Cys-113 and Cys-422 form a high affinity metalloid binding site in the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb(III) with high affinity only in the presence of Mg(2+)-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb(III) binding to purified ArsA. C113A/C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA(C113A/C422A)B genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA(C113A/C422A)B growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA(C113A/C422A)B in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.     

Selective labelling of Cys-10 on actin

Conditions are described for the selective modification of Cys-10 on actin achieved following the blocking of the more reactive Cys-374. Labelling of Cys-10 did not affect the formation of actin filaments. This residue should be capable of serving as a site for fluorescence donors or acceptors and thus will be a useful locus to probe F-actin structure.     

Low Micromolar Zinc Accelerates the Fibrillization of Human Tau via Bridging of Cys-291 and Cys-322

A hallmark of a group of neurodegenerative diseases such as Alzheimer disease is the formation of neurofibrillary tangles, which are principally composed of bundles of filaments formed by microtubule-associated protein Tau. Clarifying how natively unstructured Tau protein forms abnormal aggregates is of central importance for elucidating the etiology of these diseases. There is considerable evidence showing that zinc, as an essential element that is highly concentrated in brain, is linked to the development or progression of these diseases. Herein, by using recombinant human Tau fragment Tau_244–372 and its mutants, we have investigated the effect of zinc on the aggregation of Tau. Low micromolar concentrations of Zn²⁺ dramatically accelerate fibril formation of wild-type Tau_244–372 under reducing conditions, compared with no Zn²⁺. Higher concentrations of Zn²⁺, however, induce wild-type Tau_244–372 to form granular aggregates in reducing conditions. Moreover, these non-fibrillar aggregates assemble into mature Tau filaments when Zn²⁺ has been chelated by EDTA. Unlike wild-type Tau_244–372, low micromolar concentrations of Zn²⁺ have no obvious effects on fibrillization kinetics of single mutants C291A and C322A and double mutant C291A/C322A under reducing conditions. The results from isothermal titration calorimetry show that one Zn²⁺ binds to one Tau molecule via tetrahedral coordination to Cys-291 and Cys-322 as well as two histidines, with moderate, micromolar affinity. Our data demonstrate that low micromolar zinc accelerates the fibrillization of human Tau protein via bridging Cys-291 and Cys-322 in physiological reducing conditions, providing clues to understanding the relationship between zinc dyshomeostasis and the etiology of neurodegenerative diseases.     

Cross-linking myosin subfragment 1 Cys-697 and Cys-707 modifies ATP and actin binding site interactions.

Skeletal muscle myosin is an enzyme that interacts allosterically with MgATP and actin to transduce the chemical energy from ATP hydrolysis into work. By modifying myosin structure, one can change this allosteric interaction and gain insight into its mechanism. Chemical cross-linking with N,N'-p-phenylenedimaleimide (pPDM) of Cys-697 to Cys-707 of the myosin-ADP complex eliminates activity and produces a species that resembles myosin with ATP bound (Burke et al., 1976). Nucleotide-free pPDM-modified myosin subfragment 1 (S1) was prepared, and its structural and allosteric properties were investigated by comparing the nucleotide and actin interactions of S1 to those of pPDM-S1. The structural properties of the nucleotide-free pPDM-S1 are different from those of S1 in several respects. pPDM-S1 intrinsic tryptophan fluorescence intensity is reduced 28%, indicating a large increase of an internal quenching reaction (the fluorescence intensity of the related vanadate complex of S1, S1-MgADP-Vi, is reduced by a similar degree). Tryptophan fluorescence anisotropy increases from 0.168 for S1 to 0.192 for pPDM-S1, indicating that the unquenched tryptophan population in pPDM-S1 has reduced local freedom of motion. The actin affinity of pPDM-S1 is over 6,000-fold lower than that of S1, and the absolute value of the product of the net effective electric charges at the acto-S1 interface is reduced from 8.1 esu2 for S1 to 1.6 esu2 for pPDM-S1. In spite of these changes, the structural response of pPDM-S1 to nucleotide and the allosteric communication between its ATP and actin sites remain intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine-derived quinones affect the structure of the redox sensor DJ-1 through modifications at Cys-106 and Cys-53

The physiological role of DJ-1, a protein involved in familial Parkinson disease is still controversial. One of the hypotheses proposed indicates a sensor role for oxidative stress, through oxidation of a conserved cysteine residue (Cys-106). The association of DJ-1 mutations with Parkinson disease suggests a loss of function, specific to dopaminergic neurons. Under oxidative conditions, highly reactive dopamine quinones (DAQs) can be produced, which can modify cysteine residues. In cellular models, DJ-1 was found covalently modified by dopamine. We analyzed the structural modifications induced on human DJ-1 by DAQs in vitro. We described the structural perturbations induced by DAQ adduct formation on each of the three cysteine residues of DJ-1 using specific mutants. Cys-53 is the most reactive residue and forms a covalent dimer also in SH-SY5Y DJ-1-transfected cells, but modification of Cys-106 induces the most severe structural perturbations; Cys-46 is not reactive. The relevance of these covalent modifications to the several functions ascribed to DJ-1 is discussed in the context of the cell response to a dopamine-derived oxidative insult.     

Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents

During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimide-treated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation.     

Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents

During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimide-treated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation.     

Role of Cys-1327 and Cys-1337 in Redox Sensitivity and Allosteric Monitoring in Human Carbamoyl Phosphate Synthetase

Human carbamoyl phosphate synthetase (hCPS) has evolved critical features that allow it to remove excess and potentially neurotoxic ammonia via the urea cycle, including use of only free ammonia as a nitrogen donor, a K_m for ammonia 100-fold lower than for CPSs that also use glutamine as a nitrogen donor, and required allosteric activation by N-acetylglutamate (AGA), a sensor of excess amino acids. The recent availability of a Schizosaccharomyces pombe expression system for hCPS allowed us to utilize protein engineering approaches to elucidate the distinctive hCPS properties. Although the site of AGA interaction is not defined, it is known that the binding of AGA to CPS leads to a conformational change in which a pair of cysteine side chains become proximate and can then be selectively induced to undergo disulfide bonding. We analyzed the response of hCPS cysteine mutants to thiol-specific reagents and identified Cys-1327 and Cys-1337 as the AGA-responsive proximate cysteines. Possibly two of the features unique to urea-specific CPSs, relative to other CPSs (the conserved Cys-1327/Cys-1337 pair and the occurrence at very high concentrations in the liver mitochondrial matrix) co-evolved to provide buffering against reactive oxygen species. Reciprocal mutation analysis of Escherichia coli CPS (eCPS), creating P909C and G919C and establishing the ability of these engineered cysteine residues to share a disulfide bond, indicated an eCPS conformational change at least partly similar to the hCPS conformational change induced by AGA. These findings strongly suggested an alternative eCPS conformation relative to the single crystal conformation thus far identified.When life adapted to a terrestrial habitat, removal of excess and potentially neurotoxic ammonia by the diffusion that occurred in an aquatic habitat was no longer possible (1). Arginine biosynthetic pathways almost certainly served as the precursors for the urea cycle, the present day metabolic pathway for removal of excess ammonia, with surprisingly few changes needed for the pathway evolution (2). Carbamoyl phosphate synthetase (CPS),² the enzyme that catalyzes the entry and rate-limiting step of the urea cycle, was the site of four of these critical evolutionary changes: (a) gain of communication with a sensor of excess amino acids, N-acetylglutamate (AGA, which serves as a required allosteric activator only for urea-synthesizing CPSs), (b) a decrease in K_m for ammonia to ∼1 mm from the ∼100 mm value for other CPSs, (c) loss of interaction with glutamine to avoid competition with the preferred substrate ammonia, and (d) localization to the hepatic mitochondrial matrix to allow independent regulation relative to CPSs with other metabolic roles. Structural changes have been identified in hCPS that correlate with the latter two functional changes (3-5); the cysteine residue required for releasing ammonia from glutamine has been replaced by serine, and hCPS is synthesized with 39 N-terminal residues that serve as a mitochondrial matrix targeting signal and that are cleaved as the hCPS precursor crosses the inner mitochondrial membrane. The structural basis for the first two critical functional features, AGA interaction and high affinity for ammonia, however, have not yet been elucidated nor has it been determined whether these two features are linked.CPSs from all species with varied metabolic roles share strong sequence identity, and all appear to have the same overall domain organization (6) (Fig. 1) observed in the crystal structure of E. coli CPS (eCPS), the only solved CPS structure (7). All known CPSs appear to utilize a common mechanism (Fig. 1) to catalyze the formation of carbamoyl phosphate (CP), P_i, and two molecules of ADP from ammonia (either free in the cell or generated from the hydrolysis of glutamine), bicarbonate, and two molecules of ATP (6). A variety of studies have yielded evidence for a cycle of conformational changes accompanying the catalytic cycle (6-8). Vertebrate urea-specific CPSs, including hCPS, incorporate an additional type of conformational control in the form of dependence on the allosteric activator AGA. In the absence of AGA, a very small fraction of urea-specific CPS is in a conformation capable of catalytic activity, whereas binding of AGA yields CPS with activity equivalent to that of eCPS and other non-AGA-dependent CPSs (6). Although AGA functions as an intermediate in arginine biosynthesis in prokaryotes and lower eukaryotes, the only known function for AGA in vertebrates is activation of urea-specific CPSs (9). Co-localization of AGA synthetase in the hepatic mitochondrial matrix is consistent with this dedicated role, as is the fact that increases in the substrate glutamate signal increased degradation of proteins to yield both free amino acids and free ammonia. Allosteric activation of vertebrate AGA synthetase by arginine provides a further link to the presence of excess amino acids and the accompanying excess of free ammonia requiring detoxification.Open in a separate windowFIGURE 1.Domain and oligomeric structure of eCPS. Top panel, ribbon representation of one heterodimer of eCPS. The 42-kDa glutaminase subunit contains the glutamine binding domain (A2; violet) and domain A1 (purple) that is involved in communicating active site occupancy between the glutaminase and synthetase subunits. The 120-kDa synthetase subunit contains 4 domains. Domains B (green) and C (red) are regions of internal duplication, and each contains an ATP grasp fold. The oligomerization domain (D′, yellow) and allosteric domain (D, orange) are involved in side-by-side and end-on-end interactions, respectively. The ligands glutamine and ADP are shown in space-fill representations. The intramolecular tunnel connecting the three active sites is illustrated with arrows. Also shown are the individual chemical reactions at each of the active sites that are synchronized in the overall reaction. The domains of hCPS occur as a single polypeptide, with 1462 amino acid residues in the mature protein (from which the mitochondrial targeting signal has been removed) in contrast to the heterodimeric structure of eCPS. Bottom panel, the tetramer of the glutaminase + synthetase heterodimers. Created from PDB file 1a9x.One reporter for the AGA-activated conformation of CPS is a pair of cysteine side chains that become proximate upon AGA binding and that can then be selectively induced to undergo disulfide bonding (10, 11). In the absence of AGA, only a small fraction of CPS is in a conformation with the cysteine pair proximate. This single pair of proximate sulfhydryl groups is uniquely modified when the AGA·CPS complex is exposed to a variety of disulfide-inducing reagents, and reversible activity loss accompanies disulfide formation. The recent availability (12) of a Schizosaccharomyces pombe expression system for hCPS allowed us to utilize protein engineering approaches for elucidation of the distinctive hCPS properties. To identify the residues of the cysteine reporter group, we have analyzed the response of hCPS cysteine mutants to thiol-specific reagents. We also have examined the potential for a conformational change in eCPS that parallels the AGA-induced conformational change of hCPS. Thus far only a single conformation of eCPS has been revealed from x-ray structural analysis. Defining the cycle of conformational changes that accompany the catalytic cycle and that allow synchronization of three active sites (glutamine amidotransferase site and two distinct ATP sites) to produce CP, therefore, remains a major challenge.     

The role of Cys-298 in aldose reductase function

Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.     

Palmitoylation of either Cys-3 or Cys-5 is required for the biological activity of the Lck tyrosine protein kinase.

Palmitoylation can regulate both the affinity for membranes and the biological activity of proteins. To study the importance of the palmitoylation of the Src-like tyrosine protein kinase p56lck in the function of the protein, Cys-3, Cys-5, or both were mutated to serine, and the mutant proteins were expressed stably in fibroblasts and T cells. Both Cys-3 and Cys-5 were apparent sites of palmitoylation in Lck expressed in fibroblasts, as only the simultaneous mutation of both Cys-3 and Cys-5 caused a large reduction in the incorporation of [3H]palmitic acid. The double mutant S3/5Lck was no longer membrane bound when examined by either immunofluorescence or cell fractionation. This indicated that palmitoylation was required for association of Lck with the plasma membrane. Since the S3/5Lck protein was myristoylated, myristoylation of Lck is not sufficient for membrane binding. When Cys-3, Cys-5, or both Cys-3 and Cys-5 were changed to serine in activated F505Lck, palmitoylation of either Cys-3 or Cys-5 was found to be necessary and sufficient for the transformation of fibroblasts and for the induction of spontaneous, antigen-independent interleukin-2 production in the T-helper cell line DO-11.10. Nonpalmitoylated F505Lck exhibited little activity in vivo, where it did not induce elevated levels of tyrosine phosphorylation, and in vitro, where it was unable to phosphorylate angiotensin in an in vitro kinase assay. These findings suggest that F505Lck must be anchored stably to membranes to become activated. Because palmitoylation is dynamic, it may be involved in regulating the cellular localization of p56(lck), and consequently its activity, by altering the proximity of p56(lck) to its activators and/or targets.     

Fluorescence energy transfer between Cys-10 residues in F-actin filaments

Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-[2-((iodoacetyl)amino)-ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) as a fluorescence energy donor and 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) as the acceptor. Both labels were covalently attached to Cys-10 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Cys-10 was calculated to be 23 A. This corresponds to a distance between adjacent sites along the long pitch helix of 56.1 A and along the genetic helix of 53.3 A.     

Evidence that conserved residues Cys-62 and Cys-154 within the Azotobacter vinelandii nitrogenase MoFe protein α-subunit are essential for nitrogenase activity but conserved residues His-83 and Cys-88 are not

Metallocluster extrusion requirements, interspecies MoFe-protein primary sequence comparisons and comparison of the primary sequences of the MoFe-protein subunits with each other have been used to assign potential P-cluster (Fe-S cluster) domains within the MoFe protein. In each alpha-beta unit of the MoFe protein, alpha-subunit domains, which include potential Fe-S cluster ligands Cys-62, His-83, Cys-88 and Cys-154, and beta-subunit domains, which include potential Fe-S cluster ligands Cys-70, His-90, Cys-95 and Cys-153, are proposed to comprise nearly equivalent P-cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the alpha-subunit Cys-62, His-83, Cys-88 and Cys-154 positions by site-directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates, MoFe-protein acetylene-reduction activities, and whole-cell S = 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe-protein alpha-subunit residues, Cys-62 and Cys-154, are probably essential for MoFe-protein activity but that His-83 and Cys-88 residues are not. These results indicate either that His-83 and Cys-88 do not provide essential P-cluster ligands or that a new cluster-ligand arrangement is formed in their absence.     

Cys-140 is critical for metabotropic glutamate receptor-1 dimerization

Metabotropic glutamate receptor 1 (mGluR1) expresses at the cell surface as disulfide-linked dimers and can be reduced to monomers with sulfhydryl reagents. To identify the dimerization domain, we transiently expressed in HEK-293 cells a truncated version of mGluR1 (RhodC-R1) devoid of the extracellular domain (ECD). RhodC-R1 was a monomer in the absence or presence of the reducing agents, suggesting that dimerization occurs via the ECD. To identify cysteine residues involved in dimerization within the ECD, cysteine to serine point mutations were made at three cysteines within the amino-terminal half of the ECD. A mutation at positions Cys-67, Cys-109, and Cys-140 all resulted in significant amounts of monomers in the absence of reducing agents. The monomeric C67S and C109S mutants were not properly glycosylated, failed to reach the cell surface, and showed no glutamate response, indicating that these mutant receptors were improperly folded and/or processed and thus retained intracellularly. In contrast, the monomeric C140S mutant was properly glycosylated, processed, and expressed at the cell surface. Phosphoinositide hydrolysis assay showed that the glutamate response of the C140S mutant receptor was similar to the wild type receptor. Substitution of a cysteine for Ser-129, Lys-134, Asp-143, and Thr-146 on the C140S mutant background restored receptor dimerization. Taken together, the results suggest that Cys-140 contributes to intermolecular disulfide-linked dimerization of mGluR1.     

Interactions between cysteine residues as probes of protein conformation: the disulphide bond between Cys-14 and Cys-38 of the pancreatic trypsin inhibitor.

The rate constants for the reversible reduction by dithiothreitol of the disulphide bond linking eysteines 14 and 38 of the native pancreatic trypsin inhibitor have been measured. The results are consistent with this disulphide bond being formed as the last step in refolding of the fully reduced inhibitor.The rates of reduction of several model linear disulphides have been measured under the same conditions. A linear relationship was found between the rate of reduction and the ionization tendency of the thiol group generated.The apparent pK value of the thiol groups of cysteines 14 and 38 of the selectively reduced inhibitor were measured by the pH-dependence of their rate of alkylation with iodoacetamide. The rate of reduction of the disulphide bond between these two residues was very close to that predicted from the model compounds.The kinetics and thermodynamics of disulphide bond formation and breakage are shown to be useful for experimental determination of conformational transitions in proteins and model peptides.     

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.     

Movement of Cys-697 in myosin ATPase associated with ATP hydrolysis.

To detect movement of Cys-697 (SH2) in myosin subfragment-1 (S-1) associated with ATP hydrolysis, SH2 was labeled with the environmentally sensitive fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). Complex formation of S-1 labeled at Cys-697 with MIANS (MIANS-S-1) with adenyl-5'-yl imidodiphosphate and ADP resulted in a significant decrease in the fluorescence intensity of approximately 40 and 30%, respectively. When ATP was added to MIANS-S-1, the fluorescence intensity decreased rapidly by approximately 40%, and this fluorescence level was maintained during the steady state of ATP hydrolysis. As the substrate was used up, the fluorescence intensity increased to approximately 70% of the original value. These results together with model experiments with MIANS-N-acetylcysteine indicate that in the presence of ATP, the MIANS fluorophore attached to SH2 is located in a less hydrophobic environment than is the fluorophore in the absence of ligand and that the hydrolysis of ATP enhances hydrophobicity around the fluorophore. Acrylamide fluorescence quenching studies of MIANS-S-1 confirmed these results, indicating that addition of ATP and ADP to MIANS-S-1 results in an increase in the Stern-Volmer quenching constant of the fluorophore by factors of approximately 3 and 2.5, respectively. The present observations suggest that binding of ATP causes a movement of SH2 toward the protein surface, whereas it goes back into the protein interior after ATP hydrolysis. The results also confirmed previous observations by a chemical cross-linking approach (Hiratsuka, T. (1987) Biochemistry 26, 3168-3173).     

Ca2+ dependence of the distance between Cys-98 of troponin C and Cys-133 of troponin I in the ternary troponin complex. Resonance energy transfer measurements

We have used resonance energy transfer to study the spatial relationship between Cys-98 of rabbit skeletal troponin C and Cys-133 of rabbit skeletal troponin I in the reconstituted ternary troponin complex. The donor was introduced by labeling either troponin C or troponin I with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, while the acceptor was introduced by labeling either protein with N-[4-(dimethylamino)phenyl-4'-azophenyl]maleimide. The extent of energy transfer was determined by measuring the quenching of the donor fluorescence decay. The results indicate first that the distance between these two sites is not fixed, suggesting that the protein regions involved possess considerable segmental flexibility. Second, the mean distance between the two sites is dependent on the metal-binding state of troponin C, being 39.1 A when none of the metal-binding sites are occupied, 41.0 A when Mg2+ ions bind at the high-affinity sites, and 35.5 A when Ca2+ ions bind to the low-affinity sites. Neither the magnitude of the distances nor the trend of change with metal ions differs greatly when the locations of the probes are switched or when steady-state fluorometry was used to determine the transfer efficiency. Since the low-affinity sites have been implicated as the physiological triggering sites, our findings suggest that one of the key events in Ca2+ activation of skeletal muscle contraction is a approximately 5-A decrease in the distance between the Cys-98 region of troponin C and the Cys-133 region of troponin I.     

Temperature-induced changes in the flexibility of the loop between SH1 (Cys-707) and SH3 (Cys-522) in myosin subfragment 1 detected by cross-linking

The ability of dibromobimane to cross-link SH1 (Cys-707) in the 21-kDa C-terminal segment to SH3 (Cys-522) in the 50-kDa middle segment of the myosin S1 heavy chain has been examined as a function of nucleotide binding and temperature. The results obtained indicate that, while the reagent rapidly reacts with SH1 at both 25 and 4 degrees C, its ability to cross-link to SH3 is highly dependent on temperature. At 25 degrees C, substantial cross-linking from monofunctionally labeled SH1 to SH3 occurs, in agreement with recent work of Mornet, Ue, and Morales (1985, Proc. Natl. Acad. Sci, USA 82, 1658-1662) and of Ue (1987, Biochemistry 26, 1889-1894) and with their conclusion that a loop, allowing SH1 and SH3 to reside at the cross-linking span of dibromobimane, preexists in the protein. At 4 degrees C, however, negligible amounts of cross-linking are observed whether or not a nucleotide is present, despite indications that SH1 is labeled rapidly by the reagent at this temperature. The inability to form this cross-link is not due to an alternate cross-link between monofunctionally labeled SH1 and another thiol in the 21-kDa segment. These results indicate that this loop exists at 25 degrees C and does not exist (or exists only transiently) at the lower temperature.     

Site-directed mutagenesis of human aldolase isozymes: the role of Cys-72 and Cys-338 residues of aldolase A and of the carboxy-terminal Tyr residues of aldolases A and B

In order to elucidate the role of particular amino acid residues in the catalytic activity and conformational stability of human aldolases A and B [EC 4.1.2.13], the cDNAs encoding these isoenzyme were modified using oligonucleotide-directed, site-specific mutagenesis. The Cys-72 and/or Cys-338 of aldolase A were replaced by Ala and the COOH-terminal Tyr of aldolases A and B was replaced by Ser. The three mutant aldolases A thus prepared, A-C72A, A-C338A, and A-C72,338A, were indistinguishable from the wild-type enzyme with respect to general catalytic properties, while the replacement of Tyr-363 by Ser in aldolase A (A-Y363S) resulted in decreases of the Vmax of the fructose-1, 6-bisphosphate (FDP) cleavage reaction, activity ratio of FDP/fructose-1-phosphate (F1P), and the Km values for FDP and F1P. The wild-type and all the mutant aldolase A proteins exhibited similar thermal stabilities. In contrast, the mutant aldolase A proteins were more stable than the wild-type enzyme against tryptic and alpha-chymotryptic digestions. Based upon these results it is concluded that the strictly conserved Tyr-363 of human aldolase A is required for the catalytic function with FDP as the substrate, while neither Cys-72 nor Cys-338 directly takes part in the catalytic function although the two Cys residues may be involved in maintaining the correct spatial conformation of aldolase A. Replacement of Tyr-363 by Ser in human aldolase B lowered the Km value for FDP appreciably and also diminished the stability against elevated temperatures and tryptic digestion.(ABSTRACT TRUNCATED AT 250 WORDS)