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PubMed2000

Identification of phosphorylation sites in proteins by nanospray quadrupole ion trap mass spectrometry.

A method is described for identifying serine phosphorylation sites in proteins, based on conventional (32)P labeling followed by electrophoretic separation, 'in-gel' digestion with a protease, peptide extraction, reversed-phase high-performance liquid chromatographic separation and collection and off-line analysis of the radioactive fractions by nanospray ion trap mass spectrometry. The method was successfully applied to the identification of three phosphorylation sites in two proteins which were subjected to in vitro phosphorylation under physiological conditions. Different combinations of the various scanning modes of the ion trap, including high-resolution, multiple subfragmentation (or MS(n)) and fast scan analysis, were employed to identify the phosphopeptides, determine their sequence and localize the exact site of phosphorylation. 'Blind' fragmentation using fast scans was used to analyze a phosphopeptide which was undetectable in other scanning modes. The sequence, phosphorylation site and double cysteine modification of the potassium adduct of a peptide containing 35 residues were also determined by multiple fragmentation. The results not only support the validity of the proposed method for routine identification of phosphorylation sites, but also demonstrate the exceptional capability of off-line ion trap mass spectrometry in combination with nanospray ionization for performing very detailed studies on the structure of peptides.

Chromatography, High Pressure Liquid↗

PubMed2005

Ovarian cancer marker of 11.7 kDa detected by proteomics is a serum amyloid A1.

In this study, to reduce the number of major plasma components, we examined thermostable plasma fractions to search for a biomarker of ovarian cancer. An apparent cancer biomarker of 11.7 kDa was detected in these fractions using ProteinChip SELDI-TOF mass spectrometry system. This peak invariably appeared with another close peak of about 11.5 kDa, suggesting that it is a derivative of a larger mass molecule. Of 27 cancer plasma specimens, 15 (55.6%) demonstrated this peak pair, whereas only 2 of 34 controls specimens (5.8%) were shown to express it with low intensity. Using a method involving cysteine modification by 4-vinylpyridine (4-VP), 2-DE and HPLC, these peaks were identified by mass spectrometry as serum amyloid A1 (11.68 kDa) and its N-terminal arginine-truncated form (11.52 kDa).

Amino Acid Sequence↗

PubMed1993

Conformational and nucleic acid binding studies on the synthetic nucleocapsid protein of HIV-1.

A 55 residue peptide corresponding to the nucleocapsid protein of HIV-1 (NCp7) containing two zinc binding domains as well as three truncated peptides were synthesized by Fmoc-based solid phase synthesis using the fragment condensation approach. Circular dichroism (CD) data support a conformational model in trifluoroethanol/buffer solution consisting of two helical segments at the chain ends with two Zn-modules in the center of the molecule. CD titration experiments show that the synthetic protein binds two equivalents of Zn2+ stoichiometrically, and the Zn2+ induced conformational changes are completely reversible by addition of EDTA. NCp7 and its S-acetamidomethylated analog (NCp7-Acm), devoid of the zinc co-ordination centers, exhibit preferential binding to RNA with a Kd = approximately 10(-9) M irrespective of the cysteine modification as determined by filter binding assays. The binding affinity of the NCp7 protein to single-stranded DNA is lower than to RNA. Binding to double-stranded DNA is lower than to ssDNA. The NCp7-Acm protein exhibits reduced single-stranded DNA binding affinity compared to the unmodified protein. Nucleic acid binding analyses with the fragments of NCp7 protein suggest that two basic amino acid stretches are involved in RNA binding of the NCp7.

Amino Acid Sequence↗

PubMed1989

Evidence for an essential histidine residue in the Neurospora crassa plasma membrane H+-ATPase.

The Neurospora crassa plasma membrane H+-ATPase is rapidly inactivated in the presence of diethyl pyrocarbonate (DEP). The reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 M-1.min-1 at pH 6.9 and 25 degrees C. The difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of N-carbethoxyhistidine. No change in the absorbance of the inhibited ATPase at 278 nm or in the number of modifiable sulfhydryl groups is observed, indicating that the inhibition is not due to tyrosine or cysteine modification, and the inhibition is irreversible, ruling out serine residues. Furthermore, pretreatment of the ATPase with pyridoxal phosphate/NaBH4 under the conditions of the DEP treatment does not inhibit the ATPase and does not alter the DEP inhibition kinetics, indicating that the inactivation by DEP is not due to amino group modification. The pH dependence of the inactivation reaction indicates that the essential residue has a pKa near 7.5, and the activity lost as a result of H+-ATPase modification by DEP is partially recovered after hydroxylamine treatment at 4 degrees C. Taken together, these results strongly indicate that the inactivation of the H+-ATPase by DEP involves histidine modification. Analyses of the inhibition kinetics and the stoichiometry of modification indicate that among eight histidines modified per enzyme molecule, only one is essential for H+-ATPase activity. Finally, ADP protects against inactivation by DEP, indicating that the essential residue modified may be located at or near the nucleotide binding site.

Binding Sites↗

PubMed2000

Expression of five iduronate-2-sulfatase site-directed mutations.

Five point mutations (R88H, R88P, T118I, 959delT, R468Q) previously identified in the iduronate-2-sulfatase (IDS) gene of Italian Hunter patients were expressed in COS cells to evaluate their functional consequence on enzyme activity, processing and intracellular localization. The 88 arginine residue belongs to the CXPSR pentapeptide conserved in all human sulfatases, where cysteine modification to formylglycine is required for enzyme activity. Substitution of arginine with histidine residue resulted in 13.7% residual enzyme activity, with an apparent K(m) value (133 microM) lower than that found for the normal enzyme (327 microM), indicating a higher affinity for the substrate; substitution of arginine with proline resulted in total absence of residual activity, in agreement with the phenotypes observed in patients carrying R88H and R88P mutations. For the four missense mutations, pulse-chase labelling experiments showed an apparently normal maturation; however, subcellular fractionation demonstrated poor transport to lysosomes. Therefore, residues 88, 118 and 468 appear to be not essential for processing but important for IDS conformation.

Animals↗

PubMed1998

COS cell expression studies of P86L, P86R, P480L and P480Q Hunter's disease-causing mutations.

Three missense mutations identified in the IDS gene of our Hunter's disease patients (P86L, P480L and P480Q) and the previously described P86R mutation were expressed in COS cells to evaluate their functional consequence on iduronate-2-sulfatase (IDS) activity and processing. The 86-proline residue belongs to the highly conserved pentapeptide C-X-P-S-R in which cysteine modification to a formylglycine is required for sulfatase activity. The substitution of the 86-proline residue led to a severe mutation as no mature form was targeted to the lysosome in agreement with the severe phenotype observed in patients carrying P86L and P86R mutations. Expression studies with P480L and P480Q mutant cDNAs showed the presence of a small amount of 55 kDa mature form in the lysosomes of transfected COS cells. IDS activity of the P480L and P480Q mutants in cell extracts represents 16.6% and 5.4% of the wild-type, respectively.

Adolescent↗

PubMed2004

Theoretical investigations of the electronic structure and spectroscopy of mononuclear, non-heme [Fe-NO](6) complexes.

The unusual metal coordination and spin-state of the Fe(III) center in nitrile hydratase (NHase) has stimulated the synthesis of numerous model complexes in efforts to understand the reactivity and spectroscopic properties of the enzyme. A particular problem has been the development of model Fe(III) complexes that exhibit reversible, photolabile binding to nitric oxide (NO) in a manner similar to that observed for the NHase metal center. We now report a detailed NBO analysis of the ground-state chemical bonding in three [Fe-NO](6) complexes that exhibit different responses to irradiation, together with investigations of their spectroscopic properties using semiempirical INDO/S CI singles calculations. Our computational studies reveal a correlation between the photolability of these complexes and the existence of low-energy transitions that promote an electron into the Fe-NO pi(*) antibonding molecular orbital. In addition to providing detailed insights into how the ligand field influences the spectroscopy of these mononuclear complexes, these studies strengthen our previous conclusions regarding the role of post-translational cysteine modification in modulating the photoreactivity of the inactive, NO complex of NHase.

Journal Article↗

PubMed1994

Evidence against a role for the Kunitz domain in amyloidogenic and secretory processing of the amyloid precursor protein.

The effect of the Kunitz proteinase inhibitor (KPI) on potential beta-amyloid precursor protein (beta PP)-processing activities from control and Alzheimer's disease (AD) brains was examined using fluorogenic substrates designed to mimic the secretory and amyloidogenic cleavages in beta PP. In addition, the level of secretion of KPI-containing beta PP751 and KPI-lacking beta PP695 from transfected cells was examined to assess the effect of the KPI on beta PP secretion. beta PP751 and beta PP695, obtained from conditioned media of transfected cells, had no effect on proteinase activities against the secretory and amyloidogenic substrates in extracts from control and AD brains. At similar concentrations beta PP751, but not beta PP695, completely inhibited the activity of trypsin against these substrates. Serine proteinase inhibitors had only modest effects on activities from brain, whereas cysteine modification completely inhibited them, indicating that these proteinase activities were not of the serine type. Thus, the results do not support a role for the KPI in the secretion of beta PP or in the amyloidogenic cleavage of beta PP. The amounts of beta PP695 and beta PP751 collected from the media of transfected cells after 48 h of growth were similar, indicating an equal rate of secretion. This result suggests that the KPI domain in beta PP751 did not inhibit the secretory cleavage in transfected cells.

Aged↗

PubMed2005

The carboxyl-terminal region of cyclic nucleotide-modulated channels is a gating ring, not a permeation path.

The recent elucidation of the structure of the carboxyl-terminal region of the hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channel has prompted us to investigate a curious feature of this structure in HCN2 channels and in the related CNGA1 cyclic nucleotide-gated (CNG) channels. The crystallized fragment of the HCN2 channel contains both the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. At the center of the fourfold-symmetric structure is a tunnel that runs perpendicular to the membrane. The narrowest part of the tunnel is approximately 10 A in diameter and is lined by a ring of negatively charged amino acids: D487, E488, and D489. Many ion channels have "charge rings" that focus permeant ions at the mouth of the pore and increase channel conductance. We used nonstationary fluctuation analysis and single-channel recording, coupled with site-directed mutagenesis and cysteine modification, to determine whether this part of HCN and CNG channels might be an extension of the permeation pathway. Our results indicate that modifying charge-ring amino acids affects gating but not ion permeation in HCN2 and CNG channels. Thus, this portion of the channel is not an obligatory part of the ion path but instead acts as a "gating ring." The carboxyl-terminal region of these channels must hang below the pore much like the "hanging gondola" of voltage-gated potassium channels, but the permeation pathway must exit the protein before the level of the ring of charged amino acids.

Amino Acid Sequence↗

PubMed2006

Zinc binding to the HCCH motif of HIV-1 virion infectivity factor induces a conformational change that mediates protein-protein interactions.

Virion infectivity factor (Vif) is an accessory protein encoded by HIV-1 and is critical for viral infection of the host CD4(+) T cell population. Vif induces ubiquitination and subsequent degradation of Apo3G, a cytosolic cytidine deaminase that otherwise targets the retroviral genome. Interaction of Vif with the cellular Cullin5-based E3 ubiquitin ligase requires a conserved BC box and upstream residues that are part of the conserved H-(Xaa)(5)-C-(Xaa)(17-18)-C-(Xaa)(3-5)-H (HCCH) motif. The HCCH motif is involved in stabilizing the Vif-Cullin 5 interaction, but the exact role of the conserved His and Cys residues remains elusive. In this report, we find that full-length HIV-1 Vif, as well as a HCCH peptide, is capable of binding to zinc with high specificity. Zinc binding induces a conformational change that leads to the formation of large protein aggregates. EDTA reversed aggregation and regenerated the apoprotein conformation. Cysteine modification studies with the HCCH peptide suggest that C114 is critical for stabilizing the fold of the apopeptide, and that C133 is located in a solvent-exposed region with no definite secondary structure. Selective alkylation of C133 reduced metal-binding specificity of the HCCH peptide, allowing cobalt to bind with rates comparable to that with zinc. This study demonstrates that the HCCH motif of HIV-1 Vif is a unique metal-binding domain capable of mediating protein-protein interactions in the presence of zinc and adds to a growing list of examples in which metal ion binding induces protein misfolding and/or aggregation.

Amino Acid Motifs↗

PubMed2003

Evidence for distinct sodium-, dopamine-, and cocaine-dependent conformational changes in transmembrane segments 7 and 8 of the dopamine transporter.

Previously we obtained evidence based on engineering of Zn2+ binding sites that the extracellular parts of transmembrane segment 7 (TM7) and TM8 in the human dopamine transporter are important for transporter function. To further evaluate the role of this domain, we have employed the substituted cysteine accessibility method and performed 10 single cysteine substitutions at the extracellular ends of TM7 and TM8. The mutants were made in background mutants of the human dopamine transporter with either two (E2C) or five endogenous cysteines substituted (X5C) that render the transporter largely insensitive to cysteine modification. In two mutants (M371C and A399C), treatment with the sulfhydryl-reactive reagent [2-(trimethylammonium)-ethyl]methanethiosulfonate (MTSET) led to a substantial inhibition of [3H]dopamine uptake. In M371C this inactivation was enhanced by Na+ and blocked by dopamine. Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A399C. The present findings add new functionality to the TM7/8 region by providing evidence for the occurrence of distinct Na+-, substrate-, and perhaps inhibitor-induced conformational changes critical for the proper function of the transporter.

Amino Acid Sequence↗

PubMed2006

The cytoplasmic substrate permeation pathway of serotonin transporter.

Serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin (5-HT) and is a target for antidepressant drugs and psychostimulants. It is a member of a large family of neurotransmitter and amino acid transporters. A recent study using site-directed cysteine modification identified a helical region of the transporter with high accessibility to the cytoplasm. Subsequently, the high resolution structure of LeuT, a prokaryotic homologue, showed that the residues corresponding to this helical region are part of the fifth transmembrane domain. The accessibility of these positions is now shown to depend on conformational changes corresponding to interconversion of SERT between two forms that face the extracellular medium and the cytoplasm, respectively. Binding of the extracellular inhibitor cocaine decreased accessibility at these positions, whereas 5-HT, the transported substrate, increased it. The effect of 5-HT required the simultaneous presence of Na+ and Cl-, which are transported into the cell together (symported) with 5-HT. In light of the LeuT structure, these results begin to define the pathway through which 5-HT diffuses between its binding site and the cytoplasm. They also confirm a prediction of the alternating access model for transport, namely, that all symported substrates must bind together before translocation.

Animals↗

PubMed2006

Roles of K149, G352, and H401 in the channel functions of ClC-0: testing the predictions from theoretical calculations.

The ClC family of Cl(-) channels and transporters comprises membrane proteins ubiquitously present in species ranging from prokaryotes to mammals. The recently solved structures of the bacterial ClC proteins have provided a good model to guide the functional experiments for the eukaryotic Cl(-) channels. Theoretical calculations based on the bacterial ClC structures have identified several residues critical for the Cl(-) binding energy in the Cl(-) transport pathway. It was speculated that the corresponding residues in eukaryotic Cl(-) channels might play similar roles for the channel functions. In this study, we made a series of mutations in three such residues in eukaryotic ClC Cl(-) channels (K149, G352, and H401 in ClC-0) and studied the functional consequences on the channel properties. A cysteine modification approach was also employed to evaluate the electrostatic effects of the charge placed at these three positions. The experimental results revealed that among the three residues tested, K149 plays the most important role in controlling both the gating and the permeation functions of ClC-0. On the other hand, mutations of H401 alter the channel conductance but not the gating properties, while mutations of G352 result in very little functional consequence. The mutation of K149 into a neutral residue leucine (K149L) shifts the activation curve and leads to flickery channel openings. The anion permeability ratios derived from bi-ionic experiments are also significantly altered in that the selectivity of Cl(-) over other anions is decreased. Furthermore, removing the positive charge at this position reduces and increases, respectively, the accessibility of the negatively and positively charged methane thiosulfonate reagents to the pore. The control of the accessibility to charged MTS reagents and the regulation of the anion permeation support the idea that K149 exerts an electrostatic effect on the channel function, confirming the prediction from computational studies.

Amino Acid Substitution↗

PubMed1995

Bacterial expression, characterization and DNA binding studies on Drosophila melanogaster c-Myb DNA-binding protein.

The Drosophila Myb homologue retains an evolutionarily conserved typical sequence of three imperfect tandem tryptophan repeat units (R1-R2-R3) of 51-53 amino acids towards its N-terminus as its presumptive DNA binding domain. Using PCR amplification and the T7 expression vector pET 11d, we have overproduced this tryptophan repeat domain of Drosophila Myb in Escherichia coli and the protein has been purified. Circular dichroic measurements indicate that the protein has a high helical component (58.6%) in its overall structure. The protein is found to recognize the same cognate target sequence TAACGG, as recognized by the vertebrate proteins. The DNA binding properties of the protein have been investigated in detail by fluorescence spectroscopy taking advantage of the large number of tryptophan residues present in the protein. The fluorescence of the native Drosophila R123 was quenched when synthetic duplex DNA oligomers were added to the protein. The oligomers containing specific Myb target sites quenched the protein fluorescence to a greater extent than the non-specific DNA. Binding constants of the protein to the targets were also length dependent for smaller oligomers. Experiments with the collisional quencher acrylamide and cysteine modification reagent indicated that the specific and non-specific target sequences interact with the protein differently. In the former case both the buried and the exposed tryptophan residues were affected by DNA binding whereas in the latter only the solvent-exposed residues were involved.

Animals↗

PubMed1996

Inhibition by iodoacetamide and acetylene of the H-D-exchange reaction catalyzed by Thiocapsa roseopersicina hydrogenase.

The kinetics of H-D isotope exchange catalyzed by the thermostable hydrogenase from Thiocapsa roseopersicina have been studied by analysis of the exchange between D2 and H2O. The pH dependence of the exchange reaction was examined between pH 2.5 and pH 11. Over the whole pH range, HD was produced at a higher initial velocity than H2, with a marked optimum at pH 5.5; a second peak in the pH profile was observed at around pH 8.5. The rapid formation of H2 with respect to HD in the D2/H2O system is consistent with a heterolytic cleavage of D2 into D+ and an enzyme hydride that can both exchange with the solvent. The H-D-exchange activity was lower in the H2/D2O system than in the D2/H2O system. The other reactions catalyzed by the hydrogenase, H2 oxidation and H2 evolution, are pH dependent; the optimal pH were 9.5 for H2 uptake and 4.0 for H2 production. Treatment of the active form of hydrogenase by iodoacetamide led to a slow and irreversible inhibition of the H-D exchange. When iodo[1-14C]acetamide was incubated with hydrogenase, the radioactive labeling of the large subunit was higher for the enzyme activated under H2 than for the inactive oxidized form. Cysteine residues were identified as the alkylated derivative by amino acid analysis. Acetylene, which inhibits H-D exchange and abolishes the Ni-C EPR signal, protected the enzyme from irreversible inhibition by iodoacetamide. These data indicate that iodoacetamide can reach the active site of the H2-activated hydrogenase from T. roseopersicina. This was not found to be the case with the seleno hydrogenase from Desulfovibrio baculatus (now Desulfomicrobium baculatus). Cysteine modification by iodoacetamide upon activation of the enzyme concomitant with loss of H-D exchange indicates that reductive activation makes at least one Cys residue of the active site available for alkylation.

Acetylene↗

PubMed2006

Mouse tryptophan hydroxylase isoform 2 and the role of proline 447 in enzyme function.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin (5-HT). Once thought to be a single gene product, TPH is now known to exist in two isoforms. Isoform 1 (TPH1) is found in the pineal gland and gut, and isoform 2 (TPH2) is selectively expressed in brain. A single-nucleotide polymorphism in TPH2 results in a proline-to-arginine mutation at residue 447 and substantially lowers catalytic activity. In view of the importance of TPH in determining brain 5-HT function, we cloned TPH2 and produced the P447R mutant to assess the importance of this proline in enzyme function. Catalytically active TPH2 and the P447R mutant were expressed at the predicted subunit molecular mass of 56 kDa. The P447R mutant expressed less than 50% of the activity of TPH2. Mutation of this conserved proline in TPH1 (P403R) also resulted in an enzyme with significantly lower activity than the wild-type enzyme. The P447R mutant had a V(max) 50% lower than that of TPH2. The P447R mutation did not alter the oligomeric assembly of the protein, nor change its responsiveness to cysteine modification. The P447R mutation did not alter enzyme substrate specificity or stability, but conferred slightly enhanced sensitivity to inhibition by dopamine and diminished sensitivity to iron in catalysis. The conserved proline in TPH (residue 447 in TPH2 and 403 in TPH1) plays an important role in enzyme function by regulating V(max) of the catalytic reaction.

Animals↗

PubMed2003

Tetrahydrobiopterin prevents nitration of tyrosine hydroxylase by peroxynitrite and nitrogen dioxide.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the synthesis of the neurotransmitter dopamine. TH is inhibited and nitrated at tyrosine residues in vitro by the reactive nitrogen species peroxynitrite and nitrogen dioxide (NO2) and in vivo by drugs that damage dopamine neurons. Tetrahydrobiopterin, which is the essential cofactor for TH and is concentrated in dopamine neurons, completely blocks nitration of tyrosine residues in TH caused by peroxynitrite or NO2. Various tetrahydro- and dihydro-analogs of tetrahydrobiopterin, including 6,7-dimethyl-tetrahydropterin, 6-methyl-tetrahydropterin, 6-hydroxymethyl-tetrahydropterin, tetrahydropterin, 7,8-dihydrobiopterin, 7,8-dihydroxanthopterin, and sepiapterin, also prevent nitration of tyrosines caused by the reactive nitrogen species. Biopterin and pterin, the fully oxidized forms of the pterin molecule, fail to block peroxynitrite- or NO2-induced nitration of TH. Reduced pterins prevent neither the inhibition of TH activity nor cysteine modification caused by peroxynitrite or NO2, despite blocking tyrosine nitration. However, dithiothreitol prevents and reverses these effects on TH of tetrahydrobiopterin and reactive nitrogen species. Using an enhanced green fluorescent protein-TH fusion construct as a real-time reporter of intracellular tyrosine nitration, tetrahydrobiopterin was found to prevent NO2-induced tyrosine nitration in intact cells but to leave TH activity inhibited. These results indicate that tetrahydrobiopterin prevents the tyrosine-nitrating properties of peroxynitrite and NO2. Tetrahydrobiopterin-derived radical species formed by reaction with reactive nitrogen species may account for inhibition of TH via mechanisms that do not involve tyrosine nitration.

Animals↗

PubMed2002

OxyR: a molecular code for redox sensing?

Helmann discusses the controversy surrounding the activation of the bacterial redox-regulated transcription factor OxyR. Evidence from different sources, including crystallographic data, has led to opposing models for the chemical changes that activate OxyR. Is it an intramolecular disulfide-linkage? Is it oxidation of a single cysteine residue to a sulfenic acid? Are there different active forms depending on the type of cysteine modification: intramolecular disulfide bond, sulfenic acid, S-nitrosothiol, or mixed disulfide with glutathione? These issues are discussed in the broader context of transcriptional regulation and how particular regulators may activate distinct genetic programs depending on the precise state of the regulator produced in response to environmental cues.

Bacterial Proteins↗