Comparison between CUUG and UUCG tetraloops: thermodynamic stability and structural features analyzed by UV absorption and vibrational spectroscopy.

CUUG loop is one of the most frequently occurring tetraloops in bacterial 16S rRNA. This tetraloop has a high thermodynamic stability as proved by previous UV absorption and NMR experiments. Here, we present our results concerning the thermodynamic and structural features of the 10mer 5'-r(GCG-CUUG-CGC)-3', forming a highly stable CUUG tetraloop hairpin in aqueous solution, by means of several optical techniques (UV and FT-IR absorption, Raman scattering). UV melting profile of this decamer provides a high melting temperature (60.7 degrees C). A set of Raman spectra recorded at different temperatures allowed us to analyze the order-to-disorder (hairpin-to-random coil) transition. Assignment of vibrational markers led us to confirm the particular nucleoside conformation, and to get information on the base stacking and base pairing in the hairpin structure. Moreover, comparison of the data obtained from two highly stable CUUG and UUCG tetraloops containing the same nucleotides but in a different order permitted an overall discussion of their structural features on the basis of Raman marker evidences.

Investigating the conformational coupling between the transmembrane and cytoplasmic domains of a single-spanning membrane protein. A 1H-NMR study.

PMP1 is a 38-residue single-spanning membrane protein whose C-terminal cytoplasmic domain, Y25-F38, is highly positively charged. The conformational coupling between the transmembrane span and the cytoplasmic domain of PMP1 was investigated from 1H-nuclear magnetic resonance data of two synthetic fragments: F9-F38, i.e. 80% of the whole sequence, and Y25-F38, the isolated cytoplasmic domain. Highly disordered in aqueous solution, the Y25-F38 peptide adopts a well-defined conformation in the presence of dodecylphosphocholine micelles. Compared with the long PMP1 fragment, this structure exhibits both native and non-native elements. Our results make it possible to assess the influence of a hydrophobic anchor on the intrinsic conformational propensity of a cytoplasmic domain.

Concerted influence of key amino acids on the lipid binding properties of a single-spanning membrane protein: NMR and mutational analysis.

Finding the combinations of key amino acids involved in the interaction network underlying the interfacial features of membrane proteins would contribute to a better understanding of their sequence-structure-function relationships and the role of anionic phospholipids. To further address these questions, we performed mutational analysis associated with NMR experiments on synthetic fragments of the single-spanning membrane protein PMP1 that exhibit binding specificity for phosphatidylserine (PS). The aromatic and glutamine residues of the helix part of the PMP1 cytoplasmic domain were mutated. (1)H NMR experiments were carried out using perdeuterated DPC micelles as a membrane-like environment, in the absence and presence of small amounts of either POPC or POPS lipids. From intermolecular NOEs and chemical shift data, specific and nonspecific aspects of peptide-phospholipid interactions were distinguished. The major finding of our study is to reveal the concerted influence of a tryptophan and a glutamine residue on the interfacial conformation and lipid binding specificity of the PMP1 cytoplasmic domain.

Solution conformation of an RNA--DNA hybrid duplex containing a pyrimidine RNA strand and a purine DNA strand.

RNA--DNA hybrid duplexes are involved in transcription, replication and reverse transcription of nucleic acids. Information on such duplexes may shed some light on the mechanism of these processes. For this purpose, the influence of base composition on the structure of a polypyrimidine--polypurine RNA--DNA duplex r(cucuccuucucuu). d(GAGAGGAAGAGAA) has been studied using 1H, 31P and 13C NMR experiments, molecular modeling (JUMNA program) and NOE back-calculation methods. The resulting structure of the 13-mer hybrid duplex shows that the RNA strand is in the expected A-type conformation while the DNA strand is in a very flexible conformation. In the DNA strand, the desoxyribose sugars retain the C2'-endo B-type conformation. The duplex helical parameters (such as inclination, twist and displacement of the bases) are close to the A-type conformation. No bending was observed for the global axis curvature. The major groove width is close to the B-form value and the minor groove width is intermediate between standard values for A and B-forms. These results are in favour of the independence of minor groove size (where RNase H interacts) and the base composition of the hybrid duplexes.

Parallel and antiparallel G*G.C base triplets in pur*pur.pyr triple helices formed with (GA) third strands.

Triple helices with G*G.C and A*A.T base triplets with third GA strands either parallel or antiparallel with respect to the homologous duplex strand have been formed in presence of Na (+) or Mg(2+) counterions. Antiparallel triplexes are more stable and can be obtained even in presence of only monovalent Na(+) counterions. A biphasic melting has been observed, reflecting third strand separation around 20 degrees C followed by the duplex -> coil transition around 63 degrees C. Parallel triplexes are far less stable than the antiparallel ones. Their formation requires divalent ions and is observed at low temperature and in high concentration conditions. Different FTIR signatures of G*G.C triplets in parallel and antiparallel triple helices with GA rich third strands have been obtained allowing the identification of such base triplets in triplexes formed by nucleic acids with heterogeneous compositions. Only S-type sugars are found in the antiparallel triplex while some N-type sugar conformation is detected in the parallel triplex.

PMP1 18-38, a yeast plasma membrane protein fragment, binds phosphatidylserine from bilayer mixtures with phosphatidylcholine: a (2)H-NMR study.

PMP1 is a 38-residue plasma membrane protein of the yeast Saccharomyces cerevisiae that regulates the activity of the H(+)-ATPase. The cytoplasmic domain conformation results in a specific interfacial distribution of five basic side chains, thought to strongly interact with anionic phospholipids. We have used the PMP1 18-38 fragment to carry out a deuterium nuclear magnetic resonance ((2)H-NMR) study for investigating the interactions between the PMP1 cytoplasmic domain and phosphatidylserines. For this purpose, mixed bilayers of 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine (POPS) were used as model membranes (POPC/POPS 5:1, m/m). Spectra of headgroup- and chain-deuterated POPC and POPS phospholipids, POPC-d4, POPC-d31, POPS-d3, and POPS-d31, were recorded at different temperatures and for various concentrations of the PMP1 fragment. Data obtained from POPS deuterons revealed the formation of specific peptide-POPS complexes giving rise to a slow exchange between free and bound PS lipids, scarcely observed in solid-state NMR studies of lipid-peptide/protein interactions. The stoichiometry of the complex (8 POPS per peptide) was determined and its significance is discussed. The data obtained with headgroup-deuterated POPC were rationalized with a model that integrates the electrostatic perturbation induced by the cationic peptide on the negatively charged membrane interface, and a "spacer" effect due to the intercalation of POPS/PMP1f complexes between choline headgroups.

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn(2+) ions.

We present evidence of formation of an intramolecular parallel triple helix with T*A.T and G*G.C base triplets (where * represents the hydrogen bonding interaction between the third strand and the duplex while. represents the Watson-Crick interactions which stabilize the duplex). The third GT strand, containing seven GpT/TpG steps, targets the polypurine sequence 5'-AGG-AGG-GAG-GAG-3'. The triple helix is obtained by the folding back twice of a 36mer, formed by three dodecamers tethered by hydroxyalkyl linkers (-L-). Due to the design of the oligonucleotide, the third strand orientation is parallel with respect to the polypurine strand. Triple helical formation has been studied in concentration conditions in which native gel electrophoresis experiments showed the absence of intermolecular structures. Circular dichroism (CD) and UV spectroscopy have been used to evidence the triplex structure. A CD spectrum characteristic of triple helical formation as well as biphasic UV and CD melting curves have been obtained in high ionic strength NaCl solutions in the presence of Zn(2+) ions. Specific interactions with Zn(2+) ions in low water activity conditions are necessary to stabilize the parallel triplex.

The HIV-1(Lai) RNA dimerization. Thermodynamic parameters associated with the transition from the kissing complex to the extended dimer.

Retroviruses contain dimeric RNA consisting of two identical copies of the genomic RNA. The interaction between these two RNA molecules occurs near their 5' ends. A region upstream from the splice donor comprising an auto-complementary sequence has been identified as being responsible for the initiation of the formation of dimeric HIV-1(Lai) RNA. This region (SL1), part of the PSI encapsidation domain, can adopt a stem-loop structure. It has already been shown that this stem-loop structure can initiate the formation of two distinct dimers differing in their thermostability: a loop-loop dimer or 'kissing complex' and an extended dimer. We report here a study using UV and 1D NMR spectroscopy of the dimerization of a short oligoribonucleotide (23 nucleotides) spanning nucleotides 248-270 of the HIV-1(Lai) SL1 sequence, in order to derive the thermodynamic parameters associated with the transition from the loop-loop complex to the extended dimer. The temperature dependence of the UV absorbency shows an hypochromicity for this transition with a small enthalpy change equal to - 29.4 +/- 5 kcal x mol-1, together with a concentration independent transition which implies a monomolecular reaction. On the other hand, our NMR results don't indicate a dissociation of the GCGCGC sequence engaged in the loop-loop interaction during the rearrangement of the loop-loop complex into the extended dimer. Our data suggest that the loop-loop interaction is maintained during the temperature dependent conformational change while the intramolecular base-pairing of the stems is disrupted and then reconstituted to form an intermolecular base-pairing leading to an extended dimer.

Targeting of Pu.Py Duplexes by GA and GT Rich Oligonucleotides on Microchip and in Solution.

Abstract Formation of triple helices with GA and GT third strands has been studied. Besides the usual investigation techniques common for characterizing triple helical formation (CD spectroscopy, gel shift mobility assay, chemical probing and S1 nuclease footprinting) we have used a new technique in which targeting of polypurine sequences in duplexes was demonstrated on oligonucleotide microchips. This technique is very successful to quickly test a large number of potential triple helix formation. In this work we used oligonucleotide microairay to study the specificity of DNA duplex recognition by GA and GT strands. Generic 6-mer microchip containing all possible 4(6) = 4,096 single-stranded hexadeoxyribonucleotides immobilized within individual gel pads was applied. To study formation of intermolecular triple helices on the generic microchip, a number of Pu.Py duplexes were formed by hybridization of the mixture of purine octadeoxyribonucleotides on the microchip followed by targeting of the duplexes by GA or GT third strands. Melting behavior of the formed structures was investigated using fluorescence measurements under microscope. In solution we present the results obtained for GT triplexes and discuss the characteristics of the CD spectra. Results obtained by S1 nuclease footprinting, KMnO(4) and DMS chemical probing are consistent with the spectroscopic data reflecting triplex structure formation.

Dimer initiation sequence of HIV-1Lai genomic RNA: NMR solution structure of the extended duplex.

The genome of all retrovirus consists of two copies of genomic RNA which are noncovalently linked near their 5' end. A sequence localized immediately upstream from the splice donor site inside the HIV-1 psi-RNA region was identified as the domain responsible for the dimerization initiation. It was shown that a kissing complex and a stable dimer are both involved in the HIV-1Lai RNA dimerization process in vitro. The NCp7 protein activates the dimerization by converting a transient loop-loop complex into a more stable dimer. The structure of this transitory loop-loop complex was recently elucidated by Mujeeb et al. In work presented here, we use NMR spectroscopy to determine the stable extended dimer structure formed from a 23 nucleotides RNA fragment, part of the 35 nucleotides SL1 sequence. By heating to 90 degrees C, then slowly cooling this sequence, we were able to show that an extended dimer is formed. We present evidence for the three dimensional structure of this dimer. NMR data yields evidence for a zipper like motif A8A9.A16 existence. This motif enables the surrounding bases to be positioned more closely and permit the G7 and C17 bases to be paired. This is different to other related sequences where only the kissing complex is observed, we suggest that the zipper like motif AA.A could be an important stabilization factor of the extended duplex.

Water and ions in a high resolution structure of B-DNA.

A detailed picture of hydration and counterion location in the B-DNA duplex d(GCGAATTCG) is presented. Detailed data have been obtained by single crystal x-ray diffraction at atomic resolution (0.89 A) in the presence of Mg(2+). The latter is the highest resolution ever obtained for a B-DNA oligonucleotide. Minor groove hydration is compared with that found in the Na(+) and Ca(2+) crystal forms of the related dodecamer d(CGCGAATTCGCG). High resolution data (1.45 A) of the Ca(2+) form obtained in our laboratory are used for that purpose. The central GAATTC has a very stable hydration spine identical in all cases, independent of duplex length and crystallization conditions (counterions, space group). However, the organization of the water molecules (tertiary and quaternary layers) associated with the central spine vary in each case.

Structure of the oligonucleotide d(CGTATATACG) as a site-specific complex with nickel ions.

In this paper we explore the application of Ni2+to the crystallization of oligonucleotides. We have determined in this way the structure of a fully alternating (Y-R) decanucleotide d(CGTATATACG) by single crystal X-ray diffraction. This is the first oligonucleotide crystal structure with an alternating 5'-(TA)3-3' central part. Alternating oligonucleotides have a particular interest since they often have a unique structure. In this case the general conformation is B-like with an alternating twist and an end-to-end interaction which involves terminal guanines. The crystal belongs to space group P41212 with a = b = 52.46, c = 101.49 A. This packing imposes a 90 degrees crossing of the symmetry related helices. This is a new way of packing for decamers. The oligonucleotide structure is characterized by the specific association with seven nickel ions, involving the N7 atom of every guanine. One of the Ni2+ions is shared between two guanines of symmetry related molecules. Until now no oligonucleotide has been crystallized in the presence of this metal ion. A novel C.A.T triplet structure has also been tentatively identified.

Unusual nucleotide conformations in GNRA and UNCG type tetraloop hairpins: evidence from Raman markers assignments.

High resolution NMR data on UNCG and GNRA tetraloops (where N is any of the four nucleotides and R is a purine) have shown that they contain ribonucleosides with unusual 2'-endo/anti and 3'-endo/syn conformations, in addition to the 3'-endo/anti ones which are regularly encountered in RNA chains. In the current study, Raman spectroscopy has been used to probe these nucleoside conformations and follow the order (hairpin) to disorder (random chain) structural transitions in aqueous phase in the 5-80 degreesC temperature range. Spectral evolution of GCAA and GAAA tetraloops, as formed in very short hairpins with only three G.C base pairs in their stems (T m >60 degreesC), are reported and compared with those previously published on UUCG and UACG tetraloops, for which the syn orientation of the terminal guanine as well as the 2'-endo/anti conformation of the third rC residue have been confirmed by means of vibrational marker bands. Raman data obtained as a function of temperature show that the first uracil in the UUCG tetraloop is stacked and the two middle residues (rU and rC) are in the 2'-endo/anti conformation, in agreement with the previously published NMR results. As far as the new data concerning the GNRA type tetraloops are concerned, they lead us to conclude that: (i) in both cases (GCAA and GAAA tetraloops) the adenine bases are stacked; (ii) the second rC residue in the GCAA tetraloop has a 3'-endo/anti conformation; (iii) the sugar pucker associated with the third rA residue in both tetraloops possibly undergoes a 3'-endo/2'-endo interconversion as predicted by NMR results; (iv) the stem adopts a regular A-form structure; (v) all other nucleosides of these two GNRA tetraloops possess the usual 3'-endo/anti conformation.

Structure of the d(CGCCCGCGGGCG) dodecamer: a kinked A-DNA molecule showing some B-DNA features.

We have determined the structure of the dodecamer duplex d(CGCCCGCGGGCG)2. A careful use of the molecular replacement programme AMoRe has been essential in order to solve the structure. This dodecamer shows a unique conformation, quite different from all the previously studied oligonucleotide duplexes: the central octamer has an A conformation, but with a sharp 65 degrees kink in the centre; the terminal base-steps have a B-like conformation; the major groove is completely closed in the centre, a hollow molecule is thus found. The results obtained confirm the high degree of variability of DNA structure. A new type of kink and an intermediate A/B double-helical conformation have been found. Such intermediate conformation differs from those described in DNA polymerase complexes.

The structure of the most studied DNA fragment changes under the influence of ions: a new packing of d(CGCGAATTCGCG).

The title oligonucleotide and many related dodecamers have been extensively studied alone and as DNA-drug complexes. In practically all cases they were found to crystallize in the same space group, stabilized by interactions among the terminal guanine bases. Here we report new packing interactions (R3) in the presence of Ca2+. The oligonucleotides interact by placing their terminal guanines in the narrow groove of a neighbor molecule, an interaction which had never been found in dodecamers.

Structure of the complex between the HIV-1 nucleocapsid protein NCp7 and the single-stranded pentanucleotide d(ACGCC).

The nucleocapsid protein NCp7 of HIV-1 Mal contains two successive Zn knuckles of the CX2CX4HX4C type and plays a major role in virion morphogenesis, genomic RNA packaging and viral infectivity, mainly through single-stranded nucleic acid binding. We report here the study by 1H 2D NMR of the complex formed between the (12-53)NCp7, encompassing the two Zn knuckles, and d(ACGCC), a deoxynucleotide sequence analog corresponding to the shortest NCp7 binding site. Ten structures of the (12-53)NCp7/d(ACGCC) complex have been obtained from 607 NOE-derived distance constraints, 28 of which are intermolecular, and from molecular dynamics studies. The oligonucleotide is almost perpendicular to the sequence linking the two Zn knuckles. The Trp37 indole ring is inserted between the C2 and G3 bases and stacked on the latter. The complex is stabilized by hydrophobic interactions and hydrogen bonds, and accounts for the observed loss of virus infectivity induced by mutations in the Zn knuckle domain. Thus, the interaction between d(ACGCC) and the inactive mutant Cys23 (12-53)NCp7 was found by NMR to be completely different from that observed with the wild-type peptide. A mechanism of action for NCp7 in virus morphogenesis and replication is proposed from these results, which could facilitate the design of possible antiviral agents acting by a new mechanism.

1H- and 2H-NMR studies of a fragment of PMP1, a regulatory subunit associated with the yeast plasma membrane H(+)-ATPase. Conformational properties and lipid-peptide interactions.

PMP1 is a 38-residue polypeptide associated with the yeast plasma membrane H(+)-ATPase, found to regulate the enzyme activity. To investigate the molecular basis of the PMP1 biological function, the conformational properties of a synthetic PMP1 fragment, A18-F38, comprising the predicted C-terminal cytoplasmic domain and a part of the transmembrane anchor have been studied by 1H- and 2H-NMR spectroscopies. High resolution 1H-NMR experiments showed that, in deuterated DPC micelles, the A18-G34 segment adopts a well defined helix conformation. Our data suggest that the whole PMP1 molecule forms a unique helix whose axis might be slightly tilted with respect to the bilayer normal. Protonated DPC, DMPC and DMPS were incorporated in deuterated micelles containing the PMP1 fragment for studying lipid-peptide interactions. Unusually strong and selective intermolecular NOEs between lipid chain and peptide side chain protons, especially those of the unique Trp residue, were observed. Solid state 2H-NMR experiments performed on pure deuterated POPC and mixed deuterated POPC:POPS (5:1) bilayers revealed that the PMP1 fragment specifically interacts with negatively charged PS lipids.

A conformational equilibrium in a protein fragment caused by two consecutive capping boxes: 1H-, 13C-NMR, and mutational analysis.

The conformational properties of an 18 residues peptide spanning the entire sequence, L1KTPA5QFDAD10ELRAA15MKG, of the first helix (A-helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two successive potential capping boxes, T3xxQ6 and D8xxE11. The former corresponds to the native hydrogen bond network stabilizing the alpha helix N-terminus in the protein; the latter is a non-native capping box able to break the helix at residue D8, and is observed in the domain 2 partially folded state. Using 2D-NMR techniques, we showed that two main populations of conformers coexist in aqueous solution. The first corresponds to a single helix extending from T3 to K17. The second corresponds to a broken helix at residue Ds. Four mutants, T3A, F7A, D8A, and E11A, were designed to further analyze the role of key amino acids in the equilibrium between the two ensembles of conformers. The sensitivity of NMR parameters to account for the variations in the populations of conformers was evaluated for each peptide. Our data show the delta13Calpha chemical shift to be the most relevant parameter. We used it to estimate the population ratio in the various peptides between the two main ensembles of conformers, the full helix and the broken helix. For the WT, E11A, and F7A peptides, these ratios are respectively 35/65, 60/40, 60/40. Our results were compared to the data obtained from helix/coil transition algorithms.

Structural features of the UCCG and UGCG tetraloops in very short hairpins as evidenced by optical spectroscopy.

Structures of the UCCG and UGCG tetraloops formed in octamer ribonucleotidic hairpin sequences, i.e., 5'-r[GC(UCCG)GC]-3' and 5'-r[GC(UGCG)GC]-3', have been studied in aqueous solution by methods of optical spectroscopy. UV absorption melting profiles of these short hairpins, containing only two closing GC base pairs in the stem, are consistent with a monophasic, completely reversible order-to-disorder transition and clearly confirm their unusual structural stability (with Tm congruent with 50 degrees C). To establish structural characteristics of these tetraloops, Raman and FTIR spectroscopies have been used and vibrational conformation markers arising from the phosphate backbone and various nucleosides have been analyzed. They have been assigned on the basis of known unambiguous vibrational markers established for DNA and RNA chains. Surprisingly, they are easily transferable to short oligonucleotidic sequences. Intensities and wavenumbers of these conformation markers have been monitored in the 0-70 degrees C temperature range, i.e., in going from an ordered to a disordered structure. The main structural features of the UCCG and UGCG tetraloops are similar to those previously found in the UUCG and UACG tetraloops by means of NMR and vibrational spectroscopies, except those of the second nucleosides of the tetraloops (rC and rG, respectively) which adopt a 3'-endo/anti rather than a 2'-endo/anti conformation.

Exploring the folding pathways of annexin I, a multidomain protein. I. non-native structures stabilize the partially folded state of the isolated domain 2 of annexin I.

Proteins of the annexin family constitute very attractive models because of their four approximately 70 residue domains, D1 to D4, exhibiting an identical topology comprising five helix segments with only a limited sequence homology of approximately 30%. We focus on the isolated D2 domain, which is only partially folded. A detailed analysis of this equilibrium partially folded state in aqueous solution and micellar solution using 15N-1H multidimensional NMR is presented. Comparison of the residual structure of the entire domain with that of shorter fragments indicates the presence of long-range transient hydrophobic interactions that slightly stabilize the secondary structure elements. The unfolded domain tends to behave as a four-helix, rather than as a five-helix domain. The ensemble of residual structures comprises: (i) a set of native structures consisting of three regions with large helix populations, in rather sharp correspondence with A, B and E helices, and a small helix population in the second part of the C helix; (ii) a set of non-native local structures corresponding to turn-like structures stabilized by several side-chain to side-chain interactions and helix-disruptive side-chains to backbone interactions. Remarkably, residues involved in these local non-native interactions are also involved, in the native structure, in structurally important non-local interactions. During the folding process of annexin I, the local non-native interactions have to switch to native long-range interactions. This structural switch reveals the existence of a sequence-encoded regulation of the folding pathways and kinetics, and emphasizes the key role of the non-native local structures in this regulation.