Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.     

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.     

Identification of the cadaverine recognition site on the cadaverine-lysine antiporter CadB

Amino acid residues involved in cadaverine uptake and cadaverine-lysine antiporter activity were identified by site-directed mutagenesis of the CadB protein. It was found that Tyr(73), Tyr(89), Tyr(90), Glu(204), Tyr(235), Asp(303), and Tyr(423) were strongly involved in both uptake and excretion and that Tyr(55), Glu(76), Tyr(246), Tyr(310), Cys(370), and Glu(377) were moderately involved in both activities. Mutations of Trp(43), Tyr(57), Tyr(107), Tyr(366), and Tyr(368) mainly affected uptake activity, and Trp(41), Tyr(174), Asp(185), and Glu(408) had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the K(m) value. Mutation of Arg(299) mainly affected excretion, suggesting that Arg(299) is involved in the recognition of the carboxyl group of lysine. These results indicate that amino acid residues involved in both uptake and excretion, or solely in excretion, are located in the cytoplasmic loops and the cytoplasmic side of transmembrane segments, whereas residues involved in uptake were located in the periplasmic loops and the transmembrane segments. The SH group of Cys(370) seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys(125), Cys(389), or Cys(394) together with Cys(370). The relative topology of 12 transmembrane segments was determined by inserting cysteine residues at various sites and measuring the degree of inhibition of transport through crosslinking with Cys(370). The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII.     

Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.     

Purification and characterization of a low and a high molecular weight form of epidermal growth factor from rat urine

Two forms of epidermal growth factor (EGF) have been purified to homogeneity from rat urine by immunoaffinity chromatography and gel filtration. For one of the purified peptides the molecular mass has been determined to be 5891 by mass spectrometry. This peptide consists of 51 amino acid residues. The sequence of the first 48 amino acid residues is identical to the previously published sequence for submandibular rat EGF. The C-terminal three residues (49-51) are Trp-Trp-Lys. The other purified peptide has a molecular mass of 45 kDa as determined by SDS-polyacrylamide gel electrophoresis. The N-terminal sequence is Asn-Tyr-Lys-Asp-(Cys)-Gly-Pro-Gly-Gly-(Cys)-Gly-Ser-His-Ala. Both the high and the low molecular mass form of urinary rat EGF are able to bind to the human placenta receptor for EGF.     

Modification of the catalytic subunit of plasma fibrin-stabilizing factor under induced oxidation

For the first time, by using mass-spectrometry method, the oxidation-mediated modification of the catalytic FXIII-A subunit of plasma fibrin-stabilizing factor, pFXIII, has been studied. The oxidative sites were identified to belong to all structural elements of the catalytic subunit: the β-sandwich (Tyr104, Tyr117, and Cys153), the catalytic core domain (Met160, Trp165, Met266, Cys328, Asp352, Pro387, Arg409, Cys410, Tyr442, Met475, Met476, Tyr482, and Met500), the β-barrel 1 (Met596), and the β-barrel 2 (Met647, Pro676, Trp692, Cys696, and Met710), which correspond to 3.9%, 1.11%, 0.7%, and 3.2%, respectively, of oxidative modifications as compared to the detectable amounts of amino acid residues in each of the structural domains. Lack of information on some parts of the molecule may be associated with the spatial unavailability of residues, complicating analysis of the molecule. The absence of oxidative sites localized within crucial areas of the structural domains may be brought about by both the spatial inaccessibility of the oxidant to amino acid residues in the zymogen and the screening effect of the regulatory FXIII-B subunit.     

Molecular basis of intramolecular electron transfer in proteins during radical-mediated oxidations: computer simulation studies in model tyrosine-cysteine peptides in solution

Experimental studies in hemeproteins and model Tyr/Cys-containing peptides exposed to oxidizing and nitrating species suggest that intramolecular electron transfer (IET) between tyrosyl radicals (Tyr-O(·)) and Cys residues controls oxidative modification yields. The molecular basis of this IET process is not sufficiently understood with structural atomic detail. Herein, we analyzed using molecular dynamics and quantum mechanics-based computational calculations, mechanistic possibilities for the radical transfer reaction in Tyr/Cys-containing peptides in solution and correlated them with existing experimental data. Our results support that Tyr-O(·) to Cys radical transfer is mediated by an acid/base equilibrium that involves deprotonation of Cys to form the thiolate, followed by a likely rate-limiting transfer process to yield cysteinyl radical and a Tyr phenolate; proton uptake by Tyr completes the reaction. Both, the pKa values of the Tyr phenol and Cys thiol groups and the energetic and kinetics of the reversible IET are revealed as key physico-chemical factors. The proposed mechanism constitutes a case of sequential, acid/base equilibrium-dependent and solvent-mediated, proton-coupled electron transfer and explains the dependency of oxidative yields in Tyr/Cys peptides as a function of the number of alanine spacers. These findings contribute to explain oxidative modifications in proteins that contain sequence and/or spatially close Tyr-Cys residues.     

Oxidation of human lens recombinant alphaA-crystallin and cysteine-deficient mutants

Disulfide cross-linking, one of the results of oxidative stress, has been thought to play an important role in cataractogenesis. High molecular mass (HMM) protein aggregation also contributes to cataract development, and a prevailing speculation is that disulfide cross-linking induces HMM aggregation. However, there is no direct evidence to support this speculation. Dimerization is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. alphaA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutants, two single mutants (C131I and C142I) and one double mutant (C131I/C142I). They were subjected to H202 oxidation in an ascorbate-FeCl(3)-EDTA-H202 system. The effects of oxidation on the mutants, including changes in aggregate size and conformation, were compared with those of the wild-type alphaA-crystallin by FPLC gel filtration, absorption, fluorescence, and circular dichroism measurements. The results indicated that other amino acid residues besides Cys, such as Trp and Tyr, were also oxidized by H202. Disulfide dimerization alone seems to play a less important role in HMM aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys.     

Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis

The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66-->Asn and Asp-66-->Glu mutation remarkably decreased kinetic parameters such as Vmax and kcat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49-->Ser/Tyr and Cys-72-->Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211-->Ser/Tyr mutant enzyme was less than 10 min at 80 degrees C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211-->Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.     

Metallothionein protein variants generated in rat liver as a result of DNA and RNA ethylations by the carcinogen diethylnitrosamine

Metallothionein (MT) is a protein which contains 20 cysteine residues but no aromatic amino acids. It was tested whether treatment of male rats with the hepatocarcinogen diethylnitrosamine (DENA) could ethylate nucleic acids in such a way that protein variants containing measurable amounts of aromatic amino acid residues could be isolated from the livers of treated animals. To give a low limit of detection, the "wrong" amino acid precursors were administered in radiolabelled form at high levels of activity (7 mCi/kg each of [3H]tyrosine and [3H]phenylalanine). 11 microCi/kg [14C]cysteine was given as an internal marker for MT biosynthesis. 6 h after amino acid administration, metallothionein (MT) was isolated from the liver and extensively purified. After acid hydrolysis and collection of Cys, Tyr, and Phe from an HPLC analysis of the amino acids, the 3H/14C ratio was determined. The carcinogen-treated rats exhibited a significantly higher ratio than the vehicle-treated animals. This type of in vivo assay might find interesting applications in the investigation of nucleic acid alkylations as promutagenic lesions.     

Trends in Codon and Amino Acid Usage in Thermotoga maritima

The usage of synonymous codons and the frequencies of amino acids were investigated in the complete genome of the bacterium Thermotoga maritima using a multivariate statistical approach. The GC3 content of each gene was the most prominent source of variation of codon usage. Surprisingly the usage of UGU and UGC (synonymous triplets coding for Cys, the least frequent amino acid in this species) was detected as the second most prominent source of variation. However, this result is probably an artifact due to the very low frequency of Cys together with the nonbiased composition of this genome. The third trend was related to the preferential usage of a subset of codons among highly expressed genes, and these triplets are presumed to be translationally optimal. Concerning the amino acid usage, the hydropathy level of each protein (and therefore the frequency of charged residues) was the main trend, while the second factor was related to the frequency of usage of the smaller residues, suggesting that the cell economy strongly influences the architecture of the proteins. The third axis of the analysis discriminated the usage of Phe, Tyr, Trp (aromatic residues) plus Cys, Met, and His. These six residues have in common the property of being the preferential targets of reactive oxygen species, and therefore the anaerobic condition of T. maritima is an important factor for the amino acid frequencies. Finally, the Cys content of each protein was the fourth trend. Received: 22 June 2001 / Accepted: 1 October 2001     

Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide

Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.     

Structural insights into the second step of RNA-dependent cysteine biosynthesis in archaea: crystal structure of Sep-tRNA:Cys-tRNA synthase from Archaeoglobus fulgidus

In the ancient organisms, methanogenic archaea, lacking the canonical cysteinyl-tRNA synthetase, Cys-tRNA(Cys) is produced by an indirect pathway, in which O-phosphoseryl-tRNA synthetase ligates O-phosphoserine (Sep) to tRNA(Cys) and Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). In this study, the crystal structure of SepCysS from Archaeoglobus fulgidus has been determined at 2.4 A resolution. SepCysS forms a dimer, composed of monomers bearing large and small domains. The large domain harbors the seven-stranded beta-sheet, which is typical of the pyridoxal 5'-phosphate (PLP)-dependent enzymes. In the active site, which is located near the dimer interface, PLP is covalently bound to the side-chain of the conserved Lys209. In the proximity of PLP, a sulfate ion is bound by the side-chains of the conserved Arg79, His103, and Tyr104 residues. The active site is located deep within the large, basic cleft to accommodate Sep-tRNA(Cys). On the basis of the surface electrostatic potential, the amino acid residue conservation mapping, the position of the bound sulfate ion, and the substrate amino acid binding manner in other PLP-dependent enzymes, a binding model of Sep-tRNA(Cys) to SepCysS was constructed. One of the three strictly conserved Cys residues (Cys39, Cys42, or Cys247), of one subunit may play a crucial role in the catalysis in the active site of the other subunit.     

Structure-function relationship of [2Fe2S] ferredoxins and design of a model molecule

[2Fe2S] ferredoxins isolated from various plants and algae comprise 93–99 amino acid residues and resemble each other not only in sequences, but also in physiological functions. One of them isolated from Spirulina platensis was subjected to X-ray analysis and its three dimensional structure is now known. [2Fe2S] ferredoxins of a different type are found in halobacteria and comprise 128 amino acid residues. Both types of the [2Fe2S] ferredoxins exhibit low redox potentials. By comparing the amino acid sequences of 28 [2Fe2S] ferredoxins and the tertiary structure of S. platensis ferredoxin we predicted a common three-dimensional structure to the [2Fe2S] ferredoxins and proposed a molecular surface area to be interacting with FNR. An artificial small molecule composed of 20 amino acid residues is designed on the basis of the tertiary structure of S. platensis ferredoxin. The amino acid sequence was predicted to be ProTyrSerCysArgAlaGlyAlaCysSerThrCysAlaGly ProLeuLeuThr CysVal which should have a [2Fe2S] cluster with a low redox potential     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

家蚕滞育生物钟蛋白质Ease A4的纯化及其分子结构分析

ＥａｓｅＡ４是家蚕卵的一种滞育生物钟蛋白质．产下后２ｄ的家蚕Ｃ１０８品种滞育性卵,经过丙酮脱脂、８５℃热处理、硫酸铵沉淀和ＳｅｐｈａｄｅｘＧ－２５凝胶过滤层析初步分离,进一步经过Ｓｅｐ－ＰａｋＣ１８脱盐浓缩,ＨＰＬＣ（柱为ＹＭＣ－ＰａｃｋＰｒｏｔｅｉｎ－ＲＰ）分离,通过ＳＤＳ－ＰＡＧＥ和ＭＡＬＤＩＭＳ方法鉴定,纯化得到ＥａｓｅＡ４蛋白质．从１０ｇ蚕卵最终得到了１１．８μｇＥａｓｅＡ４．ＥａｓｅＡ４由从Ｈｉｓ到Ｔｙｒ的１５５个氨基酸残基构成,蛋白质部分的分子量为１６６０１．其２２位氨基酸残基Ａｓｎ处有一个Ａｓｎ－Ｘ－Ｔｈｒ糖基化场所,并有糖基结合在该部位,糖基的分子量约为７６０．ＥａｓｅＡ４的６１位和１５０位的两个Ｃｙｓ氨基酸残基之间存在二硫键．糖基和二硫键的存在不仅有利于酶蛋白的分离,还可能与酶活性有关     

Synthesis of side chain-protected amino acid phenylthiohydantoins and their use in quantitative solid-phase Edman degradation

Solid-phase Edman degradation of synthetic peptidyl-resins has been used advantageously to detect errors of deletion which might occur during Merrifield peptide synthesis. To facilitate complete quantitation of the resulting phenylthiohydantoin(PTH)-amino acids, the PTH derivatives of the following side chain-protected amino acid residues have been synthesized: Arg(Tos), Asp(OBzl), Cys(3,4-(CH3)2-Bzl), Glu(OBzl), Lys(2-ClZ), Ser(Bzl), Thr(Bzl), Tyr(2-BrZ), and Tyr(2,6-Cl2Bzl). For each derivative, a melting point, elemental analysis, and extinction coefficient were obtained. With these new compounds as HPLC standards, an unequivocal assignment and quantification of each side chain protected amino acid was possible. A quantitative analysis was performed for six model peptides with the general formula Ala-X-Leu-Y-Ala-Gly-NHCH2-resin (where X and Y represented different side chain-protected amino acyl residues). We have found solid-phase Edman degradation to be a useful aid for the characterization of peptides when they are used unpurified as synthetic antigens.     

Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis.

The tat gene of HIV-1 is a potent trans-activator of gene expression from the HIV long terminal repeat (LTR). To define the functionally important regions of the product of the tat gene (Tat) of HIV-1, deletion, linker insertion and single amino acid substitution mutants within the Tat coding region of strain SF2 were constructed. The effect of these mutations on trans-activation was assessed by measuring the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene linked to the HIV-LTR. These studies have revealed that four different domains of the protein that map within the N-terminal 56 amino acid region are essential for Tat function. In addition to the essential domains, an auxiliary domain that enhances the activity of the essential region has also been mapped between amino acid residues 58 and 66. One of the essential domains maps in the N-terminal 20 amino acid region. The other three essential domains are highly conserved among the various strains of HIV-1 and HIV-2 as well as simian immunodeficiency virus (SIV). Of the conserved domains, one contains seven Cys residues and single amino acid substitutions for several Cys residues indicate that they are essential for Tat function. The second conserved domain contains a Lys X Leu Gly Ile X Tyr motif in which the Lys residue is essential for trans-activation and the other residues are partially essential. The third conserved domain is strongly basic and appears to play a dual role. Mutants lacking this domain are deficient in trans-activation and in efficient targeting of Tat to the nucleus and nucleolus. The combination of the four essential domains and the auxiliary domain contribute to the near full activity observed with the 101 amino acid Tat protein.     

Re-evaluation of amino acid sequence and structural consensus rules for cysteine-nitric oxide reactivity

Nitric oxide (NO), produced in different cell types through the conversion of L-arginine into L-citrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NO-reactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acid-base catalysed Cys beta93-nitrosylation and Cys beta393NO-denitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites -1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for Cys-NO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the -2 position has been observed in some NO-reactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri- or tetra-peptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18, 691-696]. Here, we analyse the three-dimensional structure of several proteins containing NO-reactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the Cys-Sy atomic structural microenvironment rather than on the tri- or tetra-peptide sequence consensus motif.     

Lysozyme modification by the fenton reaction and gamma radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H2 O2) or hydroxyl radicals produced by y radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. Gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.