Dependence of the conformational state of the isolated adenine nucleotide carrier protein on the detergent used for solubilization

The mitochondrial adenine nucleotide (AdN) carrier can assume two conformational states that are trapped by the specific inhibitors of AdN transport carboxyatractyloside (CATR) and bongkrekic acid (BA). When the AdN carrier protein was extracted from beef heart mitochondria by the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate (CHAPS) and purified in the same detergent, the fluorescence of the tryptophanyl residue(s) of the protein was partially quenched by ATP (or ADP), but not by nontransportable nucleotides; CATR, which alone was ineffective, was able in the presence of ATP (ADP) to further quench the fluorescence, and BA reversed the quenched fluorescence to the original level. With 3'-O-naphthoyl-ATP (N-ATP) as an extrinsic fluorescence probe, it was shown that BA could release bound N-ATP but that CATR was ineffective. These results indicate that the AdN carrier in CHAPS is able to react readily with BA, but not with CATR. The opposite situation occurs with the carrier solubilized and purified in (laurylamido)-N,N-dimethylpropylamine oxide (LAPAO) [Brandolin, G., Dupont, Y., & Vignais, P.V. (1985) Biochemistry 24, 1991-1997]. These data taken together were interpreted to mean that the CATR and BA conformations of the isolated AdN carrier depend on the micellar structure in which it is embedded; the carrier in LAPAO is in the CATR conformation, and the carrier in CHAPS is in the BA conformation. For the transition between the CATR and BA conformations to occur in the carrier in CHAPS and in the carrier in LAPAO, ATP or ADP is required; nontransportable nucleotides were ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate-induced fluorescence changes of the isolated ADP/ATP carrier protein in solution

Fluorescence studies were carried out on a purified preparation of the ADP/ATP carrier protein solubilized in 3-laurylamido-N-N-dimethylpropylaminoxide. The intrinsic fluorescence of the protein was modified upon addition of ADP and ATP and specific inhibitory ligands (carboxyatractyloside and bongkrekic acid). The fluorescence was transitorily enhanced by micromolar concentrations of ADP or ATP. The rise in fluorescence lasted for 10 sec at 25°C. It was suppressed by carboxyatractyloside and on the contrary enhanced by bongkrekic acid. These data were interpreted as reflecting conformational changes probably related to the functioning of the ADP/ATP carrier. Mg⁺⁺ inhibited the ADP- or ATP-induced rise in fluorescence, indicating that the free forms (and not the Mg⁺⁺ complexed forms) of ADP and ATP are the true substrates for the ADP/ATP carrier.     

Monitoring protein conformational changes by quenching of intrinsic fluorescence

The quenching of intrinsic fluorescence of human serum albumin and pigeon liver malic enzyme by acrylamide was studied after the proteins were denatured to different stages. The progress of protein denaturation induced by guanidine hydrochloride was accompanied by increasing of effective dynamic quenching constant which provides a convenient parameter for monitoring protein conformational change.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Photolabeling of the adenine nucleotide carrier by 8-azido-ADP

[¹⁴C] 8N₃ADP was synthesized from [¹⁴C] 8BrADP. It shows atractylate sensitive specific binding to the adeninnucleotide carrier of mitochondria, but is only a weak inhibitor of the translocator. Via nitrene formation uv-irradiation allows covalent labeling of the carrier protein and induces irreversible inhibition of transport. The labeled carrier protein can be isolated; the stoichiometry of labeling is 0.5 moles N₃ADP/mole carrier subunit which has a molecularweight of 31000, suggesting a dimer structure of the carrier in situ. Labeling is specific for 8N₃ADP; 8N₃AMP is inactive as an inhibitor or as photolabel.     

Molecular aspects of the adenine nucleotide carrier from mitochondria

The ADP/ATP carrier (AAC) of mitochondria is a functionally central and characteristic component of the eukaryotic cell. By linking the thermodynamically divergent metabolites in the intra- and extramitochondrial compartments, it had to evolve with the emergence of the eukaryotic cell. Because of a number of unique properties, the AAC provided advanced insight into the molecular basis of solute transport through biomembrane carriers. With highly specific and unusually large substrates, ADP and ATP, and with high-affinity inhibitors binding selectively either from the inside or the outside, the first molecular demonstration of the single-binding-center gated pore mechanism was made. This framework can only partially be interpreted with the available yet rapidly increasing structural information on the AAC. The primary structure, first established for the AAC from beef heart mitochondria, showed a relatively wide distribution of hydrophilic residues which permits assignment of only two hydrophobic transmembrane stretches. However, a striking tripartition of the primary structure into about three 100-residue-long domains allows a more significant assignment of transmembrane elements. With alignment of these three domains for maximum conservation of structurally critical residues, each domain can be assigned to have two transmembrane alpha elements between 18 and 22 residues long. The interdomain homology between these alpha regions is low. The central regions flanked by these helices contain most of the polar residues and are significantly interdomain conserved. With lysine probes the central regions are assigned to the matrix side (m-side) and the two connecting regions as well as C and N termini to the cytosolic side (c-side). Out of the central regions a loop is assumed to protrude through the membrane, probably for lining the translocation channel. This localization of a major protein mass within the membrane agrees with hydrodynamic evidence, the carrier being an oblate ellipsoid with only about 50 A along the short axis. In accordance, the loops of domains 2 and 3 are affinity labeled by azido-ADP or azido-atractylate. Primary structures of AAC from other sources (fungi, plants) also exhibit the tripartition. The interdomain conserved residues are also interspecies conserved, thus showing that they are essential. These repeat domains have probably evolved from a common gene coding for about 100 residues. Isoforms of the AAC exist, as shown by primary structure analysis of human cDNA libraries from different organs. Three different isoforms are identified in human organs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of phospholipid-ATPase complex interaction by the adenine nucleotide carrier

(1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP and P_i are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment.     

Agonist-mediated conformational changes in acetylcholine-binding protein revealed by simulation and intrinsic tryptophan fluorescence

We delineated acetylcholine (ACh)-dependent conformational changes in a prototype of the nicotinic receptor ligand binding domain by molecular dynamics simulation and changes in intrinsic tryptophan (Trp) fluorescence. Prolonged molecular dynamics simulation of ACh-binding protein showed that binding of ACh establishes close register of Trps from adjacent subunits, Trp(143) and Trp(53), and draws the peripheral C-loop inward to occlude the entrance to the binding cavity. Close register of Trp(143) and Trp(53) was demonstrated by ACh-mediated quenching of intrinsic Trp fluorescence, elimination of quenching by mutation of one or both Trps to Phe, and decreased lifetime of Trp fluorescence by bound ACh. Occlusion of the binding cavity by the C-loop was demonstrated by restricted access of an extrinsic quencher of binding site Trp fluorescence by ACh. The collective findings showed that ACh initially establishes close register of conserved Trps from adjacent subunits and then draws the C-loop inward to occlude the entrance to the binding cavity.     

The adenine nucleotide carrier of plant mitochondria: contraction induced by adenosine diphosphate

Energised mitochondria show an ADP-induced contraction which is partially resistant to oligomycin, uncouplers or respiratory inhibitors but sensitive to atractyloside.The addition of ADP or ATP, but not AMP, to non-energised corn mitochondria induces a contraction with a K_d of approx. 1 μM. Titration of the ADP-induced contraction with atractyloside produces an inhibition curve closely resembling the atractyloside inhibition curve of phosphorylating respiration. Partial recovery of atractyloside-inhibited contraction occurs in the presence of bongkrekic acid.It is suggested that these changes reflect changes in orientation of the adenine nucleotide (AdN) carrier in the inner mitochondrial membrane.     

Substrate-induced conformational changes of the truncated catalytic domain of Geobacillus stearothermophilus lysyl-tRNA synthetase as examined by fluorescence

The substrate-induced conformational change of the truncated C-terminal catalytic domain (CAT) of Geobacillus stearothermophilus lysyl-tRNA synthetase was examined by measuring tryptophan fluorescence of the truncated CAT domain in the presence or absence of the truncated N-terminal tRNA anticodon-binding domain (TAB). The fluorescence spectrum of CAT was not changed by the addition of l-lysine or ATP, whereas the intensity increased by adding a lysyl-adenylate analogue, suggesting that the CAT fluorescence increases when lysyl-adenylate is formed in the active site of CAT in l-lysine activation. In the presence of TAB, the addition of l-lysine to CAT decreased the fluorescence, and the subsequent addition of ATP recovered partially the decreased intensity, as is similar to the case of the intact enzyme. The static parameters of the CAT-TAB complex were similar to those of the intact enzyme, suggesting that a somewhat impaired structure of CAT is repaired on the formation of the complex with TAB. The mutational analysis of the fluorescence showed that Trp314 but not Trp332 is responsible for the observed fluorescence changes. The role of the TAB domain in the intact enzyme is considered to enhance the binding efficiency of lysyl-adenylate to the CAT domain.     

A high-resolution 1H-NMR investigation of the histidine-binding protein J of Salmonella Typhimurium: Substrate-induced conformational changes

High-resolution ¹ H-NMR spectroscopy at 600 MHz has been used to investigate the conformational transitions of the histidine-binding protein J of Salmonella Typhinmrium in solution as a function of pH and of l-histidine concentration. The dissociation constant for the binding of l-histidine to histidine-binding protein J increases from 6.0 × 10^?8 to 5.1 × 10^?7 M in going from pH 5.57 to 8.00. The conformation of this protein as observed by ¹H-NMR also changes over this range of pH. However, when l-histidine is bound, the changes in conformation with pH are much smaller. Also, the pk for the single histidyl residue in histidine-binding protein J changes from 6.75 in the absence of l-histidine to 6.52 when l-histidine is bound. Earlier work in this laboratory resulted in the identification of several proton resonances believed to be at or near the l-histidine-binding site. Two of these resonances have been assigned to a tyrosine and the single histidyl residue in the histidine-binding protein J molecule.