Lack Of Cysteine Research Articles

PCNA from Thermococcus gammatolerans: A protein involved in chromosomal DNA metabolism intrinsically resistant at high levels of ionizing radiation.

Proliferating cell nuclear antigen (PCNA) is an essential protein for cell viability in archaea and eukarya, since it is involved in DNA replication and repair. In order to obtain insights regarding the characteristics that confer radioresistance, the structural study of the PCNA from Thermococcus gammatolerans (PCNATg ) in a gradient of ionizing radiation by X-ray crystallography was carried out, together with a bioinformatic analysis of homotrimeric PCNA structures, their sequences, and their molecular interactions. The results obtained from the datasets and the accumulated radiation dose for the last collection from three crystals revealed moderate and localized damage, since even with the loss of resolution, the electron density map corresponding to the last collection allowed to build the whole structure. Attempting to understand this behavior, multiple sequence alignments, and structural superpositions were performed, revealing that PCNA is a protein with a poorly conserved sequence, but with a highly conserved structure. The PCNATg presented the highest percentage of charged residues, mostly negatively charged, with a proportion of glutamate more than double aspartate, lack of cysteines and tryptophan, besides a high number of salt bridges. The structural study by X-ray crystallography reveals that the PCNATg has the intrinsic ability to resist high levels of ionizing radiation, and the bioinformatic analysis suggests that molecular evolution selected a particular composition of amino acid residues, and their consequent network of synergistic interactions for extreme conditions, as a collateral effect, conferring radioresistance to a protein involved in the chromosomal DNA metabolism of a radioresistant microorganism.

Studies on the Interaction between Poly-Phosphane Gold(I) Complexes and Dihydrofolate Reductase: An Interplay with Nicotinamide Adenine Dinucleotide Cofactor.

A class of gold(I) phosphane complexes have been identified as inhibitors of dihydrofolate reductase (DHFR) from E. coli, an enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using NADPH as a coenzyme. In this work, to comprehend the nature of the interaction at the basis of these inhibitory effects, the binding properties of bis- and tris-phosphane gold(I) chloride compounds in regards to DHFR have been studied by emission spectroscopy and spectrophotometric assays. The lack of cysteine and seleno-cysteine residues in the enzyme active site, the most favorable sites of attack of Au(I) moieties, makes this work noteworthy. The interaction with the gold compounds results into the quenching of the DHFR tryptophan’s emissions and in an enhancement of their intrinsic emission intensities. Moreover, a modulating action of NADPH is highlighted by means of an increase of the gold compound affinity toward the enzyme; in fact, the dissociation constants calculated for the interactions between DHFR and each gold compound in the presence of saturating NADPH were lower than the ones observed for the apo-enzyme. The fluorimetric data afforded to Kd values ranged from 2.22 ± 0.25 µM for (PPh3)2AuCl in the presence of NADPH to 21.4 ± 3.85 µM for 4L3AuTf in the absence of NADPH. By elucidating the energetic aspects of the binding events, we have attempted to dissect the role played by the gold phosphane/protein interactions in the inhibitory activity, resulting in an exothermic enthalpy change and a positive entropic contribution (ΔH° = −5.04 ± 0.08 kcal/mol and ΔS° = 7.34 ± 0.005 cal/mol·K).

Apolipoprotein E4 domain interaction: Synaptic and cognitive deficits in mice

Background Apolipoprotein E4 (apoE4), the major genetic risk factor for Alzheimer’s disease (AD) and other neurodegenerative diseases, has three structural and biophysical properties that distinguish it from the other isoforms—domain interaction, reduced stability, and lack of cysteine. Assessing their relative contributions to effects of apoE4-associated pathogenesis in AD is important from a mechanistic and therapeutic perspective, that is not possible using human apoE transgene or knock-in models. Methods We analyzed Arg-61 apoE mice, a gene-targeted model that selectively displays domain interaction. Results The mice displayed age-dependent loss of the synaptic protein synaptophysin in neocortex and hippocampus and had lower levels of the postsynaptic neuroligin-1. Activation of dentate gyrus granule neurons increased Arc expression 3.5-fold in wildtype mice but only 2.3-fold in Arg-61 mice. The losses of synaptic proteins caused a mild memory deficit in Arg-61 mice in the water-maze test. Since synaptic integrity requires efficient glutamate uptake, we measured astrocyte glutamate transporter 1 in the hippocampus. The level was reduced in Arg-61 mice, suggesting that inefficient glutamate uptake by astrocytes causes chronic excitotoxicity. Consistent with the reduced secretion of Arg-61 apoE by astrocytes in this model, cholesterol secretion was also reduced 34%. This reduction could also contribute to the synaptic deficits by limiting the availability of cholesterol for neuronal repair. Conclusions Domain interaction in the absence of other structural characteristics of apoE4 is sufficient to cause synaptic pathology and functional synaptic deficits, potentially associated with astrocyte dysfunction and impaired maintenance of neurons. Therapeutic targeting of domain interaction might blunt effects of apoE4 in neurodegenerative disease.

Identification of a Novel Type of Silk Protein and Regulation of Its Expression

The silk of lepidopteran insects has been studied extensively as proteins of two categories: the fibroins, which are produced in the posterior section of silk glands, and the sericins, which are secreted in the middle section. We now describe a third category that is named seroins to accentuate the fact that both the sericin- and the fibroin-producing cells participate in seroin secretion. Using a probe derived from the N-terminal sequences of a 23-kDa components of Galleria mellonella silk, we isolated silk gland-specific cDNA encoding 167 amino acids, of which 17 constitute the signal peptide. The following 14 residues match the N-terminal sequences of the 23- and 22.5-kDa silk proteines. The reaction of these proteins with concanavalin A and the presence of two glycosylation sites in the seroin peptide sequence indicate that seroin is secreted in two forms that both contain a mannose-rich sugar moiety. Seroin is distinguished from other silk proteins by high proline content (34 residues or 20.26% by weight), lack of cysteines, and the presence of two kinds of short amino acid repeats. The seroin gene is expressed in both the posterior and middle silk gland sections. The expression fluctuates during development in correlation with the feeding regime and the changes in hormone titers: seroin mRNA is high in the silk glands of feeding larvae, declines at ecdysis, reaches a maximum during cocoon spinning, and thereafter rapidly drops to an undetectable level. In vivo and in vitro experiments showed that the drop is caused by ecdysteroid hormones and is prevented by juvenile hormones. N-terminal sequencing of several silk proteins of Bombyx mori revealed that the 8- and 13-kDa proteins share 5 or 6 out of 10 identified amino acids with the N terminus of Galleria seroin and obviously represent seroin homologues. The result suggests that seroin-type proteins are a general component of lepidopteran silk.

Fluorescence studies on plasminogen activator inhibitor 1: Reactive centre cysteine mutants remain active after fluorophore attachment

To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1). Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1. The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed. The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration. The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion. The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys. No significant spectral changes could be seen when the P3cys mutant was transferred to latency. In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine. Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.

Evidence for a charge variant of Cd-BP 14, a cadmium-binding protein from Allolobophora caliginosa (Oligochaeta, Annelida)

1. 1. In Allolobophora caliginosa , a Cd-binding protein distinct in charge from Cd-BP 14, a Cd-binding protein previously isolated from the same oligochaete species [Nejmeddine et al. (1992) Comp. Biochem. Physiol. 101C, 601–605], has been purified by a three-step chromatographic procedure including gel permeation and cation-exchange chromatography. 2. 2. This Cd-binding protein exists in a monomeric form with a molecular weight of 14 kDa and does not contain carbohydrate. 3. 3. The purified protein significantly absorbed at 280 nm and its amino acid composition revealed the presence of a high level of aromatic amino acids and a lack of cysteine, indicating that the molecule is distinct from metallothioneins. 4. 4. By contrast, except for its chromatographic behavior on an ion-exchange chromatography column, the metalloprotein was found to be similar to Cd-BP 14. We thus conclude that it represents a charge-variant of Cd-BP 14.

Isolation and characterization of a Cd-binding protein from Allolobophora caliginosa (Annelida, Oligochaeta): Distinction from metallothioneins

1. One Cd-binding peak was detected after gel filtration chromatography on Sephadex G75 in an extract from Allolobophora caliginosa contaminated with Cd. 2. Two subsequent cation-exchange chromatographies allowed the isolation of a Cd-binding protein which was called Cd-BP14. This protein is a monomer with a molecular weight of 14 kDa and has an isoelectric point of 6.5. 3. Amino acid analysis showed the presence of a high level of aromatic amino acids and a lack of cysteine. 4. On the basis of these results we conclude that Cd-BP14 is different from metallothioneins described in mammals or other invertebrates.

Nuclear basic protein transition during sperm differentiation. Primary structure of the spermatid-specific protein S2 from the dog-fish Scylliorhinus caniculus.

The remodeling of nucleoproteins during dog-fish spermiogenesis involves two successive nuclear protein transitions: the first from somatic-type histones to transition proteins during the nuclear elongation of spermatids and the second leading to protamine-DNA association in mature spermatozoa. The chromatin of elongating spermatids contains two transition proteins called S1 and S2. The amino acid sequence of protein S1, a polypeptide of 87 residues was determined previously [Chauvière, M., Martinage, A., Briand, G., Sautière, P. & Chevaillier, Ph. (1987) Eur. J. Biochem. 169, 105-111]. In the present paper, we report the elucidation of the primary structure of the minor transition protein S2 established by automated Edman degradation of the protein and of its fragments generated by cleavage at methionine and aspartate residues. S2 contains 80 residues and has a molecular mass of 9726 Da. S2 is mainly characterized by a high content of basic amino acids mostly represented by lysine, a relatively high level of hydrophobic residues, the presence of six phosphorylatable residues and the lack of cysteine. Its amino acid sequence shows that the N-terminal half is highly basic, while the acidic residues are located in the C-terminal part of the protein where more diversity in amino acids is noticed. The two transition proteins S1 and S2 share striking structural similarities. Few but significative similarities have been detected with the mammalian transition protein TP1 [Kistler, W. S., Noyes, C., Hsu, R. & Heinrikson, R. L. (1975) J. Biol. Chem. 250, 1847-1853], suggesting similar functions for all these proteins in chromatin remodeling during sperm differentiation. By contrast, the two dog-fish spermatid-specific proteins are structurally unrelated to sperm protamines and cannot be considered as their precursors.

Nucleotide sequence of the P1 attachment-protein gene of Mycoplasma pneumoniae

The specific attachment of Mycoplasma pneumoniae to the respiratory ciliated epithelium is mediated by a surface protein designated P1. The nucleotide (nt) sequence of the P1 attachment-protein gene has been determined and the amino acid (aa) sequence deduced. mRNA and cDNA sequencing confirm that this gene is transcribed in M. pneumoniae. The predicted amino acid sequence matches the N-terminal 12 aa residues of P1 protein from M. pneumoniae [Jacobs et al. J. Gen. Microbiol. 133 (1987) 2233–2236] beginning with Asn at aa position 60, where aa 1 represents the first codon of the open reading frame (ORF). Notably, the Trp at aa position 69 aligns with a UGA codon deduced from the nucleotide sequence, providing supporting evidence that UGA is read as Trp rather than stop in M. pneumoniae. Analysis of the first 59 aa suggests that it is probably a leader sequence that is processed to yield the mature protein. The codons of the mature P1 protein sequence represent 1568 aa with a calculated M r of 169758. A unique feature of this protein sequence is the lack of cysteine, and this was confirmed by sodium dodecyl sulfate-polyacryamide gel electrophoresis of M. pneumoniae proteins metabolically labeled with radioactive cysteine or methionine. This study has revealed that the 4881 nt of the P1 structural gene are flanked by ORFs, and there are no obvious ribosome-binding sites or transcription termination sequences in the immediately adjacent regions. This suggests that the P1 gene is transcribed as part of a larger polycistronic message. In addition, a number of untran scribed and therefore nonfunctional P1 epitope sequences were found in the M. pneumoniae genome; their purpose remains unknown.

Purification and immunochemical properties of a wall protein antigen from Clostridium difficile ATCC 11011.

A wall-surface protein antigen, designated 32K antigen, was extracted from whole cells of Clostridium difficile strain ATCC 11011 with phosphate buffered saline and purified by ion-exchange chromatography, gel filtration, and chromatofocusing. The 32K antigen preparation was determined to be highly homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the antigen was characteristic in the predominance of the acidic amino acids, the very low contents of methionine and histidine, and the lack of cysteine. A monomeric molecular weight of the 32K antigen was estimated to be 32,000 by SDS-PAGE and 30,200 by sedimentation equilibrium. The antigen exhibited two isoelectric forms (IP, 4.12 and 3.96). Neither carbohydrate nor phosphorus was detectable in the antigen. The antigen was relatively resistant to trypsin but sensitive to pepsin. Immunoblot analysis of the wall proteins isolated from other strains of C. difficile probed with monospecific antiserum against the antigen from ATCC 11011 showed that the antigenicity of 32K wall protein was common among some of the strains containing 32K wall proteins.

Hornetin: the lethal protein of the hornet ( Vespa flavitarsus) venom

By gel permeation on a Fractogel TSK HW 50 column followed by ion-exchange chromatography on carboxymethylcellulose CM 52, a lethal protein, designated hornetin, was purified from the venom of Vespa flavitarsus. Hornetin is a highly basic protein (p I 10.2) with a molecular mass of about 32 kDa. Its amino acid composition is characterized by a high content of lysine, aspartic and glutamic acid, and is devoid of tryptophan and cysteine. The lack of cysteine in the molecule is distinct from other known vespid venom proteins of comparable size. The i.v. LD 50 of the toxin is 0.42 μg per g mouse. Assayed on the red blood cells of the mouse and guinea-pig as well as isolated nerve muscle preparations of the chick and mouse, hornetin showed direct hemolytic activity and presynaptic neurotoxicity at microgram level and displayed musculotropic effect at higher concentrations.

Characterization of the surface layer protein from Azotobacter vinelandii

The regular surface array protein (S protein) of Azotobacter vinelandii was extracted from outer membrane fragments with distilled water and further purified by gel filtration chromatography. The protein was shown to behave anomalously on sodium dodecyl sulfate polyacrylamide gels producing a number of conformational isomers. The amino acid composition of S protein was similar to that of other surface array proteins, particularly in its lack of cysteine. The theoretical monomelic molecular weight of S protein was calculated to be 60 218 based on the total amino acid composition and the apparent molecular weight determined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Circular dichroism spectra indicated that S protein was composed of approximately 35% β sheet structure, negligible α helix, with the remainder of the polypeptide backbone aperiodic in nature. The effect of the divalent cations Ca2+ and Mg2+ on the conformation of S protein was examined by circular dichroism spectroscopy; however, no conformational change was detected in response to the presence of these species nor did S-protein monomer aggregate into multimers in the presence of these cations. Purified S-protein monomer was inactive in divalent cation mediated reassembly of the S layer onto the surface of distilled water washed cells. A larger multimeric form present only in fresh preparations appeared to be the active species involved in in vitro reassembly of the A. vinelandii surface array.

Biochemical and immunological characterization of a histone variant associated with spermatogenesis in the mouse

A chromosomal histone, H2S, specific to the mouse testis has been purified. Amino acid analysis indicated lack of cysteine and a high basic amino acid content typical of histones. Specific antibodies against histones H2S have been generated in rabbits and partially purified using (NH 4) 2SO 4 precipitation and ion-exchange chromatography. Protein transfer experiments indicate presence of antigenically similar histones in the rat and rabbit testes but not in the guinea pig and dog testes. In addition, histone complement of somatic tissues such as lung, kidney, liver and spleen lacked antigenically similar proteins. Immunocytochemical studies using peroxidase-antiperoxidase complex indicated presence of immunoreactive cells in the seminiferous epithelium which were lacking in the interstitium. These data demonstrate histone H2S to be a unique histone associated with spermatogenesis in the mouse.

A short chain (pro)collagen from aged endochondral chondrocytes. Biochemical characterization.

After 5 weeks in secondary culture, chondrocytes derived from specific regions of the embryonic chick tibiotarsus secrete greater than 90% of their culture medium collagen as a short chain (SC) collagen. Quantities of the molecule, sufficient for biochemical characterization, were isolated without proteolytic treatment from the medium of such mass cell cultures. The chains, Mr = 59,000, of SC collagen are cleaved to Mr = 45,000 by limited pepsin digestion of the native molecule. These two forms of SC collagen are referred to as the 59K form and the 45K form. The CD spectrum of the 59K form confirms the presence of a triple helical domain within the molecule. The amino acid composition of the two forms of SC collagen show it to be different from any other known collagen, including the short chain collagens that have been isolated by the proteolytic extraction of cartilages. The most characteristic features of SC collagen are its high content of methionine, low level of arginine, and a lack of cysteine. The amino acid composition of the 45K form shows it to be the collagenous domain, while the differences between the 45K and 59K form presumably reflect the composition of the nonhelical domain of the 59K form. The nonhelical domain contains greatly elevated levels of aromatic amino acids which contribute to the hydrophobic character of this domain.

Purification and characterization of two basic spermatid-specific proteins isolated from the dog-fish Scylliorhinus caniculus

In dog-fish spermatid nuclei two intermediate proteins S 1 and S 2 replace histones before the setting down of protamines. These spermatid-specific proteins were isolated by carboxymethyl-cellulose chromatography and purified by high pressure liquid chromatography. S 1 and S 2 are characterized by a high content of basic residues and by the lack of cysteine and phenylalanine. The determination of their amino acid composition and of their N- and C-terminal sequences prove that each protein corresponds to a specific molecule which can be considered neither as a histone hydrolytic product nor as a protamines precursor.

Role of Calmodulin in Brain Function

This chapter reviews the salient features of calmodulin and some of the recent findings on its role in cellular processes of the brain. Calmodulin is a single polypeptide with 148 amino acids and a molecular weight of 16 700. Some 30 % of its amino acids consist of aspartate or glutamate, which accounts for the low isoelectric point (4.3). It contains no cysteine, hydroxyproline or tryptophan, but does contain a trimethylated lysine at position 115. One striking characteristic of calmodulin is its tendency to retain biologic activity after exposure to heat (99oC), urea (8 M), acidic pH and detergents. Another is its lack of both tissue and species specificity. A third is that calmodulin from a variety of sources possesses many similar physicochemical properties. Calmodulin isolated from certain sources contains no trimethylated lysine and appears to be equally active, at least in activating bovine brain phosphodiesterase. The lack of cysteine and hydroxyproline would allow calmodulin to have a flexible tertiary structure, a desirable feature for a multifunctional protein that interacts with many different receptor proteins.

The acid-soluble fraction of amyloid—a fibril forming protein

Warming of five amyloid suspensions in 0.15 M NaCl made in 0.02 M HCl at 60 °C for 1 h, resulted in solubilization of 50% of their dry weight. The acid-soluble fraction of the amyloid fiber was soluble in 0.007 M to 0.08 M HCl, contained 16% N and did not contain measurable amounts of mucopolysaccharides and free sugars. Amino acid analyses showed a predominance of alanine and aspartic acid and a lack of cysteine and methionine. The sedimentation coefficient of the acid-soluble fraction was found to be between 0.64 and 0.88 S; its molecular weight by sodium dodecyl sulfate polyacrylamide electrophoresis was estimated to be between 4000 and 7000. When the acid-soluble fraction was dialyzed against distilled water or 0.15 M saline, it precipitated in the form of fibrils or clumps depending on the initial protein concentration.