Amphipols from A to Z.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

Isotopic double-labeling of two honeybee odorant-binding proteins secreted by the methylotrophic yeast Pichia pastoris.

Odorant-binding proteins (OBPs) are soluble, low-molecular-weight proteins secreted in the sensillum lymph surrounding the dendrites of olfactory sensilla from a wide range of insect species. These proteins play a role in the solubilization, transport and/or deactivation of pheromones and odorants. In order to study the relationships between the molecular structure in solution and their ligand-binding properties, we have (13)C/(15)N-double-labeled two divergent honeybee OBPs, called ASP1 and ASP2, in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The recombinant labeled proteins produced by the methylotrophic yeast Pichia pastoris have been secreted into a buffered minimal medium using native insect signal peptide. Mass spectrometry and Edman sequencing showed a native-like processing with a labeling efficiency of secreted proteins greater than 98%. After dialysis, the recombinant proteins were purified to homogeneity by one-step reversed-phase liquid chromatography. The final yield after 4-day shake-flask liquid culture was approximately 60 and 100 mg/L for ASP1 and ASP2, respectively. The inexpensive overproduction of labeled recombinant ASP1 and ASP2 should allow NMR studies of the structures and ligand-binding analysis in order to understand the relationships between structure and biological function of these proteins.

The solution structure of an AlcR-DNA complex sheds light onto the unique tight and monomeric DNA binding of a Zn(2)Cys(6) protein.

BACKGROUND: In Aspergillus nidulans, the transcription activator AlcR mediates specific induction of a number of the genes of the alc cluster. This cluster includes genes involved in the oxidation of ethanol and other alcohols to acetate. The pattern of binding and of transactivation of AlcR is unique within the Zn(2)Cys(6) family. The structural bases for these specificities have not been analyzed at the atomic level until now. RESULTS: We have used NMR spectroscopy and restrained molecular dynamics to determine a set of structures of the AlcR DNA binding domain [AlcR(1-60)] in complex with a 10-mer DNA duplex. Analysis of the structures reveals specific interactions between AlcR and DNA common to the other known zinc clusters. In addition, the involvement of the N-terminal residues upstream of the AlcR zinc cluster in DNA binding is clearly highlighted, and the pivotal role of R6 is confirmed. Totally unprecedented specific and nonspecific contacts of two additional regions of the protein with the DNA are demonstrated. The differences with the available crystallographic structures of other zinc binuclear cluster proteins-DNA complexes are analyzed. CONCLUSIONS: The structures of the AlcR(1-60)-DNA complex provide the basis for a better understanding of some of the specificities of the AlcR system: the DNA consensus recognition sequence--usually the triplet CGG--is extended to five base pairs, AlcR acts as a monomer, and additional contacts inside and outside the DNA binding domain in the major and minor groove are observed. These extensive interactions stabilize the AlcR monomer to its cognate DNA site.

Differential chemical labeling of the AlcR DNA-binding domain from Aspergillas nidulans versus its complex with a 16-mer DNA target: identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid.

DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface. A similar result obtained with Trp53, an amino acid present at the C-terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.

15N NMR relaxation as a probe for helical intrinsic propensity: the case of the unfolded D2 domain of annexin I.

The isolated D2 domain of annexin I is unable to adopt a tertiary fold but exhibits both native and non-native residual structures. It thus constitutes an attractive model for the investigation of dynamics of partially folded states in the context of protein folding and stability. 15N relaxation parameters of the D2 domain have been acquired at three different magnetic fields, 500, 600 and 800 MHz. This enables the estimation of the contribution of conformational exchange to the relaxation parameters on the micro- to millisecond time scale, thus providing a suitable data set for the description of motions on the pico- and nanosecond time scale. The analysis of the seven spectral densities obtained (J(0), J(50 MHz), J(60 MHz), J(80 MHz), , , ) provides complementary and meaningful results on the conformational features of the D2 domain structure previously depicted by chemical shift and NOE data. Especially, residual helix segments exhibit distinct dynamical behaviors that are related to their intrinsic helical propensity. Beside the spectral density analysis, a series of models derived from the Lipari and Szabo model-free approach are investigated. Two models containing three parameters are able to reproduce equally well the experimental data within experimental errors but provide different values of order parameters and correlation times. The inability to find a unique model to describe the data emphasizes the difficulty to use and interpret the model-free parameters in the case of partially or fully unfolded proteins consisting of a wide range of interconverting conformers.

Pregnane glycosides from Caralluma russeliana.

The aerial parts of Caralluma russeliana yielded four new pregnane glycosides, russeliosides A-D (1-4), in addition to a known flavone glycoside, luteolin 4'-O-beta-D-neohesperidoside. The structures of compounds 1-4 were elucidated using a combination of spectroscopic methods.

NMR characterization of a kissing complex formed between the TAR RNA element of HIV-1 and a DNA aptamer.

This work presents the first structural analysis of an RNA-DNA complex consisting of an 18 nt RNA hairpin and a 20 nt DNA aptamer. The DNA molecule was previously selected, from a randomly synthesized library, against the transactivation response element (TAR) involved in transcriptional regulation of the HIV genome. The DNA aptamer used in the present study is an imperfect stem-loop with the sequence 5'-ACTCCCAT-3', characteristic of the selected candidates, in the apical loop. This octameric motif contains five bases complementary to the TAR loop sequence 5'-CUGGGA-3'. The use of homo- and heteronuclear NMR spectroscopy allowed assignment of the complex resonances and resolution of its secondary structure. Evidence is given for a kissing complex fold, which consists of a quasi-continuous helix formed by one stem of DNA, one stem of RNA and a central hybrid helix comprising 5 bp. Two out of helices residues of DNA and one of RNA connect the DNA-RNA loop-loop helix to the stem of either partner in the complex. In addition, two thymines of the DNA stem are engaged in a non-canonical T.T base pair.

NMR solution structure of AlcR (1-60) provides insight in the unusual DNA binding properties of this zinc binuclear cluster protein.

The three-dimensional structure of the DNA-binding domain (residues 1-60) of the ethanol regulon transcription factor AlcR from Aspergillus nidulans has been solved by NMR. This domain belongs to the zinc binuclear cluster class. Although the core of the protein is similar to previously characterized structures, consisting of two helices organized around a Zn(2)Cys(6 )motif, the present structure presents important variations, among them the presence of two supplementary helices. This structure gives new insight into the understanding of the AlcR specificities in DNA binding such as longer consensus half-sites, in vitro monomeric binding but in vivo multiple repeat transcriptional activation, either in direct or inverse orientations. The presence of additional contacts of the protein with its DNA target can be predicted from a model proposed for the interaction with the consensus DNA target. The clustering of accessible negative charges on helix 2 delineates a possible interaction site for other determinants of the transcriptional machinery, responsible for the fine tuning of the selection of the AlcR cognate sites.

A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases of higher eukaryotes possess polypeptide extensions in contrast to their prokaryotic counterparts. These extra domains of poorly understood function are believed to be involved in protein-protein or protein-RNA interactions. Here we showed by gel retardation and filter binding experiments that the repeated units that build the linker region of the bifunctional glutamyl-prolyl-tRNA synthetase had a general RNA-binding capacity. The solution structure of one of these repeated motifs was also solved by NMR spectroscopy. One repeat is built around an antiparallel coiled-coil. Strikingly, the conserved lysine and arginine residues form a basic patch on one side of the structure, presenting a suitable docking surface for nucleic acids. Therefore, this repeated motif may represent a novel type of general RNA-binding domain appended to eukaryotic aminoacyl-tRNA synthetases to serve as a cis-acting tRNA-binding cofactor.

New (15)N NMR exchange experiments for the unambiguous assignment of (1)H(N)/(15)N resonances of proteins in complexes in slow chemical exchange with free form.

The potentialities of a 2D proton-detected heteronuclear exchange experiment to assign the nitrogen and amide proton resonances in a uniformly (15)N-enriched macromolecule involved in a complex, starting from the free form assignments, are demonstrated on a protein-DNA complex. This 2D experiment is further extended to a 3D experiment in the case of severe superpositions.

Characterization of the interaction between bovine pancreatic trypsin inhibitor and thiocyanate by NMR.

The interaction between Bovine Pancreatic Trypsin Inhibitor and thiocyanate was studied using NMR spectroscopy following several experimental approaches. The chemical shift variations of the BPTI protons in the absence and in the presence of increasing thiocyanate concentrations (up to 0.2 M) were significant (> 0.05 ppm) for 30 protein protons belonging to 20 residues. The largest deviation, 0.2 ppm, was observed for the amide backbone proton of Arg42 in the absence of thiocyanate and in the presence of 40 molar equivalents of thiocyanate. The influence of the presence of thiocyanate on the electrostatic potential surrounding the protein was demonstrated by NOESY spectra selective at the water frequency: the presence of SCN- favours acid catalysed exchange and disfavours base catalysis. However, a specific effect of thiocyanate was pointed out since the comparison of the chemical shifts in the presence of 40 molar equivalents of KSCN and KCl, respectively, showed much more as well as larger deviations compared to measurements in the absence of salt. A dissociation constant, KD, for a 1/1 complex between BPTI and thiocyanate was calculated from chemical shifts measurements: KD = 89 +/- 8 mM. A second value, KD = 99 +/- 10 mM, was extracted from SC15N relaxation time measurements.

Dynamics of the DNA binding domain of the fructose repressor from the analysis of linear correlations between the 15N-1H bond spectral densities obtained by nuclear magnetic resonance spectroscopy.

The spectral densities of the backbone and arginine side chain NH bonds of the DNA binding domain of the fructose repressor (FruR) were extensively analyzed in order to extract reliable motions parameters. An accurate measurement of 15N NMR relaxation rates allowed their calculation at three frequencies, zero, omegaN, and omegaH + omegaN, using a reduced matrix approach. Linear correlations were found between J(omegaN) and J(0) and between and J(0). The analysis of the compatibility between the motions parameters obtained independently from the two correlation lines allowed further development of the linear correlation approach proposed recently [Lefèvre, J. F., Dayie, K. T., Peng, J. W., and Wagner, G. (1996) Biochemistry 35, 2674-2686]. The results demonstrate (i) the existence of a concerted motion along the whole backbone with a global correlation time equal to 5.95 ns.rad-1, and (ii) the presence of complex internal movements at an intermediate time scale around 1 ns. The extracted motion parameters have been related to those obtained with the extended Lipari and Szabo approach but are incompatible with those obtained using the usual simple Lipari and Szabo approach. They were correlated to the features of the NMR structure of FruR(1-57)*. Some residues in the turns and in the third helix experience slow motions in the micro- to millisecond time scale. Side-chain motions are not correlated to the backbone dynamics. A direct examination of spectral densities reveals a higher flexibility for the side chains of arginines that are not involved in ionic bridges.

Study of amide proton exchange in 15N-enriched cryptogein using pH-dependent off-resonance ROESY-HSQC experiments.

A new pH-dependent off-resonance ROESY-HSQC experiment has been used to characterize the degree of protection of the amide protons of cryptogein, a protein of the elicitin family, against solvent exchange. The study of the pH dependence of solvent-shielded amide protons in this protein reveals that the helices have different levels of stability. Two of the five helices exhibit strong protection of amide hydrogens against exchange with the solvent. By contrast, greater flexibility is observed in the other three helices, particularly in the C-terminal helix. These results provide information on the dynamic features of the protein and are consistent with the RMSD for the backbone atoms of residues involved in helical structures. In addition, the question of the flexibility in a hydrophobic cavity made of conserved residues, which represent a plausible binding site, is addressed by this method.

Determination of fast proton exchange rates of biomolecules by NMR using water selective diffusion experiments.

We present a new water selective pulse sequence allowing rapid determination of exchange rates of labile protons on the millisecond time scale. Using diffusion measurements, exchange rates of resolved protons can be determined without prior knowledge of relaxation parameters in a short overnight experiment. The use of a sensitive, highly selective and easy to implement water excitation scheme allows for its straightforward application to a wide range of biomolecules. The results obtained for the imino proton exchange rates of a 16 bp DNA are in strong agreement with values obtained by the classical approach of two-dimensional exchange spectroscopy.

The Aspergillus nidulans transcription factor AlcR forms a stable complex with its half-site DNA: a NMR study.

The Aspergillus nidulans transcription factor AlcR is shown by NMR and gel retardation assay to form a stable complex with oligonucleotide sequences comprising the consensus half-site 5'-TGCGG-3'. Apparent microM dissociation constants are evaluated by both methods. The measured lifetime of the complex is 74+/-7 ms at 20 degrees C with the following DNA sequence: 5'-C1G2T3G4C5G6G7A8T9C10-3'. The major chemical shift variations upon binding involve both the two adjacent GC pairs (G6 and G7) and, clearly, the AT pairs at both ends of the consensus sequence (T3 and A8), suggesting additional contacts of the protein with the DNA. This extensive and strong interaction with the half-site is another example of the variability in contacts of the fungal DNA-binding proteins containing Zn2Cys6 domains with their consensus sites. It is the first demonstration that a binuclear cluster protein can bind to DNA as a monomer with strong affinity.

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea.

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.

A study of protein-water exchange through the off-resonance ROESY experiment: application to the DNA-binding domain of AlcR.

In this communication a new NMR experiment for the safe observation and quantification of water-protein exchange phenomena is presented. It combines a water-selective pulse, offering chemical shift-based separation, and the off-resonance ROESY dynamic filter, which permits the elimination of the unwanted intramolecular dipolar cross relaxation of protein protons. Moreover, pulsed field gradients are used for the suppression of radiation damping and the solvent signal. The straightforward incorporation of this sequence in heteronuclear experiments is demonstrated for the case of the DNA-binding domain of the alcohol regulator protein.

Use of BOP-Cl in the presence of Boc-amino monothioacids for the thioacylation of imino acid residues.

BOP-Cl was found to be an efficient coupling reagent for the introduction of thiopeptide bonds on imino acid residues (Pro, Sar). Boc-amino monothioacids were coupled at moderate temperature (0 degree C-RT) with fair yields and with retained optical purity. The mechanism of the coupling reaction is discussed.