The structure of mitochondrial creatine kinase and its membrane binding properties

The biochemical and biophysical characterization of the mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle is reviewed with emphasis on the structure of the octameric oligomer by electron microscopy and on its membrane binding properties. Information about shape, molecular symmetry and dimensions of the Mi-CK octamer, as obtained by different sample preparation techniques in combination with image processing methods, are compared. The organization of the four dimeric subunits into the Mi-CK complex as apparent in the end-on projections is discussed and the consistently observed high binding affinity of the four-fold symmetric end-on faces towards many support films and towards each other is outlined. A study on the oligomeric state of the enzyme in solution and in intact mitochondria, using chemical crosslinking reagents, is presented together with the results of a search for a possible linkage of Mi-CK with the adenine nucleotide translocator (ANT). The nature of Mi-CK binding to model membranes, demonstrating that rather the octameric than the dimeric subspecies is involved in lipid interaction and membrane contact formation, is resumed and put into relation to our structural observations. The findings are discussed in light of a possiblein vivo function of the Mi-CK octamer bridging the gap between outer and inner mitochondrial membranes at the contact sites.     

Undecagold cluster labeling of proteins at reactive cysteine residues.

Undecagold cluster labeling of reactive cysteine residues in numerous proteins has allowed the labeled sites to be identified by electron microscopy, providing high-resolution information on the location and orientation of subunits in oligomeric enzymes, virus capsids, crystalline sheets of membrane proteins, and muscle thin filaments. The range of applications of undecagold cluster labeling has been greatly extended by the availability of site-directed mutagenesis to introduce cysteine residues at sites of interest. In this paper I discuss factors that can influence the extent and specificity of labeling, methods for biochemical analysis of undecagold-labeled proteins, and the effects of undecagold cluster labeling on biological activity.     

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.     

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.     

The local electrostatic environment determines cysteine reactivity of tubulin

Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [14C]iodoacetamide, N-[(14)C]ethylmaleimide, or IAEDANS ([5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios 1 cysteine/dimer always leads to labeling of 6-7 cysteine residues. These have been identified as Cys-305alpha, Cys-315alpha, Cys-316alpha, Cys-347alpha, Cys-376alpha, Cys-241beta, and Cys-356beta by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347alpha as the most reactive cysteine of tubulin. Using the 3.5-A electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 A of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

The selective blocking of the polymerization reaction of striated muscle actin leading to a derivative suitable for crystallization. Modification of Tyr-53 by 5-diazonium-(1H)tetrazole.

The polymerization reaction of rabbit muscle actin was completely inhibited by reaction of one amino acid side chain per protein monomer with 5-diazonium-(1H)[14C]tetrazole. A tryptic peptide fingerprint showed a single peptide labeled by the reagent. The peptide was isolated and the labeled amino acid identified by amino acid analysis as Tyr-53. This side chain is not accessible to the reagent in F-actin. The modification is compared to similar inhibitions by other reagents.     

Covalent modification of a highly reactive and essential lysine residue of ribulose-1,5-bisphosphate carboxylase/oxygenase activase.

Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. Incubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-1 s-1 compared to 21 M-1 s-1 for sulfo-NHS-acetate. Inactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mol of AMCA per mol of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acid-lysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of K247 accounted for 1 mol of AMCA incorporated per mol of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS- and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the gamma-phosphate showed that K247 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of K247 is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

Excimer fluorescence of pyrene-maleimide-labeled tubulin.

Excimer-forming cysteines in tubulin are detected by the presence of excimer fluorescence in N-(1-pyrenyl)maleimide-labeled tubulin. The ratio of excimer/monomer fluorescence of labeled protein remained unchanged upon its dilution. These results indicating that both partner of each pair(s) of cysteine are located in the same subunit. The excimer fluorescence is insensitive to prior treatment of tubulin with either colchicine or GTP, indicating that pairs of cysteines protected by those drugs are not involved in excimer formation. This excimer fluorescence of N-(1-pyrenyl)maleimide-labeled tubulin disappeared upon treatment with SDS, guanidinium chloride (GdmCl) and urea. Studies with GdmCl induced unfolding of N-(1-pyrenyl)maleimide-labeled tubulin showed that the loss of excimer fluorescence precedes subunit dissociation. The loss of both colchicine-binding activity and the excimer fluorescence with increasing temperature indicates a major conformational change of the tubulin molecule at elevated temperatures.     

Localization of a reactive exofacial sulfhydryl on the glucose carrier of human erythrocytes

Tryptic digestion studies of the human erythrocyte glucose carrier have shown that a reactive and transport-sensitive exofacial sulfhydryl is located in the carboxy-terminal half of the molecule, corresponding to Cys347, Cys421, or Cys429. In the present studies, the erythrocyte glucose carrier labeled on the exofacial sulfhydryl with bis(maleimidomethyl) ether-L-[35S]cysteine was chemically cleaved, either at tryptophans by N-bromosuccinimide or at nonalkylated cysteines by 2-nitro-5-thiocyanobenzoic acid. The resulting fragments were separated by linear gradient polyacrylamide gel electrophoresis, and the labeled fragments were identified by their apparent molecular weight, and by immunoblotting with antibodies to specific regions of the carrier protein. All of the labeled fragments were recognized by an antibody to the carboxy terminus of the carrier, but not by an antibody to a cytoplasmic loop on the C-terminal half of the carrier. The labeled exofacial sulfhydryl was assigned to Cys429, since this is the only residue of the three possibilities which is beyond the expected cleavage sites of the two reagents in the carrier sequence. These results concur with the predictions of hydropathy analysis and will be relevant for studies of how modification of this sulfhydryl affects carrier function, particularly since several other known carrier isoforms lack a corresponding cysteine.     

Identification of free and [Fe2S2]-bound cysteine residues of adrenodoxin

Bovine adrenodoxin was labeled with 5-iodoacetamidofluorescein to determine which of the five cysteines is free and which participate in iron coordination. Native protein was labeled at two stoichiometries, 0.15:1 and 1:1, both of which produced a single fluorescent product. Labeled tryptic peptides were isolated from both preparations and identified as residues 90-98 with 5-acetamidofluorescein cysteine at residue 95. From the preparation labeled at 0.15:1 stoichiometry, the fraction of tryptic peptide containing nonlabeled cysteines 92 and 95 was isolated and identified; this peptide was shown to be absent in the sample labeled at 1:1 stoichiometry. 5-Acetamidofluorescein-labeled adrenodoxin supported electron transport with adrenodoxin reductase and cytochromes P-450sec and P-45011 beta, demonstrating that labeling occurred without disruption of the iron-sulfur center. These results identify cysteine 95 as the most reactive and single free thiol in native adrenodoxin and imply the role of cysteine residues 46 [corrected], 52, 55, and 92 in iron-sulfur coordination.     

Effect of different fixatives on Con A surface receptors of mouse peritoneal macrophages

The effects of glutaraldehyde, formaldehyde, or osmium tetroxide fixation on the number of labeled Con A surface receptors on mouse peritoneal macrophages were compared. Gold-labeled Con A receptors were found to be isolatedly arranged and evenly distributed on cell surfaces independent of the fixative used. Only cells preincubated with Con A and subsequently fixed by osmium tetroxide showed arrangement of labeled receptors in clusters. Significant differences were found in the number of Con A receptors per cell depending on the fixative used. The fluorescence intensity of FITC-Con A staining was detected spectrophotometrically, the characteristic X-rays of gold-labeled Con A receptors were determined by means of electron beam-induced X-ray microanalysis. The experimental results obtained both at light and electron microscopic level pointed to formaldehyde being the best fixative also for this purpose.     

Effect of different fixatives on Con A surface receptors of mouse peritoneal macrophages

Summary The effects of glutaraldehyde, formaldehyde, or osmium tetroxide fixation on the number of labeled Con A surface receptors on mouse peritoneal macrophages were compared. Gold-labeled Con A receptors were found to be isolatedly arranged and evenly distributed on cell surfaces independent of the fixative used. Only cells preincubated with Con A and subsequently fixed by osmium tetroxide showed arrangement of labeled receptors in clusters. Significant differences were found in the number of Con A receptors per cell depending on the fixative used. The fluorescence intensity of FITC-Con A staining was detected spectrophotometrically, the characteristic X-rays of gold-labeled Con A receptors were determined by means of electron beam-induced X-ray microanalysis. The experimental results obtained both at light and electron microscopic level pointed to formaldehyde being the best fixative also for this purpose.     

Herpes simplex virus capsid structure: DNA packaging protein UL25 is located on the external surface of the capsid near the vertices

UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.     

The region between transmembrane domains 1 and 2 of the reduced folate carrier forms part of the substrate-binding pocket

A functional cysteine-less form of the hamster reduced folate carrier protein was generated by alanine replacement of the 14 cysteine residues. The predicted 12-transmembrane topology was examined by replacing selected amino acids, predicted to be exposed to the extracellular or cytosolic environments, with cysteines. The location of these cysteines was defined by their accessibility to biotin maleimide in the presence or absence of specific blocking agents. Amino acids predicted to be exposed to the extracellular environment (S46C, S179C, L300C, Y355C, and K430C) could be labeled with biotin maleimide; this modification could be blocked by prior treatment with nonpermeable reagents. Amino acids predicted to be within the cytosol (S152C, Cys224, and L475C) could be labeled only after streptolysin O permeabilization. In addition, the cysteine-less reduced folate carrier was exploited to evaluate a potential substrate-binding domain as suggested by previous studies. Nineteen cysteine replacements were generated between residues 39 and 75, a region located between the first and second transmembrane segments. From the biotinylation of these sites and the ability of various reagents to block this labeling, it appears that L41C, E45C, S46C, T49C, I66C, and L70C are exposed to the extracellular environment, whereas Q54C, Q61C, and T63C are slightly less accessible. Cysteines 39, 42, 44, 47, 51, and 73 were inefficiently biotinylated, suggesting that these sites are located in the membrane or within a tightly folded domain of the protein. Furthermore, biotinylation of cysteines 41, 46, 49, 70, and 71 could be prevented by prior treatment with either methotrexate or folinic acid, indicating that these sites form part of a substrate-binding pocket.     

Morphometric analysis of gold particles in low-label cell compartments.

Quantitative estimation of the binding of gold-conjugated ligands to various cell organelles has become a commonly used method to quantify the amount of ligand-binding sites associated with those organelles. However, often a small percentage of organelles is labeled or the density of gold labeling is low. We have defined the "gold-labeled region" as a zone that has a boundary defined by the localization of the outermost gold particles. Such a phenomenon was recently observed in a study of the internalization of gold-labeled native surfactant into lamellar bodies of cultured pulmonary type II cells. We estimated the size and density of gold-labeled regions in lamelar bodies using a simple stereological approach and demonstrated that the low percentage of gold-labeled organelles can be explained as a result of the probability of random selecting through the labeled areas. Our method, which permits use of transmission electron microscopy to calculate the true parameters of gold-labeled regions, can significantly facilitate analyses of ligand binding to various cell compartments.     

Ethylmalonic Acid Inhibits Mitochondrial Creatine Kinase Activity from Cerebral Cortex of Young Rats in Vitro

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an inherited metabolic disorder biochemically characterized by tissue accumulation of predominantly ethylmalonic acid (EMA) and clinically by neurological dysfunction. In the present study we investigated the in vitro effects of EMA on the activity of the mitochondrial (Mi-CK) and cytosolic (Cy-CK) creatine kinase isoforms from cerebral cortex, skeletal muscle, and cardiac muscle of young rats. CK activities were measured in the mitochondrial and cytosolic fractions prepared from whole-tissue homogenates of 30-day-old Wistar rats. The acid was added to the incubation medium at concentrations ranging from 0.5 to 2.5 mM. EMA had no effect on Cy-CK activity, but significantly inhibited the activity of Mi-CK at 1.0 mM and higher concentrations in the brain. In contrast, both Mi-CK and Cy-CK from skeletal muscle and cardiac muscle were not affected by the metabolite. We also evaluated the effect of the antioxidants glutathione (GSH), ascorbic acid, and a-tocopherol and the nitric oxide synthase inhibitor L-NAME on the inhibitory action of EMA on cerebral cortex Mi-CK activity. We observed that the drugs did not modify Mi-CK activity per se, but GSH and ascorbic acid prevented the inhibitory effect of EMA when co-incubated with the acid. In contrast, L-NAME and -tocopherol could not revert the inhibition provoked by EMA on Mi-CK activity. Considering the importance of CK for brain energy homeostasis, it is proposed that the inhibition of Mi-CK activity may be associated to the neurological symptoms characteristic of SCAD deficiency.     

Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferase activity of the S1 subunit of pertussis toxin

Sulfhydryl-alkylating reagents are known to inactivate the NAD glycohydrolase and ADP-ribosyltransferase activities of the S1 subunit of pertussis toxin, a protein which contains two cysteines at positions 41 and 200. It has been proposed that NAD can retard alkylation of one of the two cysteines of this protein (Kaslow, H.R., and Lesikar, D.D. (1987) Biochemistry 26, 4397-4402). We now report that NAD retards the ability of these alkylating reagents to inactivate the S1 subunit. In order to determine which cysteine is protected by NAD, we used site-directed mutagenesis to construct analogs of the toxin with serines at positions 41 and/or 200. Sulfhydryl-alkylating reagents reduced the ADP-ribosyltransferase activity of the analog with a single cysteine at position 41; NAD retarded this inactivation. In contrast, sulfhydryl-alkylating reagents did not inactivate analogs with serine at position 41. An analog with alanine at position 41 possessed substantial ADP-ribosyltransferase activity. We conclude that alkylation of cysteine 41, and not cysteine 200, inactivates the S1 subunit of pertussis toxin, but that the sulfhydryl group of cysteine 41 is not essential for the ADP-ribosyltransferase activity of the toxin. These results suggest that the region near cysteine 41 contributes to features of the S1 subunit important for ADP-ribosyltransferase activity. Using site-directed mutagenesis, we found that changing aspartate 34 to asparagine, arginine 39 to lysine, and glutamine 42 to glutamate had little effect on ADP-ribosyltransferase activity. However, substituting an asparagine for the histidine at position 35 markedly decreased, but did not eliminate, ADP-ribosyltransferase activity. Chou-Fasman analysis predicted no significant modifications in secondary structure of the S1 peptide with the change of histidine 35 to asparagine. Thus, histidine 35 may interact with a substrate of the S1 subunit without being essential for catalysis.     

Immunocytochemical localization of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum

Cells of Methanobacterium thermoautotrophicum were fixed with glutaraldehyde, sectioned and labeled with antibodies against the subunit of component C (=methyl-CoM reductase) of methyl-CoM reductase system and with colloidal gold-labeled protein A. It was found that the gold particles were located predominantly in the vicinity of the cytoplasmic membrane, when the cells were grown under conditions where methyl-CoM reductase was not overproduced. This finding confirms the recent data obtained with Methanococcus voltae showing via the same immunocytochemical localization technique that in this organism methyl-CoM reductase is membrane associated.