A study of the subcellular localisation of an ethylene binding site in developing cotyledons of Phaseolus vulgaris L. by high resolution autoradiography

Use was made of light microscopy and high resolution electron microscope autoradiography to determine the subcellular localisation of a binding site with a high affinity and specificity for ethylene in developing cotyledons of Phaseolus vulgaris L. The results indicate that the binding site is located on the endoplasmic reticulum and protein body membranes, confirming previous studies using cellular fractionation and marker enzymes.     

Inhibitors of photosystem II and the topology of the herbicide and QB binding polypeptide in the thylakoid membrane

The folding through the thylakoid membrane of the D-1 herbicide binding polypeptide and of the homologous D-2 subunit of photosystem II is predicted from comparison of amino acid sequences and hydropathy index plots with the folding of the subunits L and M of a bacterial photosystem. As the functional amino acids involved in Q and Fe binding in the bacterial photosystem of R. viridis, as indicated by the X-ray structure, are conserved in the homologous D-1 and D-2 subunits of photosystem II, a detailed topology of the binding niche of Q_B and of herbicides on photosystem II is proposed. The model is supported by the observed amino acid changes in herbicide tolerant plants and algae. These changes are all in the binding domain on the matrix side of the D-1 polypeptide, and turn out to be of functional significance in the Q_B binding.New inhibitors of Q_B function are described. Their chemical structure, i.e. pyridones, quinolones, chromones and benzodiones, contains the features of the phenolic type herbicides. Their essential elements, -charges at particular atoms, QSAR and steric requirements for optimal inhibitory potency are discussed and compared with the classical herbicides of the urea/triazine type.     

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [¹⁴C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [¹⁴C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m^–2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.Abbreviations AdN adenine nucleotide - Chl chlorophyll - DTT dithiothreitol - FCCP carbonylcianide p-trifluoromethoxyphenilhydrazone - P_i inorganic orthophosphate - PMS phenazine methosulfate - TLC thin-layer chromatography - Tricine N-[tris(hydroxymethyl)methyl] glycine     

Distinct affinity and effector residues in the binding site for a regulatory ligand

A hypothesis concerning two distinct classes of amino acid residues in some regulatory binding sites is proposed. The affinity residues are those that are unable to transduce the ligand information signal but are responsible for overcoming the barrier for the attachment of a ligand to its binding site while the effector residues transfer the binding signal to the other functional part of the protein, which then undergoes a non-equilibrium energetic cycle induced by interaction with the ligand.As an example, the purine nucleotide inhibition of H⁺ transport through the uncoupling protein of brown adipose tissue mitochondria is discussed; there is a concentration range in which the nucleotide is bound but does not inhibit H⁺ transport. This is interpreted in terms of inaccessibility of the effector residues inducing H⁺ transport inhibition below a certain threshold concentration.     

Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry

Nano-electrospray ionization time-of-flight mass spectrometry (ESI-MS) was used to study the conformational consequences of metal ion binding to the colicin E9 endonuclease (E9 DNase) by taking advantage of the unique capability of ESI-MS to allow simultaneous assessment of conformational heterogeneity and metal ion binding. Alterations of charge state distributions on metal ion binding/release were correlated with spectral changes observed in far- and near-UV circular dichroism (CD) and intrinsic tryptophan fluorescence. In addition, hydrogen/deuterium (H/D) exchange experiments were used to probe structural integrity. The present study shows that ESI-MS is sensitive to changes of the thermodynamic stability of E9 DNase as a result of metal ion binding/release in a manner consistent with that deduced from proteolysis and calorimetric experiments. Interestingly, acid-induced release of the metal ion from the E9 DNase causes dramatic conformational instability associated with a loss of fixed tertiary structure, but secondary structure is retained. Furthermore, ESI-MS enabled the direct observation of the noncovalent protein complex of E9 DNase bound to its cognate immunity protein Im9 in the presence and absence of Zn(2+). Gas-phase dissociation experiments of the deuterium-labeled binary and ternary complexes revealed that metal ion binding, not Im9, results in a dramatic exchange protection of E9 DNase in the complex. In addition, our metal ion binding studies and gas-phase dissociation experiments of the ternary E9 DNase-Zn(2+)-Im9 complex have provided further evidence that electrostatic interactions govern the gas phase ion stability.     

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.     

Modeling the quinone-B binding site of the photosystem-II reaction center using notions of complementarity and contact-surface between atoms

Functional identity and significant similarities in cofactors and sequence exist between the L and M reaction center proteins of the photosynthetic bacteria and the D1 and D2 photosystem-II reaction center proteins of cyanobacteria, algae, and plants. A model of the quinone (Q_B) binding site of the D1 protein is presented based upon the resolved structure of the Q_B binding pocket of the L subunit, and introducing novel quantitative notions of complementarity and contact surface between atoms. This model, built -without using traditional methods of molecular mechanics and restricted to residues in direct contact with Q_B, accounts for the experimentally derived functional state of mutants of the Dl protein in the region of Q_B. It predicts the binding of both the classical and phenol-type PSII herbicides and rationalizes the relative levels of tolerance of mutant phenotypes. © 1995 Wiley-Liss, Inc.     

Common conformational changes in flavodoxins induced by FMN and anion binding: the structure of Helicobacter pylori apoflavodoxin

Flavodoxins, noncovalent complexes between apoflavodoxins and flavin mononucleotide (FMN), are useful models to investigate the mechanism of protein/flavin recognition. In this respect, the only available crystal structure of an apoflavodoxin (that from Anabaena) showed a closed isoalloxazine pocket and the presence of a bound phosphate ion, which posed many questions on the recognition mechanism and on the potential physiological role exerted by phosphate ions. To address these issues we report here the X-ray structure of the apoflavodoxin from the pathogen Helicobacter pylori. The protein naturally lacks one of the conserved aromatic residues that close the isoalloxazine pocket in Anabaena, and the structure has been determined in a medium lacking phosphate. In spite of these significant differences, the isoallozaxine pocket in H. pylori apoflavodoxin appears also closed and a chloride ion is bound at a native-like FMN phosphate site. It seems thus that it is a general characteristic of apoflavodoxins to display closed, non-native, isoalloxazine binding sites together with native-like, rather promiscuous, phosphate binding sites that can bear other available small anions present in solution. In this respect, both binding energy hot spots of the apoflavodoxin/FMN complex are initially unavailable to FMN binding and the specific spot for FMN recognition may depend on the dynamics of the two candidate regions. Molecular dynamics simulations show that the isoalloxazine binding loops are intrinsically flexible at physiological temperatures, thus facilitating the intercalation of the cofactor, and that their mobility is modulated by the anion bound at the phosphate site.     

A possible calcium binding site in D1 protein: A fluorescence and FTIR study of the interaction between lanthanides and a synthetic peptide

A peptide ranging from residue 229 to 240 of the D₁ protein of Photosystem (PS) II was synthesized and lanthanides were used as candidates of calcium. Fluorescence and FTIR spectroscopy were used to test the conformational adaptation after lanthanide additions. Fluorescence spectroscopy showed that the synthetic peptide provides lanthanide binding site, and that glutamic acids are involved in lanthanide binding. Resolution enhancement techniques were combined with band curve-fitting procedures to quantitate the FTIR spectral information from the amide 1 bands. The relative areas of these component bands indicate that lanthanide induced a substantial decrease in the amount of unordered structure and turns, while a corresponding increase in the amount of -helix and open loop was also observed. This indicates that a relatively compact structure of the synthetic peptide is formed if lanthanides are applied. The results may reflect on the physiological and biochemical function of calcium in PS II, including preventing D₁ from trypsin digestion.Abbreviations DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea - FTIR Fourier transform infrared - FSD Fourier self-deconvolution - PS Photosystem - Q_B Secondary plastoquinone electron acceptor of PS II     

ATP hydrolysis-dependent conformational changes in the extracellular domain of ABCA1 are associated with apoA-I binding

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high-density lipoprotein (HDL) metabolism. Although it is predicted that apolipoprotein A-I (apoA-I) directly binds to ABCA1, the physiological importance of this interaction is still controversial and the conformation required for apoA-I binding is unclear. In this study, the role of the two nucleotide-binding domains (NBD) of ABCA1 in apoA-I binding was determined by inserting a TEV protease recognition sequence in the linker region of ABCA1. Analyses of ATP binding and occlusion to wild-type ABCA1 and various NBD mutants revealed that ATP binds equally to both NBDs and is hydrolyzed at both NBDs. The interaction with apoA-I and the apoA-I-dependent cholesterol efflux required not only ATP binding but also hydrolysis in both NBDs. NBD mutations and cellular ATP depletion decreased the accessibility of antibodies to a hemagglutinin (HA) epitope that was inserted at position 443 in the extracellular domain (ECD), suggesting that the conformation of ECDs is altered by ATP hydrolysis at both NBDs. These results suggest that ATP hydrolysis at both NBDs induces conformational changes in the ECDs, which are associated with apoA-I binding and cholesterol efflux.     

Identification of an alcohol binding site in the first cysteine-rich domain of protein kinase Cdelta

Protein kinase C (PKC) is an important signal transduction protein whose cysteine-rich regulatory domain C1 has been proposed to interact with general anesthetics in both of its diacylglycerol/phorbol ester-binding subdomains, the tandem repeats C1A and C1B. Previously, we identified an allosteric binding site on one of the two cysteine-rich domains, PKCdelta C1B. To test the hypothesis that there is an additional anesthetic site on the other cysteine-rich subdomain, C1A, we subcloned, expressed in Escherichia coli, purified, and characterized mouse PKCdelta C1A. Octanol and butanol both quenched the intrinsic fluorescence of PKCdelta C1A in a saturable manner, suggesting the presence of a binding site. To locate this site, PKCdelta C1A was photolabeled with three diazirine-containing alkanols, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry revealed that at low concentrations all three photoincorporated into PKCdelta C1A with a stoichiometry of 1:1 in the labeled fraction, but higher stoichiometries occurred at higher concentrations, particularly with azibutanol. Photocomplexes of PKCdelta C1A with azioctanols were separated from the unlabeled protein by HPLC, reduced, alkylated, digested with trypsin, and sequenced by mass spectrometry. All the azioctanols photolabeled PKCdelta C1A at residue Tyr-29, corresponding to Tyr-187 of the full-length PKCdelta, and at a neighboring residue, Lys-40, suggesting there is an alcohol site in this vicinity. In addition, Glu-2 was photolabeled more efficiently by 3-azibutanol than by the azioctanols, suggesting the existence of a second, smaller site.     

The crystal structure of the carbohydrate-recognition domain of the glycoprotein sorting receptor p58/ERGIC-53 reveals an unpredicted metal-binding site and conformational changes associated with calcium ion binding

p58/ERGIC-53 is a calcium-dependent animal lectin that acts as a cargo receptor, binding to a set of glycoproteins in the endoplasmic reticulum (ER) and transporting them to the Golgi complex. It is similar in structure to calcium-dependent leguminous lectins. We have determined the structure of the carbohydrate-recognition domain of p58/ERGIC-53 in its calcium-bound form. The structure reveals localized but large conformational changes in relation to the previously determined metal ion-free structure, mapping mostly to the ligand-binding site. It reveals the presence of two calcium ion-binding sites located 6A apart, one of which has no equivalent in the plant lectins. The second metal ion-binding site present in that class of lectins, binding Mn(2+), is absent from p58/ERGIC-53. The absence of a short loop in the ligand-binding site in this protein suggests that it has adapted to optimally bind the high-mannose Man(8)(GlcNAc)(2) glycan common to glycoproteins at the ER exit stage.     

The role of aspartate-235 in the binding of cations to an artificial cavity at the radical site of cytochrome c peroxidase.

The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically bind substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818). Proposals that a nearby carboxylate, Asp-235, and competing monovalent cations should modulate the affinity of the W191G cavity for ligand binding are addressed in this study. Competitive binding titrations of the imidazolium ion to W191G as a function of [K+] show that potassium competes weakly with the binding of imidazoles. The dissociation constant observed for potassium binding (18 mM) is more than 3,000-fold higher than that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unperturbed by replacement of the carboxylate. Refined crystallographic B-values of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essential feature of the cation binding determinant and indicate that this carboxylate plays a critical role in stabilizing the formation of the Trp-191 radical cation.     

A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.

The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.     

Mechanism of interaction of PITPalpha with membranes: conformational changes in the C-terminus associated with membrane binding

Eukaryotic phosphatidylinositol transfer proteins (PITPs) are composed predominantly of small ( approximately 32 kDa) soluble proteins that bind and transfer a single phospholipid, normally phosphatidylinositol or phosphatidycholine. Two forms, PITPalpha and PITPbeta, which share approximately 80% amino acid sequence similarity, are known. Rat PITPalpha was labeled at specific single reactive Cys residues with I-AEDANS and used to examine PITP-membrane interactions. Upon binding to phospholipid vesicles, PITP labeled with AEDANS at the C-terminus, a region postulated to be involved in membrane binding, shows significant decreases in both steady-state and dynamic fluorescence anisotropy. In contrast, PITPs labeled with AEDANS at sites located distal to the C-terminus show increases in both steady-state and dynamic anisotropy. These results suggest that interaction of PITP with membrane surfaces leads to significant alterations in conformation and perhaps melting of the C-terminal helix.     

Positions of disulfide bonds and N-glycosylation site in juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues. It contains four Cys residues forming two disulfide bridges. In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G. mellonella was determined, using electrospray mass spectrometry. The MS analysis was performed on tryptic digests of JHBP. Our results show that the disulfide bridges link Cys10 and Cys17, and Cys151 and Cys195. Of the two potential N-glycosylation sites in JHBP, Asn4, and Asn94, only Asn94 is glycosylated. This site of glycosylation is also found in the fully biologically active recombinant JHBP expressed in the yeast Pichia pastoris.     

Methionine sulfoxidation of the chloroplast small heat shock protein and conformational changes in the oligomer

The small heat shock proteins (sHsps), which counteract heat and oxidative stress in an unknown way, belong to a protein family of sHsps and alpha-crystallins whose members form large oligomeric complexes. The chloroplast-localized sHsp, Hsp21, contains a conserved methionine-rich sequence, predicted to form an amphipatic helix with the methionines situated along one of its sides. Here, we report how methionine sulfoxidation was detected by mass spectrometry in proteolytically cleaved peptides that were produced from recombinant Arabidopsis thaliana Hsp21, which had been treated with varying concentrations of hydrogen peroxide. Sulfoxidation of the methionine residues in the conserved amphipatic helix coincided with a significant conformational change in the Hsp21 protein oligomer.     

The differences in the microenvironment of the two tryptophan residues of the glutamine-binding protein from Escherichia coli shed light on the binding properties and the structural dynamics of the protein

Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel beta-strands. The large domain is similar to the small domain but it contains two additional alpha-helices and three more short antiparallel beta-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP.