Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods.

A screening procedure for protein-protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophoresis (ACE) was established. GFP was fused as a fluorescent indicator to the C-terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster. Cyclophilins (Cyps) belong to the ubiquitously distributed enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPlase activity of the GFP fused rDmCyp20 as well as the high affinity to CsA remain intact. Using native gel electrophoresis and ACE mobility-shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protein p24 of HIV-1 was detectable in the case of rDmCyp20 fused to the fluorescent tag. For the p24 / rDmCyp20-GFP binding an ACE method was established which allowed to determine a dissociation constant of Kd = 20+/-1.5 x 10(-6) M. This result was verified by size-exclusion chromatography and is in good agreement with published data for the nonfused protein. Moreover the fusion protein was utilized to screen rDmCyp20-protein interactions by capillary electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility-shift assays using native gel electrophoresis with fluorescence imaging and ACE with laser-induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for moderate protein-protein interactions in biological samples.

Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter.

To elucidate the decisive structural factors relevant for dipeptide-carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (Ki) of 0.15-1.2 mM and 0.08-9.5 mM, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N-terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa-Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24-70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1-0.16 mM. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, Arg-Pro and Pro-Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa-Ala dipeptides support this conclusion. Potential substrates or inhibitors of peptide transport were found among Xaa-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.

Interaction of cyclophilin and cyclosporins monitored by affinity capillary electrophoresis.

The affinity capillary electrophoretic separation of the complex of the enzyme cyclophilin (Cyp) with the immunosuppressive drug cyclosporin A (CsA) from uncomplexed Cyp and CsA in phosphate buffer (pH 8) under non-denaturing conditions by equilibrium-mixture analysis is reported. Using a new approach combining mobility-shift analysis and electrophoretically mediated microanalysis the binding constant of rhCyp18 to CsA and derivatives was estimated.

Separation of cis/trans conformers of human and salmon calcitonin by low temperature capillary electrophoresis.

Conformer-specific recognition of peptides and proteins has often been proved with the aid of indirect methods. Here we provide an analytical approach for a direct investigation of separated isomers. Cis/trans conformers of the peptide hormones human (hCT) and salmon (sCT) calcitonin exhibit different migration properties in capillary zone electrophoresis at subambient temperatures. Calcitonin consists of 32 amino acids with two proline residues incorporated. It is the longest unstructured peptide for which a conformer separation by capillary electrophoresis has yet been achieved. Lowering the temperature yielded a splitting into two and three peaks for sCT and hCT, respectively, in acidic buffer. The peak ratios of 66:34 for sCT and 71:23 for hCT are in good agreement with the conformer distribution previously reported from nuclear magnetic resonance (NMR) studies. The addition of different organic modifiers (5-20% v/v) to the running buffer does not improve the separation. The observed merging of conformer peaks in buffer containing 20% v/v 2,2,2-trifluoroethanol (TFE) is attributed to structure formation.

Evidence for the absolute conformational specificity of the intestinal H+/peptide symporter, PEPT1.

This study was initiated to determine whether the intestinal H+/peptide symporter PEPT1 differentiates between the peptide bond conformers of substrates. We synthesized a modified dipeptide where the peptide bond is replaced by the isosteric thioxo peptide bond. The Ala-Pro derivative Ala-psi[CS-N]-Pro exists as a mixture of cis and trans conformation in aqueous solution and is characterized by a low cis/trans isomerization rate. The compound was recognized by PEPT1 with high affinity. The Ki value of Ala-psi[CS-N]-Pro for the inhibition of the uptake of radiolabeled glycylsarcosine in Caco-2 cells was 0.30 +/- 0.02 mM, determined in solution with 96% trans conformation. In contrast, the Ki value was 0.51 +/- 0.02 mM when uptake media with 62% trans conformer were used. We conclude that only the trans conformer interacts with the transport system. From our data, a significant affinity of the cis conformer at PEPT1 cannot be derived. In a second approach, conformer-specific uptake of Ala-psi[CS-N]-Pro was studied by analyzing the intracellular content of Caco-2 cells following transport as well as the composition of the extracellular medium using capillary electrophoresis. The percentage of trans conformer that was 62% in the uptake medium increased to 92% inside the cells. This is the first direct evidence that an H+/peptide cotransport system selectively binds and transports the trans conformer of a peptide derivative.

Kinetic study on the cis-trans isomerization of peptidyl-proline dipeptides.

The equilibrium and kinetic parameters of cis-trans interconversion of dipeptides containing peptidyl-proline moiety were investigated using the in-column incubation method with both CZE and HPLC and the ad hoc dissolution method. The use of the latter was possible because the conformational make-up of the solid peptides, and consequently of their ad hoc solution, was sufficiently different from that of the solution at equilibrium. This method with 1H-NMR and CZE analyses yielded very similar results for the cis-trans isomerization of Phe Pro in aqueous sodium borate, pH 8.4, at 10 degrees C with an average value of 0.34 and 6.6 x 10(-5) s-1 for the equilibrium and rate constant, respectively. The in-column incubation method is performed by CZE or HPLC so that the conformers are separated in the first half of the column and then incubated in column where they interconvert and reach equilibrium. Subsequent separation in the second half of the column yielded four peaks. Thus by measuring the conformer composition as a function of the reaction time, the rate constant can be evaluated. The forward rate constant of 1.42 x 10(-4) s-1 determined by the CZE in-column incubation method for Phe-Pro isomerization at 10 degrees C was twice of the value obtained by the ad hoc dissolution method. It is believed that the inner wall of fused-silica capillaries has a catalytic effect on the isomerization. Computer simulation was also employed to gain further insight on the catalytic activity of the capillary inner wall on such isomerization. Whereas the experimental and simulation profiles of Phe-Pro in aqueous borate buffer, pH 8.4, with a 37 cm long capillary were in excellent agreement, a four times faster interconversion rate had to be used to match the experimental profile obtained with a 57 cm long capillary under otherwise identical conditions. The catalytic effect of the octadecyl silica stationary phase on the isomerization was confirmed by the in-column incubation method with HPLC. The overall rate of the cis-trans isomerization of Phe-Pro, which entails the reaction both on the stationary phase and in the mobility phase, was about six times faster at 0 degree C than the rate measured by NMR in free solution using the mobile phase containing 65% (v/v) sodium phosphate, pH 6.5, and 35% (v/v) methanol. The results presented here serve as a caveat that the effect of the wall in CZE or the stationary phase in HPLC on the reaction cannot be ignored.

Isolation and identification of peptide conformers by reversed-phase high-performance liquid chromatography and NMR at low temperature.

Peptide conformers with one or more rotationally hindered peptide bonds due to the presence of proline and/or another N-substituted amino acid residue in the molecule were separated by reversed-phase chromatography at low temperatures, isolated and identified by NMR. The scope of this investigation included the cis-trans isomers of the dipeptides Leu-Pro, Phe-Pro and Tyr-Pro as well as conformers of opioid peptides containing proline and/or the proline-like Tic (1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid) residues: Tyr-Pro-Phe (beta-casomorphin 1-3 fragment), Tyr-Tic-Phe-Phe, Try-Pro-Phe-Pro-Gly (beta-casomorphin-5), Tyr-Tic-Phe-Phe-Val-Val-Gly-NH2 and Tyr-Tic-Phe-Gly-Tyr-Pro-Ser-NH2. Chromatography with micropellicular and totally porous octadecylated silica stationary phases and aqueous methanol under isocratic elution conditions resulted in well separated peaks of the rotational isomers at sufficiently low temperatures. Preparative RP-HPLC was carried out with eluents containing water and methanol, both deuterated, and the effluent fractions containing each isomer were collected for further investigation. The conformational states of the peptide isomers upon separation were conserved by storing the effluent fractions in liquid nitrogen. The Leu-Pro, Phe-Pro, Tyr-Pro and Tyr-Pro-Phe conformers were identified by one- and two-dimensional NMR spectroscopy at -15 degrees C. Upon comparing the NMR spectra of the isomers, for these peptides the retention order of the conformers was unambiguously established: in each case the trans, conformer is eluted before the cis conformer. On the basis of NMR data obtained the beta-casomorphin-5, which contains two proline residues, the elution order of its four conformers was established by NMR spectroscopy of the fractions obtained by RP-HPLC at low temperature as trans-trans (least retained), trans-cis, cis-cis and cis-trans (most retained).

Capillary zone electrophoresis at subzero temperatures. I. Separation of the cis and trans conformers of small peptides.

The cis-trans conformers of two dipeptides, Phe-Pro and Leu-Pro, and two opioid heptapeptides containing one or two proline residues were separated by capillary zone electrophoresis (CZE) in borate buffer at low temperatures down to -17 degrees C. At temperatures near ambient, the relaxation time of the cis-trans isomerization is on the time-scale of minutes for the dipeptides and thus commensurate with the migration times in CZE under usual operating conditions. The conformers of both dipeptides could be separated with baseline resolution below 10 degrees C in neat aqueous 100 mM sodium borate (pH 8.4). The conformer peaks on the electropherograms were identified by using authentic samples of the cis and trans forms of Phe-Pro and Leu-Pro that were obtained by reversed-phase HPLC at 0 degree C, validated by NMR spectroscopy and stored in liquid nitrogen. The interplay of the electrophoretic migration and on-column isomerization reaction in CZE of Phe-Pro under various conditions was analyzed in the light of the Damköhler number (Da). The results showed that besides employing low temperature increasing the voltage and/or decreasing the capillary length also reduce the magnitude of Da to bring about the separation of interconverting species. In this work the use of low temperature in this work was preferred due to the experimental simplicity. The separation of cis-trans conformers of two opioid heptapeptides was carried out by CZE at subzero temperatures with aqueous sodium borate containing 23% (v/v) glycerol at pH* 11.3 as measured with a glass electrode. The two conformers of Tyr-Pro-Phe-Asp-Val-Val-Gly-NH2 were baseline separated at -12 degrees C and the four conformers of Tyr-Pro-Phe-Gly-Tyr-Pro-Ser-NH2 due to the presence of two peptidyl-proline bonds in the molecule, were also resolved at -12 degrees C. From the electrophoretic mobilities, the hydrodynamic radii of the cis-trans conformers of the dipeptides Phe-Pro and Leu-Pro were estimated. In both cases, the trans isomers had 1.3 times greater Stokes radii than the cis conformers. This agrees with the observed migration order and molecular modeling results. The hydrodynamic radii of the Phe-Pro conformers were smaller than those of the Leu-Pro isomers despite the lower molecular mass of the latter. The results demonstrate that CZE is suitable for measuring certain molecular properties and suggest that the methods introduced here are applicable to the study of other systems of interconverting conformers.