Intra- and intermolecular charge-transfer interactions between terminal electron donor and acceptor attached to poly(γ-benzyl-L-glutamate) in solution

Poly(γ-benzyl-L -glutamate) having a terminal dimethylaminoanilide group as an electron donor (D) and a terminal 3,5-dinitrobenzoyl group as an electron acceptor (A) (A-[Glu(OBzl)]_n-D) was synthesized by the N-carboxyanhydride method. Polymer samples were fractionated by gel chromatography and their number-average degrees of polymerization n were determined by the absorbances of the terminal chromophores. These polymers in chloroform and dimethylformamide solutions showed a charge-transfer (CT) absorption band around 455 nm, and the fraction of the polymer forming the CT complex was evaluated as a function of the chain length. CT absorption for short chains (n = 5 ～ 20) was attributed to intramolecular CT complex in which the A-[Glu(OBzl)]_n-D chain takes cyclic conformations. An optimum chain length for the intramolecular CT was found to be n ? 10, where the [Glu(OBzl)]_n chain may most easily bend back to form cyclic conformations. Stronger CT absorption observed for longer chains than n = 20 was shown to be intermolecular, and an intermolecular head-to-tail aggregation was found to be a cause of the strong CT interaction. All helical A-[Glu(OBzl)]_n-D chains were found to form the head-to-tail dimers in chloroform solution.     

Evidence for three alpha subunits in one molecule of F1-ATPase from thermophilic bacterium PS3.

The numbers of sulfhydryl residues in F₁-ATPase of thermophilic bacterium PS3 and its isolated subunits were analyzed with Ellman's reagent. This F₁-ATPase contained three sulfhydryl residues and no disulfide bridge. Of the five kinds of subunits of the F₁-ATPase, only the α subunit contained one sulfhydryl residue. So there are three α subunits in one molecule of the F₁-ATPase.     

A quantitating reagent for methanethiolation of protein sulfhydryl groups

p-Nitrophenoxycarbonyl methyl disulfide has been synthesized for use as a quantitating agent for methanethiolation of protein sulfhydryl groups. This reagent reacts specifically and quantitatively with cysteine residues of proteins to yield an unsymmetrical disulfide containing a CH₃S group and concomitantly releases the chromophore, p-nitrophenol. Titration of the sulfhydryl groups of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with this reagent has been studied. Incorporation of CH₃S as measured by the release of p-nitrophenol paralleled the loss of sulfhydryl group dependent activity of the enzyme. The enzyme was found inactive on modification of four of the eight sulfhydryl groups present in the enzyme. Stability of p-nitrophenoxycarbonyl methyl disulfide has also been studied in different buffer systems. The rate of decomposition of the p-nitrophenyl ester due to hydrolysis was found negligible below a pH of 8.0 compared to its rate of reaction with free sulfhydryl groups.     

Distribution of free sulfhydryl and disulfide groups among platelet membrane proteins.

Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polycrylamide gel electrophoresis. Platelet membranes treated with N-ethyl(1-14C)maleimide, phenyl(203Hg)mercuric acetate and p-chloro(203Hg)mercuribenzoate showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from greater than 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with 14C-labeled 5,5'-dithio-bis(2-nitrobenzoic acid) resulted changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon, 5,5'-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.     

Studies of the function and location of two cysteines in the beta 2 subunit of tryptophan synthase.

Our findings that the apo β₂ subunit of tryptophan synthase of Escherichia coli is inactivated by the modification of one sulfhydryl residue per monomer by nitrothiocyanobenzoic acid and is reactivated by removal of the CN group indicate that the reactive sulfhydryl residue (SH-I) is essential for catalytic activity. SH-I is shown to be the same residue which was previously found to react with bromoacetylpyridoxamine phosphate and different from a sulfhydryl (SH-II) which reacts with N-ethylmaleimide in the presence of pyridoxal phosphate. The results of partial tryptic digestions of β₂ subunit labeled selectively at SH-I or SH-II show that both sulfhydryl residues are located in the F₁ fragment which also contains the pyridoxal phosphate binding site.     

EXPERIMENTAL HYBRIDIZATION OF XANTHIUM STRUMARIUM (COMPOSITAE) FROM ASIA AND AMERICA. II. SESQUITERPENE LACTONES OF F1 HYBRIDS

Effects of hybridization on sesquiterpene lactones in Xanthium strumarium were studied for clues to the relationship of Old and New World populations. In crosses between indigenous Asiatic plants in the “strumarium” morphological complex and various American complexes that produce xanthinin as a major sesquiterpene lactone, the F₁ hybrids contained xanthinin and the related compounds, xanthanol, xanthatin, and xanthinosin. In other crosses with various American complexes that produce xanthumin, the stereoisomer of xanthinin, the F₁ hybrids contained xanthinin and xanthumin as well as xanthinin-related compounds and their stereoisomers, xanthumanol, deacetoxylxanthumin, and tomentosin. The Asiatic plants of “strumarium” from Hong Kong involved in the crosses produce approximately equal percentages of xanthinin, xanthatin and xanthinosin, but those from India contain only xanthinosin. The putative introduction of the American morphological complex, “chinense,” contains xanthumin as the major component but shows chemical diversity that indicates genes derived from the indigenous “strumarium” complex.     

Distribution of free sulfhydryl and disulfide groups among platelet membrane proteins

Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet membranes treated with $\text{N-}\text{ethyl}\text{[1?}{}^{14}\text{C}\text{]}\text{maleimide}$, phenyl[²⁰³Hg]mercuric acetate and $\text{p-}\text{chloro}\text{[}{}^{203}\text{Hg}\text{]}\text{mercuribenzoate}$ showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from > 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with ¹⁴C-labeled 5,5′-dithio-bis(2-nitrobenzoic acid) resulted in changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon 5,5′-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.     

Crystal structure of TGF-β2 refined at 1.8 Å resolution

The crystal structure of TGF-β2 has been refined using data collected with synchrotron radiation (CHESS) to 1.8 Å resolution with a residual R (= ∑ | |F_o| ? |F_c| | /∑ |F_o|) factor of 17.3%. The model consists of 890 protein atoms from all 112 residues and 59 water molecules. The monomer of TGF-β2 assumes a rather extended conformation and lacks a well-defined hydrophobic core. Surface accessibility calculations show only 44% of the nonpolar surface is buried in the monomer. In contrast, 55.8% of the nonpolar surface area is buried when the two monomers from a dimer, a typical value for globular proteins. This includes a 1300 Ų buried interface area that is largely hydrophobic. Sequence comparisons using a profile derived from the refined TGF-β2 structure suggest that the cluster of four disulfides (three intramonomeric disulfide bonds 15–78, 44–109, 48–111 forming a disulfide knot, and one intermonomeric disulfide 77–77) together with the extended β strand region constitutes the conserved structural motif for the TGF-β superfamily. This structural motif, without the 77–77 disulfide bond, defines also the common fold for a general family of growth factors, including the nerve growth factor and platelet-derived growth factor families. The fold is conserved only at the monomer level, while the active forms are dimers, suggesting that dimerization plays an important role in regulating the binding of these cytokines to their receptors and in modulating the biological responses. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy

Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ_D) of ～200 µsec for Rhodamine‐labeled IgG, corresponding to a hydrodynamic radius (R_H) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ_D under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation‐dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. © 2014 Wiley Periodicals, Inc.     

Domain structure of fibronectin and its relation to function. Disulfides and sulfhydryl groups.

Hamster cell fibronectin is a glycoprotein consisting of two 230,000-dalton subunits in a disulfide-bonded dimer. The molecule is composed of domains which can be separated by partial proteolytic cleavage. The carbohydrates, disulfide bonds, and a single free sulfhydryl group per chain are distributed nonuniformly among these regions. All the interchain disulfides are within 10,000 daltons of the end of the molecule and are removed by mild proteolysis which also generates 200,000- and 25,000-dalton fragments which do not contain interchain disulfides. The 200,000-dalton fragment contains all or most of the carbohydrate side chains, and the free sulfhydryl group, but is relatively poor in cystine. The 25,000-dalton fragment is carbohydrate-free and cystine-rich but has no free sulfhydryl groups. There is heterogeneity in carbohydrate content among the monomeric chains of intact fibronectin and the 200,000-dalton fragments. The gelatin binding site of fibronectin is in the 200,000 fragment. Intact disulfide bonds are required for binding of fibronectin to cells and to gelatin and blockage of the free sulfhydryl groups prevents binding of fibronectin to cells, suggesting that intermolecular disulfide bonding may be important.     

Texture of SS Cross-linked Gelatin Gels

Relations among sensory properties, physical characteristics and the kinds of force supporting gel structure were studied by using thiolated gelatin.

Breaking stress of the gelatin gel was greatly influenced by contents of disulfide bonds in the gel. Young’s modulus of the gel was affected by its temperature. As to sensory properties of the gelatin gel, “hardness” highly correlated with Young’s modulus and “brittleness” correlated with breaking stress.

Therefore, it could be concluded that in the case of the gelatin gel, “hardness” was mainly attributed to hydrogen bonds and “brittleness” was to chemical bonds like disulfide linkages.     

Cadmium affects the redox state of rat liver glucocorticoid receptor

It has previously been documented that cadmium displays high affinity for protein thiol groups and induces an impairment of glucocorticoid receptor (GR) cellular functions. The present study examined the possibility that cadmium exerts these effects on GR activity by disturbing the receptor's redox equillibrium. To that end, the influence of cadmium on the rat liver GR potential to form intramolecular and intermolecular disulfide bonds under nonreducing conditions and under oxidizing conditions produced by the addition of hydrogen peroxide (H₂O₂) to the cytosol was examined by nonreducing SDS-PAGE and immunoblotting. The results show that cadmium inhibits formation of disulfide bonds within the GR both in the absence and in the presence of H₂O₂. The creation of intermolecular disulfide linkages between the apo-GR and associated heat shock proteins Hsp90 and Hsp70, which was evident in the presence of H₂O₂, was also significantly impaired after cadmium administration. These observations are consistent with the assumption that cadmium affects the redox state of the receptor, possibly by binding to its sulfhydryl groups. This revised version was published online in July 2006 with corrections to the Cover Date.     

The chain effect. Part III. Induction of secondary structure in copolymerizations

Studies of the properties of copolymers of N-carboxy sarcosine anhydride with other N-carboxy α-amino acid anhydrides have shown that the secondary structure may be a function of the type of initiator used. In particular, when polysarcosine is the initiator, and the “chain effect” becomes possible, blocklike character appears in the copolymer. This is the result of selective and rapid polymerization of N-unsubstituted N-carboxy α-amino acid anhydrides by the chain-effect mechanism. In suitable circumstances, therefore, the latter may be used to induce order into copolymerizations of this type.     

Effects of freezing and hardening on the sulfhydryl groups of protein fractions from cabbage leaves

Disc electrophoresis was used to separate water soluble proteins from hardy, non-hardy, and frost killed cabbage (Brassica oleracea var. capitata) leaves. Amidoschwarz staining failed to reveal any new bands as a result of hardening although the relative amounts of proteins in individual bands changed. Sulfhydryl groups in the protein bands were stained with 2,2-dihydroxy-6,6-dinaphthyl disulfide and labeled with ¹⁴C p-chloromercuribenzoate. Significant decreases in the sulfhydryl content of the total water soluble protein were found during hardening and as a result of frost death. The decrease during hardening was paralleled by a significant increase in the water soluble protein. There was a significant increase in the sulfhydryl content per unit high molecular weight protein but a decrease in the sulfhydryl content per total protein as a result of frost death. This was interpreted as evidence for intermolecular disulfide bond formation during freezing.     

The subunit interface of the Escherichia coli ribosome: Identification of proteins at the interface between the 30 S and 50 S subunits by crosslinking with 2-iminothiolane

The 70 S ribosomes of Escherichia coli were treated with 2-iminothiolane with the resultant addition of 110 sulfhydryl groups per ribosome. The modified ribosomes were oxidized to promote disulfide bond formation, some of which formed intermolecular crosslinks. About 50% of the crosslinked 70 S ribosomes did not dissociate when exposed to low concentrations of magnesium in the absence of reducting agent. Dissociation took place in the presence of reducing agents, which indicated that the subunits had become covalently linked by disulfide linkages. Proteins extracted from purified crosslinked 70 S ribosomes were first fractionated by polyacrylamide/urea gel electrophoresis. The proteins from sequential slices of these gels were analyzed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Monomeric proteins derived from crosslinked dimers appeared below the diagonal containing non-crosslinked proteins, since the second electrophoresis, but not the first, is run under reducing conditions to cleave the crosslinked species. Final identification of the proteins in each dimer was made by radioiodination of the crosslinked proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis in the presence of non-radioactive total 70 S proteins as markers. This paper describes the identification of 23 protein dimers that contained one protein from each of the two different ribosomal subunits. The proteins implicated must have some part of their structure in proximity to the other ribosomal subunit and are therefore defined as “interface proteins”. The group of interface proteins thus defined includes 50 S proteins that are part of the 5 S RNA: protein complex and 30 S proteins at the initiation site. Correlations between the crosslinked interface proteins and other functional data are discussed.     

Mitochondrial adenosine triphosphatase. Location of sulfhydryl groups and disulfide bonds in soluble enzyme from beef heart.

The soluble beef heart mitochondrial ATPase (F1) contains eight sulfhydryl groups and two disulfide bonds. N-Ethylmaleimide has been used to radioactively label the sulfhydryl groups before and after cleavage of the disulfide bonds by dithiothreitol. After subjecting the labeled protein to polyacrylamide gel electrophoresis in sodium dodecyl sulfate and measuring radioactivity in each of the separated subunits the location of all the sulfhydryl groups and the disulfide bonds may be specified. The conclusions are supported by direct examination of depolymerized, unreduced, enzyme by polyacrylamide gel electrophoresis. The results also indicate that current ideas regarding the overall subunit structure of this enzyme may be incorrect, and this is discussed in light of new data presented here.     

Redox-active bis-cysteinyl peptides. II. Comparative study on the sequence-dependent tendency for disulfide loop formation

Bis (cysteinyl) octapeptides related to the active sites of the oxidoreductases protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their propensity to form the intramolecular 14-membered disulfide ring in oxidation experiments. The rank order of percentage of cyclic monomer formed in aqueous buffer (pH 7.0) at 10^?3 M concentration was found to be very similar, but opposite to that of the K_ox and, correspondingly, of the redox potentials of the native enzymes. Attempts to induce intrinsic conformational preferences of the peptides by addition of trifluoroethanol led to enhancements of β-turn structures as reflected by the CD and Fourier transform ir spectra. The induced secondary structure, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppress the differences by significantly increasing the preference for cyclic monomers (≈ 90%). Similarly, operating under denaturing conditions, i.e., in 6M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the remaining peptides, again a strong increase of cyclic monomer contents was observed that could not be correlated with dissolution of β-sheet type aggregates. The CD spectra are more consistent with the presence of ordered structure to some extent, possibly resulting from an hydrophobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol disulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the redox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strongly, but apparently only in the constraint form of the three-dimensional structure as similar exchanges in the excised fragments did not produce the expected effect. This observation contrasts with numerous reports that the conformation of short disulfide loops is mainly dictated by the amino acid sequence. © 1994 John Wiley & Sons, Inc. © 1994 John Wiley & Sons, Inc.     

Cytogenetic studies in Trifolium sp. related to berseem

Summary In a cytogenetic study of five annual Trifolium spp. the genetical relationships between (1) T. scutatum Boiss. and (2) T. plebeium Boiss., and between them and (3) T. carmeli Boiss., (4) T. echinatum M. B. and (5) T. latinum Seb., were studied in intra- and interspecific F₁ and F₂ hybrids. Germination, seedlings development, pollen fertility and chromosome configurations during meiosis and seedset were recorded. All the T. scutatum x T. plebeium F₁ were green, but the hybrids between either T. plebeium or T. scutatum and T. carmeli, T. echinatum or T. latinum were mainly albinos. Those that were green had a very high rate of mortality and the survivors produced many albino seedlings. Pollen fertility was about 60% in scutatum x plebeium F₁s, and one of their eight bivalents was heteromorphic. Hybrids between either of these and T. carmeli, T. echinatum or T. latinum had 30–50% stainable pollen and had several multivalents, suggesting the existence of a system of chromosomal translocations in these species. Seed set was roughly correlated with pollen stainability. The nature of the isolation mechanisms operating between these species is discussed.Contribution from the Volcani Center, Agricultural Research Organization, Bet Dagan, Israel. 1973 Series, No. 186-E. The work reported here was financed by Grant FG-IS-222 from the U.S. Department of Agriculture under P. L. 480.     

l-THREONINE DEAMINASE IN MARINE PLANKTONIC ALGAE. II. DISULFIDE AND SULFHYDRYL GROUP REQUIREMENTS OF ENZYME ACTIVITY IN TWO CRYPTOPHYTES1

The “biosynthetic”l threonine (deaminating) dehydratase of 2 cryptophytes (Chroomonas salina and Hemiselmis virescens) showed sensitive inhibition from all thiols tested (dithiothreitol, cysteine, etc.) but no effect from ascorbic acid or reduced NAD. By contrast, the enzyme activities from 5 noncryptophyceaen unicellular algae (2 cyanophytes, 1 rhodophyte, 1 diatom, 1 chlorophyte) were generally not affected by any of these reagents. The thiol reagent inhibition of the cryptophyte enzymes (1) achieved saturation with 60–70% reduction in activity, (2) was considerably reduced by pretreatment of the enzymes with l -threonine and l -isoleucine, and (3) was partially reversed by subsequent treatment with arsenite and exposure to air. It was deduced that such inhibitions were caused by thiol-specific reduction of enzyme-protein disulfide groups essential for the full expression of activity and that these groups were susceptible to ready reductive cleavage and oxidative restoration. This disulfide requirement, unique to the cryptophytes, may be the first recorded case of such a property of threonine dehydratase from all forms of life hitherto studied. The additional activity requirement of the cryptophyte enzymes for sulfhydryl groups (which requirement was common to all the algal enzymes) was confirmed (1) by the study of their sensitivity to inhibition from mercurials and disulfide-sulfhydryl exchanging reagents, and (2) by the partial reversal of these inhibitions from subsequent treatment with dithio-threitol. Both cryptophyte enzymes were densitized to feedback inhibition from l -isoleucine by prior exposure to subinhibitory concentrations of HgCl₂ or dithiodipyridine.     

Cellular compartmentation of two species of δ-aminolevulinic acid synthetase in a facultative photohetero-trophic bacterium (Rps. spheroides Y.)

Both species of δ-aminolevulinic acid (ALA) synthetase appear compartmentalized in $\text{Rps. spheroides}$ Y. The ALA synthetase, “F_I”, activity is correlated with dark respiratory metabolism and is a cytoplasmic “soluble” enzyme. The other enzyme, “F_II”, induced only in light, is in chromatophores and its activity is correlated with photometabolism.