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PubMed2005

Cysteine modification by lipid peroxidation products inhibits protein disulfide isomerase.

A proteomic approach was applied to mitochondrial protein isolated from the livers of rats fed a combination high-fat and ethanol diet to identify proteins modified by 4-hydroxynonenal (4-HNE). Using this approach, the endoplasmic reticulum chaperone, protein disulfide isomerase (PDI), which participates in the maturation of newly synthesized proteins through promoting correct disulfide formation, was consistently found to be modified by 4-HNE. Further mass spectral analysis of PDI isolated from the animals revealed modification of an active site Cys residue thought to be involved in client protein binding. To test the hypothesis that 4-HNE inhibits the chaperone, purified bovine PDI was treated with concentrations of 4-HNE ranging from 20 to 200 microM (10-100-fold molar excess aldehyde), resulting in 14-56% inhibition, respectively. Similar treatments with the lipid peroxidation products acrolein (ACR) and 4-oxononenal (4-ONE) resulted in 60 and 100% inhibition, respectively, suggesting inactivation of the chaperone via Cys modification. Thiol sensitivity was confirmed through concentration-dependent inhibition of PDI by the Cys modifier N-ethylmaleimide (NEM). While some degree of sensitivity to these lipid aldehydes is suggested by the data, when compared to inactivation of other proteins by 4-HNE, PDI has demonstrated a relative resistance. It was also observed that physiologic (e.g., 4 mM) concentrations of GSH were capable of removing the 4-HNE adducts, likely serving as a protective mechanism against inactivation by 4-HNE and other lipid peroxidation products. However, because an active site Cys was found to be modified by 4-HNE on PDI in vivo, it is possible that the protective effect of GSH on the chaperone decreases under conditions of sustained oxidative stress, such as during chronic alcohol consumption, as GSH is depleted. The data presented here thus suggest potential impairment of an important molecular chaperone during oxidative stress.

Acrolein↗

PubMed2004

Chemical modification of cysteine residues is a misleading indicator of their status as active site residues in the vitamin K-dependent gamma-glutamyl carboxylation reaction.

The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to gamma-carboxy-glutamic acid by the integral membrane enzyme, gamma-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys(323) and Cys(343). According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys(323) and Cys(343) may disrupt the three-dimensional structure, resulting in inactivation.

Amino Acid Sequence↗

PubMed2003

Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump.

N-ethylmaleimide (NEM) impairs the ATPase function of N-terminal NBD of Candida drug resistance gene product Cdr1p. To identify the reactive cysteine(s) for such a contribution, we adopted a three-arm approach that included covalent modification, cysteine mutagenesis, and structure homology modeling. The covalent modification results clearly indicate the ability of NEM and iodoacetic acid (IAA) to potently inhibit the ATPase activity of N-terminal NBD. Since this domain contains five cysteine residues in its sequence, we mutated each and found four of these (C325A, C363A, C402A, and C462A) to stay sensitive to NEM/IAA modification and influence ATPase activity, while C193A mutation completely abrogated the catalytic function. The structural homology modeling data further validate these biochemical findings by ruling out any plausible interactions within the cysteine residues, and deriving the importance of Cys-193 in lying at a bond length clearly feasible to interact with ATP and divalent cation to critically influence ATP hydrolysis.

Adenosine Triphosphatases↗

PubMed1999

Modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class I-restricted viral determinants.

In studying the subdominant status of two cysteine-containing influenza virus nuclear protein (NP) determinants (NP39-47 and NP218-226) restricted by H-2Kd, we found that the antigenicity of synthetic peptides was enhanced 10-100-fold by treatment with reducing agents, despite the fact that the affinity for Kd was not enhanced. Reducing agents also markedly enhanced the immunogenicity of cysteine-containing peptides, as measured by propagation of long-term T cell lines in vitro. Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39-47 and NP218-226. We found similar effects for two widely studied, cysteine-containing peptides from lymphocytic choriomeningitis virus. The major modifications of cysteine-containing synthetic peptides are cysteinylation and dimerization occurring through cysteine residues. We demonstrate that both of these modifications occur in cells synthesizing a cytosolic NP218-226 minigene product and, further, that T cells specific for cysteinylated NP218-226 are induced by influenza virus infection in mice, demonstrating that this modification occurs in vivo. These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral peptides and that this must be considered in studying the status of such peptides in immunodominance hierarchies.

Amino Acid Sequence↗

PubMed1987

[Chemical modification of cysteine residues of cholesterol-hydroxylating cytochrome P-450. Identification of the cysteine residue participating in the formation of a proximal ligand].

A chemical modification of cysteine residues in mitochondrial cytochrome P-450scc from adrenal cortex has been carried out. Cysteine residues in this hemoprotein were shown to form two pools: one available to the chemical modification, which does not affect spectral and functional properties of the cytochrome and another accessible for the modification only after the protein inactivation and the heme removal. The proximal ligand in the polypeptide chain of cytochrome P-450scc was localized. Cys422 was shown to be involved in the heme coordination and Cys264 was found to be exposed and accessible to sulfhydryl reagents. The data obtained are discussed in terms of the functional role of cysteine residues in monooxygenase catalysis.

Adrenal Cortex↗

PubMed2004

Structure-function relations of the first and fourth predicted extracellular linkers of the type IIa Na+/Pi cotransporter: I. Cysteine scanning mutagenesis.

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693-705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

Amino Acid Sequence↗

PubMed1998

Influence of chemical modification of cysteine and histidine side chains upon subunit reassembly of alpha crystallin.

Alpha crystallin, the important multimeric structural protein of mammalian eye lens, is an assembly composed of 30 alpha-A and 10 alpha-B subunits. The influence of either partial or complete chemical modification of two important amino acid side chains, cysteine and histidine, upon the integrity of native alpha crystallin assembly and also upon the mode of subunit reassembly has been investigated. It has been found that chemical modification of surface-exposed cysteine and histidine side chains does not affect the subunit-subunit interactions stabilizing the native aggregate. Cysteine modifications, either partial or complete, unlike histidine modifications, do not seem to affect the backbone conformation of the subunits refolded after denaturation. Both cysteine and histidine modifications, however, affect the packing of the refolded structural elements forming the tertiary structure of the subunits and also the mode of oligomeric reorganization. The most striking effect of histidine modification is the considerable increase in size of the aggregates upon reassociation of the modified subunits. The chaperone activity, however, has been found to remain almost unaffected in spite of these chemical modifications.

Chromatography, Gel↗

PubMed1990

Alkylation of cysteine 82 of p11 abolishes the complex formation with the tyrosine-protein kinase substrate p36 (annexin 2, calpactin 1, lipocortin 2).

p36 (annexin 2) is the major cytoplasmic target of the src tyrosine-kinase and forms in vitro and in vivo a stable tetrameric complex in which two p36 polypeptides interact with a dimer of a unique p11 polypeptide. p11 belongs into the superfamily of EF-hand proteins. Upon mild cysteine modification conditions, both cysteines (position 61 and 82) of the free p11 become substituted, and the ability to form the p36.p11 complex is lost. Under the same conditions, the 2 cysteines of p11 incorporated into the complex display differential reactivity. Here, cysteine 61 is fully substituted while cysteine-82 is protected. p11 derivatives substituted only on cysteine 61 retain binding activity for p36 unless cysteine 82 is substituted by a second cycle of modification of the isolated p11. Thus, the C-terminal extension protruding from the second EF-hand of the p11 molecule (residues 77-96) is important for the interaction with p36. As a consequence of our analysis, we report a new separation of p36 and p11 from the p36.p11 complex. This is based on a reversible cysteine modification and thus is an alternative to the denaturation and renaturation cycle used previously.

Alkylation↗

PubMed1991

Inhibition of ADP-induced platelet shape change and aggregation by o-phthalaldehyde: evidence for covalent modification of cysteine and lysine residues.

Platelets play a major role in the hemostatic process following vascular injury. Chemical modification of cysteine and/or lysine residues in platelet proteins has been shown to cause loss of platelet aggregation induced by diverse agonists; however, these investigations have not addressed the identity of the specific proteins affected. o-Phthalaldehyde (OPTH) is a unique chemical modification reagent that forms and permits the identification of fluorescent isoindole derivatives with proteins by covalently and simultaneously modifying closely spaced cysteine and lysine residues. We found that OPTH inhibited platelet aggregation induced by ADP, collagen, and U46619 (an analog of prostaglandin H2), but had minimal effect on platelet aggregation induced by thrombin, plasmin, chymotrypsin, A23187 (a calcium ionophore), PMA (phorbol 12-myristate 13-acetate), and PMA + A23187. Since platelet aggregation induced by ADP, collagen, and U46619 has been shown to involve binding of endogenous or exogenous ADP to the platelet receptor, our further studies focused on platelet aggregation induced by ADP. OPTH inhibited ADP-induced shape change and aggregation in a concentration-dependent manner. The second-order rate constant for the inhibition of ADP-induced platelet shape change (Ksc = 1.0 X 10(3) M-1 s-1) was lower than that for aggregation (Kagg = 5.4 X 10(3) M-1 s-1). Fluorescence excitation and emission spectra of OPTH-platelet adduct exhibited maxima at 346 and 437 nm, respectively, consistent with the formation of an isoindole derivative(s). The nonpenetrating thiol-specific reagent, p-chloromercuribenzenesulfonate (pCMBS) (0.8 mM), is known to block the inhibition of stimulated adenylate cyclase induced by ADP but not the ADP-induced platelet shape change. The inhibition of ADP-induced platelet shape change (Ksc = 1.5 X 10(3) M-1 s-1) by OPTH was not affected by pCMBS. OPTH, at concentrations (15-50 microM) that inhibited ADP-induced platelet aggregation and shape change did not raise the intracellular levels of adenosine cyclic 3',5'-monophosphate (cAMP) in platelets nor did it impair the ability of iloprost (a stable analog of prostaglandin I2) to raise the platelet cAMP level. Thus, OPTH under these conditions did not interact with platelet adenylate cyclase. 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been previously shown to inhibit ADP-induced platelet shape change and aggregation by covalently modifying aggregin (Mr = 100 kDa), a putative ADP receptor on platelet surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗

PubMed1996

Probing the active site of Tritrichomonas foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase using covalent modification of cysteine residues.

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) of Tritrichomonas foetus was inactivated by the thiol reagents iodoacetate and 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2). Iodoacetate inactivates the enzyme in a time-dependent and concentration-dependent manner that follows pseudo-first-order kinetics. However, the observation that total inactivation with iodoacetate was not achieved suggests that none of the reactive cysteine residues is directly involved in the catalytic activity of the enzyme. Nbs2 caused 50% inactivation rapidly, which was followed by gradual modifications of an additional three cysteine residues leading to complete enzyme inactivation. Analysis of the inactivation using the method developed by Tsou (1962) revealed that modification of two cysteine residues by Nbs2 is sufficient to impair the HGXPRTase activity. Tryptic digestion of HGXPRTase labeled with iodo[2-14C]acetic acid, followed by fractionation of the digest by HPLC and sequence analysis of the labeled peptides allowed the identification of Cys71, Cys129, Cys132, and Cys148 as the reactive cysteine residues. GMP and 5-phosphoribosyl-1-diphosphate provided complete protection against HGXPRTase inactivation by iodoacetate and against carboxymethylation of Cys129, Cys132, and Cys148, Cys71 was not protected by either substrate against iodoacetate, but its carboxymethylation caused no loss in enzyme activity either. There was also no substrate protection of Cys71 against Nbs2, which, however, caused 50% inactivation of the enzyme. Replacing the thionitrobenzoate (Nbs) moiety from Cys71 with cyanide resulted in a gradual recovery of the enzyme activity, which indicates that a steric hindrance at the active site was introduced by Nbs but removed by cyanide. Thus, our results demonstrate that although the reactive cysteine residues in HGXPRTase are not directly involved in the catalytic activity, modification of cysteine residues 129, 132, and 148 by iodoacetate or Nbs2 hinders substrate binding which can, in turn, protect the cysteine residues from modifications. The substrate protection of Cys129 and Cys148 is probably also indicative of a conformational change in the protein structure brought about by substrate binding.

Amino Acid Sequence↗

PubMed1994

Modification of cysteine residues within G(o) and other neuronal proteins by exposure to nitric oxide.

Nitric oxide (NO), a free-radical gas produced endogenously by some neurons, functions as a diffusible intercellular messenger and appears to play a role in activity-dependent modification of synaptic efficacy in the mammalian CNS. The molecular targets and mechanisms of action of NO in neurons remain largely uncharacterized. Employing in vitro brain slices and isolated synaptosomes, we show here that exposure to exogenous or endogenously generated NO results in the modification of cysteine residues within neuronal proteins, as revealed by reduced binding of agents which react with cysteine sulfhydryls. In particular, exposure of synaptosomes to NO inhibits subsequent thiol-linked ADP-ribosylation of the heterotrimeric G-protein, G(o), by pertussis toxin. Our results demonstrate directly that NO may exert its neuronal effects through modification of protein cysteine thiols, and identify G(o) as a potential synaptic target of NO.

Animals↗

PubMed1993

Amino acid exchange and covalent modification by cysteine and glutathione explain isoforms of fatty acid-binding protein occurring in bovine liver.

A unique property of the liver-type member of the family of fatty acid-binding proteins is the heterogeneic pattern observed upon isolation, which can only partly be ascribed to the state of lipidation. Here we unraveled the structural basis of the heterogeneity of delipidated liver-type fatty acid-binding protein (L-FABP). Charge fractions of L-FABP focusing at pH 6.0 and at pH 7.0/7.1 were first isolated from bovine liver. Upon reduction, however, two distinct isoforms, namely pI 6.0 L-FABP and pI 7.0 L-FABP, were observed. From these isoforms peptides were generated enzymically and chemically by four independent methods. Peptides were separated by reverse phase high performance liquid chromatography and analyzed by Edman degradation and plasma desorption mass spectrometry. The complete amino acid sequences of the isoforms were established; they consist of 127 amino acids and each is N-terminally blocked with an acetyl group. The difference between pI 6.0 L-FABP and pI 7.0 L-FABP was attributed to an asparagine-aspartate exchange at position 105. When tryptic peptides of the pH 7.0/7.1 fraction were analyzed, discrepancies between sequence and mass data of the peptides containing at position 69 the sole cysteine of L-FABP led to the disclosure of a cysteinylation occurring at this position and giving rise to the slightly more basic pH 7.1 species. Moreover, chemical modification studies revealed that a part of the pH 6.0 fraction was pI 7.0 L-FABP that was glutathionylated at Cys69. Neither modification, however, prevented the binding of fatty acids. Together amino acid exchange and covalent modification of cysteine entirely explain the heterogeneity of L-FABP from bovine liver.

Amino Acid Sequence↗

PubMed2001

Partially carboxymethylated feather keratins. 2. Thermal and mechanical properties of films.

Free cysteine thiol groups of keratin extracted from chicken feathers were partially carboxymethylated with iodoacetic acid (25-76% cysteine modification). Stable dispersions were used for the preparation of films by solution casting. Glycerol was used as a plasticizer (0.05-0.47 g/g of keratin), and films were stored at a constant relative humidity (20, 30, 50, 70, or 90%). The degree of crystallinity in the films was higher when more cysteine residues were carboxymethylated. The films displayed an optimum in mechanical properties at approximately 50% cysteine carboxymethylation. The tensile strength at this optimum was 25 MPa, the E modulus, 350 MPa, and the elongation at break, 50%. Probably, this optimum was the result of both a decreasing amount of disulfide bonds and an increasing degree of crystallinity for higher degrees of cysteine modification. The influences of a higher amount of glycerol and of different storage conditions on the mechanical properties of films from keratin with a defined degree of cysteine modification were also investigated.

Animals↗

PubMed2004

Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties.

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross-linking revealed that this modification makes the acid-induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid-induced unfolding, and is more resistant to oxidation-induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper-induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.

Cysteine↗

PubMed2005

Selective side-chain modification of cysteine and arginine residues blocks pathogenic activity of HIV-1-Tat functional peptides.

Extracellular Tat protein of HIV-1 activates virus replication in HIV-infected cells and induces a variety of host factors in the uninfected cells, some of which play a critical role in the progression of HIV infection. The cysteine-rich and arginine-rich basic domains represent key components of the HIV-Tat protein for pathogenic effects of the full-length Tat protein and, therefore, could be ideal candidates for the development of a therapeutic AIDS vaccine. The present study describes selective modifications of the side-chain functional groups of cysteine and arginine amino acids of these HIV-Tat peptides to minimize the pathogenic effects of these peptides while maintaining natural peptide linkages. Modification of cysteine by introducing either a methyl or t-butyl group in the free sulfhydryl group and replacing the guanidine group with a urea linkage in the side chain of arginine in the cysteine-rich and arginine-rich Tat peptide sequences completely blocked the ability of these peptides to induce HIV replication, chemokine receptor CCR-5 expression, and NF-kappaB activity in monocytes. Such modifications also inhibited angiogenesis and migration of Kaposi's sarcoma cells normally induced by Tat peptides. Such chemical modifications of the cysteine-rich and arginine-rich peptides did not affect their reactivity with antibodies against the full-length Tat protein. With an estimated 40 million HIV-positive individuals worldwide and approximately 4 million new infections emerging every year, a synthetic subunit HIV-Tat vaccine comprised of functionally inactive Tat domains could provide a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV disease.

Arginine↗

PubMed1993

Reversible modification of cysteine residues of NADPH-cytochrome P-450 reductase.

A reversible chemical modification of SH-groups of NADPH-cytochrome P-450 reductase is the subject of the present study. The enzyme was modified using first biradical RS-SR (R being the imidazolidine derivative) and a new affinity reductase inhibitor beta-cystamine adenosine diphosphate (ANSSN). These reagents were shown to be covalently bound to reductase SH-groups via the reaction of thiol-disulfide exchange resulting in the loss of reducing activity for cytochrome c. NADP+ protected reductase from inactivation and decreased the extent of the modification by RS-SR. The modification of reductase was reversible: the modified enzyme was partially reactivated with glutathione and dithiothreitol. The method proposed can be used to study both the reductase structure and the reversible inhibition of microsomal monooxygenase systems.

Adenosine Diphosphate↗

PubMed1975

Chemical modification of bovine heart mitochondrial malate dehydrogenase. Selective modification of cysteine and histidine.

Bovine mitochondrial malate dehydrogenase (EC 1.1.1.37) was inactivated by the specific modifications of a single histidine residue upon reaction with iodoacetamide. NADH protected against this loss of activity and reaction with the histidine residue, suggesting that the histidine is at the NADH binding site. N-Ethylmaleimide also modified the enzyme by reacting with 1 sulfhydryl residue. The reaction rate with N-ethylmaleimide was increased by decreasing the pH from neutrality or by the addition of urea. NADH protected against the modification of the sulfhydryl group under all the conditions tested, again suggesting active site specificity for this inactivation. This enzyme has a subunit weight of 33,000 and is a dimer. The native malate dehydrogenase will bind only 1 mol of NADH and it is thus assumed that there is only a single active site per dimer.

Amino Acids↗

PubMed2000

Modification of cysteine residues by N-ethylmaleimide inhibits annexin II tetramer mediated liposome aggregation.

A role of cysteine residues in annexin II tetramer (AIIt)'s function was investigated using the sulfhydryl reagent N-ethylmaleimide (NEM). Incubation of AIIt with NEM resulted in a dose-dependent inhibition of AIIt-mediated liposome aggregation and loss of sulfhydryl groups of AIIt. The concentration effecting 50% inhibition was 0.18 mM. The inhibition was observed in all Ca2+ concentrations tested (1-1000 microM). NEM had no effects on liposome aggregation mediated by other annexins (I, III, and IV), indicating that the inhibitory effect caused by NEM modification is specific to AIIt. The NEM-treated AIIt still can bind to liposomes. However, once AIIt was bound to membrane, the cysteine residues were protected from NEM modification. Our results suggest that cysteine residues are critical for AIIt-mediated liposome aggregation.

Animals↗