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PubMed1989

Tryptic phosphopeptides from whole casein. II. Physicochemical properties related to the solubilization of calcium.

Casein phosphopeptides (CPP) were produced by tryptic hydrolysis of sodium caseinate and further purified by precipitation and chromatography on QAE-Sephadex A-25. Their physico-chemical properties were compared with the properties of an enzymically dephosphorylated equivalent preparation (DPP). Binding of Ca2+ to the peptides was measured using a Ca selective electrode and was found to increase with pH and to show 1/1 stoicheiometry Ca/Porg in CPP at pH 6.5 and 7.6. Klotz plots indicated equivalent binding sites at these two pH values, but some heterogeneity was seen at pH 3.5. In contrast, DPP did not bind significant amounts of Ca2+. CPP effectively inhibited the formation of insoluble calcium phosphates at different Ca/P ratios. The effective CPP concentration was 10 mg/l and complete stability of calcium phosphate solutions was obtained at about 100 mg/l. This stabilizing effect was dependent on the presence of organic P.

Calcium↗

PubMed2003

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures.

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.

Amino Acid Sequence↗

PubMed1999

Data-dependent modulation of solid-phase extraction capillary electrophoresis for the analysis of complex peptide and phosphopeptide mixtures by tandem mass spectrometry: application to endothelial nitric oxide synthase.

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) of peptides in conjunction with automated sequence database searching of the resulting collision-induced dissociation (CID) spectra has become a powerful method for the identification of purified proteins or the components of protein mixtures. The success of the method is critically dependent on the manner by which the peptides are introduced into the mass spectrometer. In this report, we describe a capillary electrophoresis-based system for the automated, sensitive analysis of complex peptide mixtures. The system consists of an ESI-MS/MS instrument, a solid-phase extraction (SPE)-capillary zone electrophoresis (CZE) device for peptide concentration and separation, and an algorithm written in Instrument Control Language (ICL) which modulates the electrophoretic conditions in a data-dependent manner to optimize available time for the generation of high-quality CID spectra of peptides in complex samples. We demonstrate that the data-dependent modulation of the electric field significantly expands the analytical window for each peptide analyzed and that the sensitivity of the SPE-CZE technique is not noticeably altered by the procedure. By applying the technique to the analysis of in vivo phosphorylation sites of endothelial nitric oxide synthase (eNOS), we demonstrate the power of this system for the MS/MS analysis of minor peptide species in complex samples such as phosphopeptides generated by the proteolytic digestion of a large protein, eNOS, phosphorylated at low stoichiometry.

Amino Acid Sequence↗

PubMed1995

Structural and dynamic characterization of the phosphotyrosine binding region of a Src homology 2 domain--phosphopeptide complex by NMR relaxation, proton exchange, and chemical shift approaches.

Arginine side chains are often involved in protein--protein and protein--nucleic acid interactions. Due to a number of factors, resonance assignment and detection of NOEs involving the arginine side chains via standard NMR techniques can be difficult. We present here an approach to characterization of the interaction between a phosphopeptide (pY1021) and four arginine residues that line the phosphotyrosine-binding pocket of the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2). Previously published [Pascal, S. M., et al. (1994) Cell 77, 461] NOE data provide a partial description of this interaction, including contacts between the aliphatic region of Arg 59 and the phosphotyrosine (pTyr) aromatic ring. Further characterization has now been accomplished by using 15N and 13C NMR relaxation studies of arginine N episilon and C zeta spins, respectively, and proton exchange rates of arginine H episilon nuclei. Differences between the chemical shifts of the arginine guanidino groups of the free SH2 domain in imidazole and phosphate buffers or in complex with pY1021 have provided insight into specific interactions with the phosphate and the aromatic ring of the pTyr. The resulting data are consistent with the most stable hydrogen bonds to phosphate donated by the Arg 39 epsilon-NH and the two Arg 37 eta-NH2 groups and with pTyr aromatic ring interactions involving the Arg 39 and possibly the Arg 18 guanidino groups.

Amino Acid Sequence↗

PubMed1994

Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation.

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Amino Acid Sequence↗

PubMed1990

Phosphopeptide mapping of Avena phytochrome phosphorylated by protein kinases in vitro.

We previously demonstrated that protein kinases are useful probes of conformational changes that occur upon photoconversion of phytochrome [Wong, Y.-S., Cheng, H.-C., Walsh, D. A., & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097]. Here we present phosphopeptide analyses of oat phytochrome phosphorylated by three mammalian protein kinases and by a polycation-stimulated, phytochrome-associated protein kinase. Phosphorylation of the Pr form by the cAMP-dependent protein kinase occurs predominantly on Ser17 while Ser598 is the preferred phosphorylation site on Pfr. The cGMP-dependent and Ca2(+)-activated, phospholipid-dependent protein kinases, which phosphorylate only the Pr form of phytochrome, recognize the same region on the phytochrome polypeptide as the cAMP-dependent protein kinase. Polycation-stimulated phytochrome phosphorylation reveals that, in contrast to the mammalian enzymes, the plant kinase recognizes the serine-rich, blocked N-terminus of phytochrome. The potential regulatory role of phytochrome phosphorylation, particularly in the structurally conserved serine/threonine-rich N-terminal region of the phytochrome polypeptide, is suggested by these results.

Amino Acids↗

PubMed2006

Rat organic anion transporting protein 1A1 (Oatp1a1): purification and phosphopeptide assignment.

Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated or singly or doubly phosphorylated forms and sensitive to alkaline phosphatase treatment. The beta-elimination reaction resulted in a mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosphorylated peptide, while phosphorylation at S634 and S635 accounted for all doubly phosphorylated peptide. These findings identify the site of oatp1a1 phosphorylation and demonstrate that it is an ordered process, in which phosphorylation at S634 precedes that at S635. The mechanism by which phosphorylation results in loss of transport activity in hepatocytes remains to be established. Whether phosphorylation near the C-terminus inhibits C-terminal oligomerization of oatp1a1, required for normal transport function, can be speculated upon but is as yet unknown.

Alkaline Phosphatase↗

PubMed1996

Alternative modes of tyrosyl phosphopeptide binding to a Src family SH2 domain: implications for regulation of tyrosine kinase activity.

Src homology 2 (SH2) domains interact with proteins containing phosphorylated tyrosine residues and as such play a key role in mediating tyrosine kinase signal transduction. Determination of how these interactions maintain specificity is central to understanding the mechanism of this intracellular signal processing. In the Src family tyrosine kinases specificity is enhanced by a form of regulation based on binding of a phosphotyrosine, pY, and its proximal amino acid sequence from the C-terminus to the SH2 domain of the same protein (autoregulation) or to a similar protein (homodimeric regulation). Activation of the protein is accomplished by removal of this regulatory interaction by competition from a "specific" interacting ligand. We adopt the SH2 domain from a member of the Src family, Fyn (whose predominant physiological role is in initiation of signals from the T-cell receptor complex), to explore the differences in structural, thermodynamic, and kinetic determinants of regulatory and specific interactions using tyrosyl phosphopeptides based on the C-terminus and on a putative physiological interacting species from the hamster middle-sized tumor antigen. The specific peptide interacts with micromolar affinity via embedding the pY and an isoleucine residue (in the pY + 3 position) in two deep pockets. This leads to a large favorable enthalpic contribution to free energy. The regulatory peptide interacts in the pY pocket which forms a pivot for the rest of the molecule which is dynamic. These structural data for the regulatory peptide are supported by the observation of a more favorable entropic term and a complex mode of binding revealed by kinetic analysis.

Amino Acid Sequence↗

PubMed1997

Changes in the NMR-derived motional parameters of the insulin receptor substrate 1 phosphotyrosine binding domain upon binding to an interleukin 4 receptor phosphopeptide.

Proteins recognize ligands by forming specific intermolecular interactions that often involve solvent exposed residues. Changes in the motional properties of these residues upon binding can affect the conformational entropy of the system and thus are related to the energetics of binding. The role that dynamics plays in ligand recognition can be investigated by comparing the motional properties of a free and ligated protein. NMR relaxation studies are well suited for examining changes in dynamics, especially for motions on a nanosecond to picosecond time scale. Recently, we determined the solution structure of the phosphotyrosine binding (PTB) domain of the insulin receptor substrate (IRS-1) complexed to a tyrosine-phosphorylated peptide derived from the interleukin 4 (IL-4) receptor [Zhou et al., (1996) Nat. Struct. Biol. 3, 388-393]. The peptide binds tightly to the protein in a surface exposed pocket, resulting in the partial burial of many protein residues. Using NMR relaxation studies, the dynamics of the backbone nitrogens of IRS-1 PTB domain were studied in both the free protein and the protein when complexed to the IL-4 receptor phosphopeptide. The backbone nitrogens of many residues that make important contacts to the ligand are motionally restricted in the free and complexed protein. Additional residues become motionally restricted only after ligand binding, including several residues that do not make any direct contacts with the ligand. These observed changes in the dynamics are compared to structural features of the complex.

Amino Acid Sequence↗

PubMed1997

Heteronuclear NMR studies of the combined Src homology domains 2 and 3 of pp60 c-Src: effects of phosphopeptide binding.

The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assignments were obtained using heteronuclear triple-resonance experiments in conjunction with 15N-separated nuclear Overhauser effect spectroscopy (NOESY) data. A modified three-dimensional 13CO-15N-1H spectral correlation experiment [(HACA)CO(CA)-NH] with improved sensitivity is presented that provided additional sequential information and resolved several ambiguities. Chemical shifts and sequential- and medium-range NOE cross peaks indicate that the structures of both the SH3 and SH2 portions of the polypeptide are very similar to those of the isolated SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQpYEEIPIYL, induces large chemical shift changes at several locations in the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects are all involved in peptide binding or undergo significant conformational changes upon binding. However, subtle changes of both 1H and 15N chemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communication.

Amino Acid Sequence↗

PubMed1999

Sulfhydryl reactivity demonstrates different conformational states for arrestin, arrestin activated by a synthetic phosphopeptide, and constitutively active arrestin.

The sulfhydryl groups of the three cysteines in bovine arrestin react with DTNB very slowly (over a period of several hours). In the presence of the synthetic phosphopeptide comprising the fully phosphorylated carboxyl-terminal 19 amino acids of bovine rhodopsin, the reactivity of one of the sulfhydryls was enhanced while that of another was greatly reduced. Since this synthetic peptide was shown to activate arrestin with respect to its binding to unphosphorylated, light-activated rhodopsin, the reactivity of the sulfhydryl groups of a constitutively active R175Q arrestin mutant was examined. All three of the sulfhydryl groups of the mutant arrestin R175Q reacted rapidly with DTNB, but not as rapidly as with SDS-denatured arrestin. The arrestin mutant R175Q bound to light-activated, unphosphorylated rhodopsin in ROS disk membranes. The arrestin mutant R175Q also inhibited the light-activated PDE activity with an IC50 of 1.3 microM under the experimental conditions that were used. These data indicate that each of these forms of arrestin is a different conformation. The activated conformation of arrestin that binds to phosphorylated rhodopsin in vivo may be yet another conformation. We conclude that arrestin is a flexible molecule that is able to attain several different conformations, all of which are able to attain the activated functional state of arrestin.

Animals↗

PubMed2002

Hierarchy of simulation models in predicting structure and energetics of the Src SH2 domain binding to tyrosyl phosphopeptides.

Structure and energetics of the Src Src Homology 2 (SH2) domain binding with the recognition phosphopeptide pYEEI and its mutants are studied by a hierarchical computational approach. The proposed structure prediction strategy includes equilibrium sampling of the peptide conformational space by simulated tempering dynamics with the simplified, knowledge-based energy function, followed by structural clustering of the resulting conformations and binding free energy evaluation of a single representative from each cluster, a cluster center. This protocol is robust in rapid screening of low-energy conformations and recovers the crystal structure of the pYEEI peptide. Thermodynamics of the peptide-SH2 domain binding is analyzed by computing the average energy contributions over conformations from the clusters, structurally similar to the predicted peptide bound structure. Using this approach, the binding thermodynamics for a panel of studied peptides is predicted in a better agreement with the experiment than previously suggested models. However, the overall correlation between computed and experimental binding affinity remains rather modest. The results of this study show that small differences in binding free energies between the Ala and Gly mutants of the pYEEI peptide are considerably more difficult to predict than the structure of the bound peptides, indicating that accurate computational prediction of binding affinities still remains a major methodological and technical challenge.

Crystallography, X-Ray↗

PubMed2005

Control of protein-protein interactions: structure-based discovery of low molecular weight inhibitors of the interactions between Pin1 WW domain and phosphopeptides.

Interactions involving phosphorylated Ser/Thr-Pro motifs in proteins play a key role in numerous regulatory processes in the cell. Here, we investigate potential ligands of the WW binding domain of Pin1 in order to inhibit protein-protein interactions between Pin1 and phosphopeptides. Our structure-based strategy implies the synthesis of analogues of the Ac-Thr(PO(3)H(2))-Pro-NH(2) dipeptide and relies on high resolution NMR spectroscopy to accurately measure the affinity constants even in the high micromolar range.

Binding Sites↗

PubMed1997

Discovery of 3-aminobenzyloxycarbonyl as an N-terminal group conferring high affinity to the minimal phosphopeptide sequence recognized by the Grb2-SH2 domain.

The observation that anthranilic acid as N-terminal group produces a dramatic increase of the binding affinity of the phosphopeptide sequence Glu-pTyr-Ile-Asn for the Grb2-SH2 domain was rationalized by molecular modeling. The model, which invokes a stacking interaction between the N-terminal group and the SH2 domain residue Arg alpha A2, was subsequently used to design the 3-aminobenzyloxycarbonyl N-terminal group. The latter confers high affinity (IC50 = 65 nM in an ELISA assay) to the minimal sequence pTyr-Ile-Asn recognized by the Grb2-SH2 domain.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Synthesis and immunogenic properties of phosphopeptides related to the human insulin receptor.

Two phosphoserine tetradecapeptides corresponding to sequences 987-1000 (peptide pSer994) and 1017-1030 (peptide pSer1023/1025) from the human insulin receptor involved in the regulation of its activity were successfully synthesized using Fmoc-based chemistry. Phosphorylation was performed by post-assembly phosphitylation followed by oxidation. The selective phosphorylation of Ser residues was achieved incorporating into the peptide chain the Ser (Trt) derivative and t-Bu blocking groups at sites other than those intended to be phosphorylated. The Trt group was selectively removed with dichloroacetic acid while under this condition t-Bu protecting groups remained unaltered. Following conjugation to keyhole limpet hemocyanin phosphopeptides were used as immunogens to generate sequence-specific phosphoserine antibodies. Peptide pSer994 induced antibodies in New Zealand white rabbits which discriminated between the phosphorylated and nonphosphorylated forms of the peptide, thus representing promising candidates to recognize signaling pathways associated to the regulation of the human insulin receptor.

Amino Acid Sequence↗

PubMed2004

Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1.

The BRCT repeats in BRCA1 are essential for its tumor suppressor activity and interact with phosphorylated protein targets containing the sequence pSer-X-X-Phe, where X indicates any residue. The structure of the tandem BRCA1 BRCT repeats bound to an optimized phosphopeptide reveals that the N-terminal repeat harbors a conserved BRCT phosphoserine-binding pocket, while the interface between the repeats forms a hydrophobic groove that recognizes the phenylalanine. Crystallographic and biochemical data suggest that the structural integrity of both binding sites is essential for peptide recognition. The diminished peptide-binding capacity observed for cancer-associated BRCA1-BRCT variants may explain the enhanced cancer risks associated with these mutations.

Amino Acid Sequence↗

PubMed2004

Inhibition of hydroxyapatite formation by osteopontin phosphopeptides.

Osteopontin (OPN) is an acidic phosphoglycoprotein that is believed to function in the prevention of soft tissue calcification. In vitro studies have shown that OPN can inhibit the formation of hydroxyapatite (HA) and other biologically relevant crystal phases, and that this inhibitory activity requires phosphorylation of the protein; however, it is not known which phosphorylated residues are involved. We have synthesized peptides corresponding to four phosphoserine-containing sequences in rat OPN: OPN7-17, containing phosphoserines 10 and 11; OPN41-52, containing phosphoserines 46 and 47; OPN248-264, containing phosphoserines 250, 257 and 262; and OPN290-301, containing phosphoserines 295-297. The abilities of these peptides to inhibit de novo HA formation were determined using a constant-composition autotitration assay. All four OPN phosphopeptides caused a dose-dependent increase in nucleation lag time, but did not significantly affect subsequent formation of the crystals. However, OPN41-52 (inhibitory constant 73.5 min/microM) and OPN290-301 (72.2 min/microM) were approx. 4 times more potent inhibitors than OPN7-17 (19.7 min/microM) and OPN247-264 (16.3 min/microM). 'Scrambling' the amino acid sequence of OPN290-301 resulted in decreased potency (45.6 min/microM), whereas omission of the phosphate groups from this peptide caused a greater decrease (5.20 min/microM). These findings have identified phosphorylated sequences that are important for the ability of rat bone OPN to inhibit HA crystal formation, and suggest that negative-charge density is an important factor in this activity.

Amino Acid Sequence↗

PubMed1987

A monoclonal antibody to the phosphorylated form of phenylalanine hydroxylase. Definition of the phosphopeptide epitope.

Monoclonal antibody PH7 has specificity for the phosphorylated form of the human liver phenylalanine hydroxylase and negligible reactivity towards the dephosphorylated form of the native enzyme by enzyme-linked immunoassay. PH7 binds specifically to the phosphorylated form of the liver enzyme after SDS/polyacrylamide-gel electrophoresis and transfer to nitrocellulose. Competitive blocking assays have been applied in conjunction with reversed-phase h.p.l.c. of purified tryptic fragments of human liver phenylalanine hydroxylase to localize the epitope. The major immunoreactive tryptic peptide cross-reacting with PH7 had an amino acid analysis corresponding to the first 41 amino acids of the human liver phenylalanine hydroxylase sequence and included the serine residue that is thought to be the phosphorylation site. The monoclonal antibody recognized the phosphorylated form of the synthetic decapeptide corresponding to the local phosphorylation-site sequence Gly-Leu-Gly-Arg-Lys-Leu-Ser(P)-Asp-Phe-Gly, but not the dephosphodecapeptide. Thermolysin digestion of the peptide demonstrated the monoclonal antibody bound to the pentapeptide Leu-Ser(P)-Asp-Phe-Gly. Monoclonal antibody PH7 recognized the phosphodecapeptide at concentrations 10(3)-fold higher than with phenylalanine hydroxylase, compared with 10(4)-10(7)-fold higher for other phosphopeptides and phosphoproteins. The results demonstrate that monoclonal antibody PH7 has specificity for the phosphorylated form of phenylalanine hydroxylase at the phosphorylation site.

Amino Acids↗