH 4, indicating that an enthalpy/entropy compensation had occurred. The efficiencies of energy transfer determined with both fluorescence intensity and lifetime measurements were in reasonably good agreement. The transfer efficiency dropped from about 60% under folding conditions to roughly 20% when the derivatives were unfolded with disulfide bonds intact and was further reduced to 5% when the disulfide bonds were reduced. The interprobe separation distance was estimated to be 35 +/- 2 A under folding conditions. The contribution to the interprobe distance resulting from the finite size of the probes was treated by using simple geometric considerations and a rotational isomeric state model of the donor probe linkage. With this model, the estimated average interprobe distance of 36 A is in excellent agreement with the experimental result cited above.     

Selective fluorescent labeling of amino groups of bovine pancreatic trypsin inhibitor by reductive alkylation

Bovine pancreatic trypsin inhibitor (BPTI) was reductively alkylated with 2-methoxy-1-naphthyl aldehyde and sodium cyanoborohydride (NaCNBH₃). All five possible derivatives, each labeled at one of the primary amino groups of BPTI, were obtained. The distribution of yields of the various derivatives can be controlled by changing the reaction conditions. Products were identified by high-performance liquid chromatography (HPLC) tryptic peptide mapping. This procedure was used for the preparation of three pure 2-methoxy-1-naphthyl-methylenyl-BPTI (MNA-BPTI) derivatives. Purification was achieved by means of affinity chromatography and HPLC. The spectral characteristics of the probe, notably monoexponential decay with a lifetime of 6.8 ± 0.1 ns and moderate limiting fluorescence polarization, P = 0.3 ± 0.015, make it a very useful donor in energy-transfer measurements.     

Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins

Two distinct sequences of amino acids, RGDS and HHLGGAKQAGDV, each inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to the platelet membrane glycoprotein IIb-IIIa complex. We have employed radiolabeled, photoactivatable aryl azide derivatives of the two sequences to explore the relationship between the binding sites for these peptides on the glycoprotein IIb-IIIa complex. Each probe specifically labeled only the glycoprotein IIb-IIIa complex of intact platelets. Since each peptide inhibited labeling of the receptor complex by the other, the peptides compete for binding sites on the receptor complex. However, the binding sites do not appear to be identical. Whereas the RGDS probe specifically labeled both glycoproteins IIb and IIIa, the HHLGGAKQA-GDV probe specifically labeled only glycoprotein IIb.     

Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry.

Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.     

Reduced bovine pancreatic trypsin inhibitor has a compact structure

The conformation of reduced bovine pancreatic trypsin inhibitor (R-BPTI) under reducing conditions was monitored by measurements of nonradiative excitation energy-transfer efficiencies (E) between a donor probe attached to the N-terminal Arg1 residue and an acceptor attached to one of the lysine residues (15, 26, 41, or 46) [Amir, D., & Haas, E. (1987) Biochemistry 26, 2162-2175]. High-excitation energy-transfer efficiencies that approach those found in the native state were obtained for the reduced labeled BPTI derivatives in 0.5 M guanidine hydrochloride (Gdn.HCl) and 4 mM DTT. Unlike the dependence expected for a random coil chain, E does not decrease as a function of the number of residues between the labeled sites. The efficiency of energy transfer between probes attached to residues 1 and 15 in the reduced state is higher than that found for the same pair of sites in the native state or reduced unfolded (in 6 M Gdn.HCl) state. This segment also shows high dynamic flexibility. These results indicate that the overall structure of reduced BPTI under folding (but still reducing) conditions shows a high population of conformers with interprobe distances similar to those of the native state. Reduced BPTI seems to be in a molten globule state characterized by a flexible, compact structure, which probably reorganizes into the native structure when the folding is allowed to proceed under oxidizing conditions.     

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.     

Protein components of the erythromycin binding site in bacterial ribosomes

Two derivatives of erythromycin have been prepared carrying either an aryl azide or a 4-nitroguaiacol as a photoreactive group. Both derivatives bind to the specific erythromycin ribosomal site as shown by saturation and competition studies. The derivatives were isotopically labeled either with tritium or with 125I, and radioactivity is covalently incorporated to the ribosome upon irradiation at the appropriate wavelength. The ribosomal proteins labeled were identified by either mono- and two-dimensional gel electrophoresis or high performance liquid chromatography. It has been found that protein L22 is the protein mainly, and under some conditions exclusively, labeled by the erythromycin derivatives. These results were confirmed using ribosomes from erythromycin-resistant mutants having a protein L22 with modified electrophoretical mobility. Protein L15 is also labeled in both cases, and the aromatic azide derivative labels to a lesser extent proteins L2 and L4. Competition experiments with erythromycin indicate that labeling in protein L22, and probably in L15, is specific, while the specificity of labeling in proteins L2 and L4 is questionable. These results indicate that the erythromycin derivatives label different ribosomal proteins than the spiramycin type of macrolides (Tejedor, F., and Ballesta, J.P.G. (1985) Biochemistry 24, 467) suggesting that the binding sites of both macrolide types are probably not identical.     

Design consideration and probes for fluorescence resonance energy transfer studies

Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-beta-(2-naphthyl)alanine (I-Nal), were characterized using single cysteine mutants of Escherichia coli adenylate kinase. Together with two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetamide and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (either native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of structural transitions in proteins by means of fluorescence resonance energy-transfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 A in labeled protein derivatives is described. The charged groups present in the probes facilitate the conjugation reaction and improve postlabeling purification. General considerations for design of charged probes and site-directed labeling for applications of FRET methods in studies of protein structure and dynamics are presented.     

Separation of dimethylaminoazobenzenethiohydantoin amino acids by high-performance liquid chromatography at low picomole concentrations

The separation of all common dimethylaminoazobenzenethiohydantoin (DABTH) amino acids derived from modified Edman sequencing can be achieved by using high-performance liquid chromatography. All derivatives, including DABTH-Ile and DABTH-Leu, can be readily separated in a solvent mixture of sodium acetate buffer and 1% ethylene dichloride in acetonitrile. The high absorbance of the DABTH amino acids at 436 nm makes possible the quantitative determination of these derivatives at picomole concentrations in a relatively short time (30–40 min).     

Scaling up a microbore separation for semipreparative analysis: differential recoveries of radiolabeled amino acids

Application of a high-sensitivity microbore system designed to separate and quantify nonderivatized amino acids by anion exchange chromatography and amperometric detection for determination of amino acid-specific activities in biological samples requires high capacity to recover sufficient labeled material for adequate count statistics. Scale up from a low (25-1000 pmol) to a high (500-15,000 pmol) working range was achieved by use of a thick working electrode gasket to reduce sensitivity and eliminate peak splitting and tailing and by modification of the wash procedure to eliminate carryover. Analysis of recoveries of labeled amino acids revealed that specific amino acids are either selectively retained on the column or partially degraded during analysis and that assessment of purities of labeled compounds and metabolic labeling patterns requires careful analysis of recoveries of labeled compounds in the appropriate eluate fraction.     

Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.     

Detection of local structures in reduced unfolded bovine pancreatic trypsin inhibitor.

The structure of BPTI and reduced BPTI in concentrated guanidinium HCl (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Interprobe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1-n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2-methoxy-naphthyl-1-methylenyl group (MNA) at the N-terminal amino group of arg1 and 7-(dimethylamino)-coumarin-4-yl-acetyl residue (DA-coum) at one of its epsilon-NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states. The N-terminal-segment, residues 1-15, is in an extended conformation (with an average interprobe distance of 34 +/- 2 A) in the native state. Upon unfolding by reduction with DTT or beta-mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced average interprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N-terminus and the turn of the twisted beta sheet element (residues 18-35), increased upon unfolding. At -30 degrees C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 +/- 20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 +/- 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation. Thus local structures seem to exist in reduced denatured BPTI even under denaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded proteins and for search for folding initiation sites (FISs) and early folding intermediates.     

The aggregation state of spin-labeled melittin in solution and bound to phospholipid membranes: evidence that membrane-bound melittin is monomeric

Spin-labeled derivatives of the bee venom protein, melittin, were obtained by reacting on the average one of the four amino groups of the protein with succinimidyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylate. All 16 statistically possible reaction products with 0, 1, 2, 3 or 4 spin labels per protein were then separated in a single pass with reversed phase high performance liquid chromatography. With the help of trypsin digestion and diode array detection it was possible to assign the primary structure of all 16 eluting fractions. All fractions with only one spin label per protein were purified for electron paramagnetic resonance measurements. The labeling sites cover different regions of the protein: one is at the N-terminus, one at lysine-7, and two are near the C-terminus at lysine-21 and lysine-23, respectively. This set of specifically labeled melittins was used to study the structure and dynamics of melittin in aqueous solutions and when bound to neutral or negatively charged membranes. In aqueous solution a reduction in rotational correlation time and appearance of spin-spin interaction was observed during salt-induced transition from a random coil monomer to a mostly alpha-helical tetramer. Membrane binding to phospholipid bilayers in low or high ionic strength was reflected only in a further decrease in mobility. The absence of any spin interaction in the membrane-bound state suggests that melittin is monomeric under these conditions. All derivatives were able to detect these structural changes, but melittin labeled at the N-terminal amino group was especially valuable. Because of postulated intramolecular hydrogen bonding, this label reflects directly the motion of the entire protein or tetramer. Broadening experiments with chromium oxalate show that all labeled sites are at least partially exposed to the aqueous phase when melittin is bound to membranes. This suggests that an alpha-helical melittin monomer binds to membranes with its axis parallel to the membrane surface.     

Changes in retention behavior of fluorescently labeled proteins during ion-exchange chromatography caused by different protein surface labeling positions

Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.     

Structural mapping of chloroplast coupling factor

Fluorescence resonance energy transfer measurements have been used to investigate the spatial relationships between the nucleotide binding sites and the gamma-subunit of the H+-ATPase from chloroplasts and the orientation of these sites with respect to the membrane surface. Fluorescent maleimides reacted covalently at specific sulfhydryl sites on the gamma-subunit served as energy donors. One sulfhydryl site can be labeled only under energized conditions on the thylakoid membrane surface (light site). The two gamma-sulfhydryls exposed after catalytic activation served as a second donor site (disulfide site). In one set of experiments, the nucleotide analogue 2'(3')-(trinitrophenyl)adenosine triphosphate, selectively bound at each of the three nucleotide binding sites of the solubilized coupling factor, was used as an energy acceptor; in another, octadecylrhodamine with its acyl chain inserted in the vesicle bilayer and the rhodamine fluorophore exposed along the membrane surface was the energy acceptor. The distance between the sulfhydryl and disulfide sites was also obtained by sequentially labeling the sites with coumarin (donor) and fluorescein (acceptor) maleimide derivatives, respectively. The results indicate that all three nucleotide sites are approximately equal to 50 A from the light-labeled gamma-sulfhydryl. Two of the nucleotide sites are very far from the gamma-disulfide (greater than 74 A), while the third site, which binds nucleotides reversibly under all conditions, is 62 A from this sulfhydryl. The light-labeled sulfhydryl and disulfide sites are about 42-47 A apart. Finally, the distance of closest approach between the membrane surface of the reconstituted system and the gamma-disulfide is 31 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internal architecture of the core nucleosome: fluorescence energy transfer studies at methionine-84 of histone H4

Chicken histone H4, labeled separately at Met-84 with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, was reassociated with unlabeled histones H2A, H2B, and H3 and 146 base pairs of DNA to produce fluorescently labeled nucleosomes having physical characteristics virtually the same as those of native core particles. Four types of particles were prepared containing respectively unlabeled H4, dansylated H4, fluoresceinated H4, and a mixture of the two labeled H4 molecules. Quantitative singlet-singlet energy-transfer measurements were carried out to determine changes in the distance between the two Met-84 H4 sites within the same nucleosome following conformational transitions which we have reported earlier. In the ionic strength range 0.1-100 mM NaCl, the distance between these sites is less than 2 nm except at 1 mM. Between 100 and 600 mM monovalent salt the distance separating the donor and acceptor fluors at Met-84 H4 increases to 3.8 nm. The conformational change centered around 200 mM NaCl is cooperative. Our results and those of others indicate that there is little unfolding of the histone octamer, at least around Met-84 H4, in the entire ionic strength range studied. A mechanism involving the rotation of the globular portion of H4 is proposed to account for this transition which occurs at physiological ionic strengths.     

Quantitative determination of phenyl isothiocyanate-derivatized amino sugars and amino sugar alcohols by high-performance liquid chromatography

Simple and rapid methods for the preparation of phenylthiocarbamyl (PTC) derivatives of amino sugars and amino sugar alcohols and their quantitative determination with high sensitivity (less than 10 pmol) by C18 reversed-phase high-performance liquid chromatography are described. Rapid sample preparation of the phenyl isothiocyanate (PITC)-derivatized amino sugars and amino sugar alcohols was achieved by a simple extraction of the reaction mixture with chloroform to remove the excess PITC and its adducts. Baseline separation of the PTC derivatives of amino sugars and amino sugar alcohols was obtained within 30 min, using a simple solvent system consisting of 0.2% each of n-butylamine, phosphoric acid, and tetrahydrofuran. The mobile phase containing n-butylamine, in conjunction with a C18 stationary phase, mimics the conditions for the separation of carbohydrates on an amino-bonded column. GlcNH2 and GalNH2 derived from the initial protein-sugar linkages were also separated from the amino acids for quantitative estimation of sugar chains in glycoproteins. Amino sugar alcohols gave single reaction products with PITC while the reaction with amino sugars was accompanied by the formation of secondary products. Apparently the secondary products were formed in an acid-catalyzed intramolecular cyclization of the PTC-hexosamines involving the aldehyde functional group. Conditions were developed to stop the transformations and maintain the stability of PTC derivatives for their convenient determination by HPLC.     

Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

Background

Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling.

Methodology

A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein.

Conclusions

We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.