Dominique Gerard

Conformational behaviour of the active and inactive forms of the nucleocapsid NCp7 of HIV-1 studied by 1H NMR

The nucleocapsid protein NCp7 of the human immunodeficiency virus type I (HIV-1) is a 72 amino acid peptide containing two zinc fingers of the type CX2CX4HX4C linked by a short basic sequence 29RAPRKKG35. NCp7 was shown to activate in vitro both viral RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In order to clarify the possible structural role of the zinc fingers in the various functions of NCp7, complete sequence specific 1H NMR assignment of the entire protein was achieved by two-dimensional NMR experiments. Moreover, to characterize the role of the peptide linker in NCp7 folding, a synthetic analogue with an inversion of Pro31 configuration was studied by NMR and fluorescence techniques. Several long range NOEs implying amino acid protons from the folded zinc fingers and the spacer, such as Ala25 and Trp37, Phe16 and Trp37, Arg32 and Trp37, Lys33 and Trp37, Cys18 and Lys33 disappeared in the D-Pro31 (12-53)NCp7, confirming the spatial proximity of the two CCHC boxes observed in the (13-51)NCp7. This was also confirmed by iodide fluorescence quenching experiments. The N and C-terminal parts of NCp7 displayed a large flexibility except for two short sequences Tyr56 to Gly58 and Tyr64 to Gly66, which seemed to oscillate between random-coil and helical conformations. The biological relevance of the structural characteristics of NCp7 was studied in vitro and in vivo. Substitution of Pro31 by D-Pro31 in the active (13-64)NCp7 peptide led to a severe reduction of dimerization in vitro. Moreover, site-directed mutagenesis substituting Leu for Pro31 resulted in the formation of non-infectious and immature viral particles. These results suggest that the spatial proximity of the zinc fingers induced by the peptide linker, plays a critical role in encapsidation of genomic RNA and morphogenesis of HIV-1 infectious particles.

Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein

The nucleocapsid protein NCp7, which is the major genomic RNA binding protein of human immunodeficiency virus type 1, plays an important role in several key steps of the viral life cycle. Many of the NCp7 activities, notably the nucleic acid annealing and the genomic RNA wrapping ones, are thought to be linked to a nonspecific binding of NCp7 to its nucleic acid targets. The mechanism of these activities is still debated but several clues are in favor of an intermediate aggregation of nucleic acids by NCp7. To check and characterize the nucleic acid aggregating properties of NCp7, we investigated the interaction of NCp7 with the model RNA homopolymer, polyA, by quasielastic light scattering and optical density measurements. The ordered growth of monodisperse large particles independently of the nucleic acid size and the almost complete covering of polyA by NCp7 strongly suggested an ordered aggregation mechanism. The aggregate kinetics of growth in the optimum protein concentration range (> or = 2 microM) were governed by a so-called Ostwald ripening mechanism limited by transfer of NCp7-covered polyA complexes from small to large aggregates. The aggregation process was strongly dependent on both Na+ and Mg2+ concentrations, the optimum concentrations being in the physiological range. Similar conclusions held true when polyA was replaced by 16S + 23S ribosomal RNA, suggesting that the NCp7 aggregating properties were only poorly dependent on the nucleic acid sequence and structure. Finally, as in the NCp7 annealing activities, the basic regions of NCp7, but not the zinc fingers, were found critical in nucleic acid aggregation. Taken together, our data indicate that NCp7 is a highly efficient nucleic acid aggregating agent and strengthen the hypothesis that aggregation may constitute a transient step in various NCp7 functions.

Properties and growth mechanism of the ordered aggregation of a model RNA by the HIV-1 nucleocapsid protein: An electron microscopy investigation

NCp7, the nucleocapsid protein of the human immunodeficiency virus type 1, induces an ordered aggregation of RNAs, a mechanism that is thought to be involved in the NCp7-induced promotion of nucleic acid annealing. To further investigate this aggregation the morphology and the properties of the NCp7-induced aggregates of the model RNA homoribopolymer, polyA, were investigated by electron microscopy in various conditions. In almost all the tested conditions, the aggregates were spherical and consisted of a central dense core surrounded by a less dense halo made of NCp7-covered polyA molecules. The formation of these aggregates with a narrow distribution of sizes constitutes a distinctive feature of NCp7 over other single-stranded nucleic acid binding proteins. In most conditions, at the shortest times that can be reached experimentally, all the polyA molecules were already incorporated in small aggregates, suggesting that the nucleation step and the first aggregation events took place rapidly. The aggregates then orderly grew with time by fusion of the smaller aggregates to give larger ones. The aggregate halo was important in the fusion process by initiating the bridging between the colliding aggregates. In the presence of an excess of protein, the aggregates grew rapidly but were loosely packed and dissociated easily, suggesting adverse protein-protein interactions in the aggregates obtained in these conditions. In the presence of an excess of nucleotides, the presence of both amorphous nonspherical and slowly growing spherical aggregates suggested some changes in the mechanism of aggregate growth due to an incomplete covering of polyA molecules by NCp7. Finally, we showed that in the absence of added salt, the aggregate fusions were unfavored but not the initial events giving the first aggregates, the reverse being true in the presence of high salt concentrations (> or = 300 mM).

Terbium as luminescent probe of calmodulin calcium-binding sites: Domains I and II contain the high-affinity sites

Terbium has been shown to be a useful tool in the study of protein calcium binding sites [l]. Due to its ionic radius, its coordination number between 6 and 8 and its strong propensity for oxygen donor groups, this trivalent ion can replace calcium in many biological systems [2]. In addition, terbium has the convenient property of being highly luminescent when it binds to a protein close to an aromatic residue, as a result of a dipole-quadrupole energy-transfer process. By contrast the free ion is only poorly luminescent. Calmodulin, the ubiquitous and multifunctional calciumdependent regulator [3-5lexhibits 4 calcium binding sites among which sites III and IV contain one tyrosine residue each (tyrosine 99 in site III and tyrosine 138 in site IV [6]). No other tyrosyl residue is present in the molecule. Upon calcium binding, calmodulin undergoes a conformational transition which is necessary for its biological activity [7-91. Here, calmodulin conformational changes were induced by Ca*+ and Tb* and studied by monitoring tyrosine fluorescence, since calmodulin contains no tryptophan residues. Two calcium binding sites were shown to be involved in tyrosine fluorescence changes. A study of terbium emission indicates these high affinity sites to be sites I and II, in contrast to troponin C where the high affinity sites are sites III and IV.

Terbium binding to octopus calmodulin provides the complete sequence of ion binding

Calmodulin, the ubiquitous and multifunctional calcium-dependent regulator [ 1,2], exhibits 4 calciumbinding sites. For each of these sites, Ca*+ compete with Mg*” and K’ (J. Haiech, C. B. Klee, J. G. D., in preparation). Binding of Ca” to calmodulin induces a conformational change which was observed by different techniques, CD and UV difference spectra 131, NMR [4] or tyrosine fluorescence (151, M. C. K., J. G. D., D. G., in preparation}. Ca*-loaded calmodulin is able to interact with and activate a number of enzymes (reviewed [ 1,2]). It is still a matter of speculation whether all enzymes are activated by calmodulin when loaded with 4 Ca2+ or whether calmodulin can activate some enzymes after binding 2 or 3 Ca2+ only. It is therefore important to determine the sequence of ion binding to the 4 domains recognized in the primary structure of the protein [6]. Homologous calmodulins in spite of a very low rate of evolution [7] exhibit amino acid substitutions that may be useful in this respect. For instance, mammalian calmodulins contain two tyrosyl residues at positions 99 (domain HI) and 138 (domain IV) [6]. We have recently shown that ‘high affinity’ sites are sites I and II, since Tb3+ luminescence increases only after binding of 2 Tbs+ equiv~ents. The 2 first Tb3+ therefore bind to sites that lack tyrosine, namely domains I

A comparison of the fluorescence properties of TMA-DPH as a probe for plasma membrane and for endocytic membrane

In earlier studies, the fluorescence probe 1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene (TMA-DPH) was shown to interact with living cells by instantaneous incorporation into the plasma membrane, according to a water (probe not fluorescent)/membrane (probe highly fluorescent) partition equilibrium. This made it interesting both as a fluorescence anisotropy probe for plasma membrane fluidity determinations and as a quantitative tracer for endocytosis and intracellular membrane traffic. In order to ascertain the limiting concentrations for its use in these applications, we performed a systematic study of its fluorescence properties (intensity, lifetime, anisotropy) in the plasma membrane and in endocytic membranes of intact L929 mouse fibroblasts. Some of the experiments were repeated on mouse-bone-marrow-derived macrophages and on phospholipidic LUV to confirm the results. Rather unexpectedly, it was observed that: (i) the incorporation of TMA-DPH into the membranes, monitored by UV absorption measurements, remained proportional to the probe concentration over the wide range explored (5 x 10(-7) M-2.5 x 10(-5) M); (ii) however, concerning fluorescence, quenching effects occurred in the membranes above certain critical concentrations. These effects were shown to result from Förster-type resonance auto-transfer; (iii) strikingly, the critical concentrations were considerably higher in early-endocytic-vesicle membranes than in the bulk plasma membrane. It was established that membrane fluidity was involved and this was confirmed by the parallel study on phospholipidic vesicles. Potential applications of these properties as a novel approach for evaluating membrane fluidity are suggested.

Purification, characterization and ion binding properties of human brain S100b protein

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.

Zinc-dependent affinity chromatography of the S100b protein on phenyl—Sepharose: A rapid purification method

A rapid high resolution method of purification of the Trp‐containing S100 proteins (S100a, S100a′) and of the S100b protein has been developed. The principle of this method is based on the fact that S100b protein becomes highly hydrophobic upon Zn2+ binding, whereas S100a and S100a′ are not affected. On an affinity chromatography of phenyl—Sepharose column, S100b is selectively bound in presence of zinc, whereas the Trp‐containing S100 patients are quickly eluted. The S100b protein is further eluted with a buffer containing EDTA.

Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea‐sodium dodecyl sulfate‐polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal‐free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCI, rat brain S100b protein is characterized by two high‐affinity Ca2+ binding sites with a KD of 2 + 10−5M and four lower affinity sites with KD about 10−4M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low‐affinity calcium binding sites whereas similar Ca2+ ‐induced conformational changes were observed for both proteins. In the presence of 120 mM KCI rat brain S100b protein bound two Zn2+‐ ions/mol of protein with a KD of 10 −7M and four other with lower affinity (KD+ 10−6M). The occupancy of the two high‐affinity Zn2+ binding sites was responsible for most of the Zn2+‐induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed. Such results significantly differ from that obtained with bovine S100b protein, which on binding of eight Zn2+ equivalents/mol undergoes maximal conformational changes associated with a sixfold increase of the tyrosine fluorescence quantum yield. Finally, we report in this work that zinc binding on rat S100b protein regulates calcium binding by increasing the calcium affinity of the protein and reducing the antagonistic effect of K+ on calcium binding.

INFLUENCE OF THE LOCATION OF TRYPTOPHANYL RESIDUES IN PROTEINS ON THEIR PHOTOSENSITIVITY

The environmental effect on Trp residues photolysis was investigated on four proteins containing a single Trp residue in environments of various polarities: glucagon (exposed residue), nuclease (partially buried residue), RNase T1 (fully buried residue) and melittin (exposed or partially buried residue depending on the salt concentration). Direct photolysis was performed in neutral N2‐saturated phosphate solution at 20°C using 302 nm monochromatic light. Tryptophan loss was monitored by both absorption and fluorescence spectroscopy and by amino acid analysis. The results suggest that tryptophan photodegradation depends on the location of the residue in the protein, with regard to the exposure to the aqueous medium and to the neighbouring amino acids in the primary amino acid sequence and in the three dimensional structure. Photochemical products were not analysed but fluorescence spectra indicate that they vary with protein.