Probing water accessibility in HET-s(218-289) amyloid fibrils by solid-state NMR.

Despite the importance of protein fibrils in the context of conformational diseases, information on their structure is still sparse. Hydrogen/deuterium exchange measurements of backbone amide protons allow the identification hydrogen-bonding patterns and reveal pertinent information on the amyloid ß-sheet architecture. However, they provide only little information on the identity of residues exposed to solvent or buried inside the fibril core. NMR spectroscopy is a potent method for identifying solvent-accessible residues in proteins via observation of polarization transfer between chemically exchanging side-chain protons and water protons. We show here that the combined use of highly deuterated samples and fast magic-angle spinning greatly attenuates unwanted spin diffusion and allows identification of polarization exchange with the solvent in a site-specific manner. We apply this measurement protocol to HET-s(218-289) prion fibrils under different conditions (including physiological pH, where protofibrils assemble together into thicker fibrils) and demonstrate that each protofibril of HET-s(218-289), is surrounded by water, thus excluding the existence of extended dry interfibril contacts. We also show that exchangeable side-chain protons inside the hydrophobic core of HET-s(218-289) do not exchange over time intervals of weeks to months. The experiments proposed in this study can provide insight into the detailed structural features of amyloid fibrils in general.

Atomic-resolution three-dimensional structure of HET-s(218-289) amyloid fibrils by solid-state NMR spectroscopy.

We present a strategy to solve the high-resolution structure of amyloid fibrils by solid-state NMR and use it to determine the atomic-resolution structure of the prion domain of the fungal prion HET-s in its amyloid form. On the basis of 134 unambiguous distance restraints, we recently showed that HET-s(218-289) in its fibrillar state forms a left-handed ß-solenoid, and an atomic-resolution NMR structure of the triangular core was determined from unambiguous restraints only. In this paper, we go considerably further and present a comprehensive protocol using six differently labeled samples, a collection of optimized solid-state NMR experiments, and adapted structure calculation protocols. The high-resolution structure obtained includes the less ordered but biologically important C-terminal part and improves the overall accuracy by including a large number of ambiguous distance restraints.

A combined solid-state NMR and MD characterization of the stability and dynamics of the HET-s(218-289) prion in its amyloid conformation.

The three-dimensional structure of amyloid fibrils of the prion-forming part of the HET-s protein [HET-s(218-289)], as determined by solid-state NMR, contains rigid and remarkably well-ordered parts, as witnessed by the narrow solid-state NMR line widths for this system. On the other hand, high-resolution magic-angle-spinning (HRMAS) NMR results have shown that HET-s(218-289) amyloid fibrils contain highly flexible parts as well. Here, we further explore this unexpected behaviour using solid-state NMR and molecular dynamics (MD). The NMR data provide new information on order and dynamics in the rigid and flexible parts of HET-s(218-289), respectively. The MD study addresses whether or not small multimers, in an amyloid conformation, are stable on the 10 ns timescale of the MD run and provides insight into the dynamic parameters on the nanosecond timescale. The atom-positional, root-mean-squared fluctuations (RMSFs) and order parameters S(2) obtained are in agreement with the NMR data. A flexible loop and the N terminus exhibit dynamics on the ps-ns timescale, whereas the hydrophobic core of HET-s(218-289) is rigid. The high degree of order in the core region of HET-s(218-289) amyloids, as observed in the MD simulations, is in agreement with the narrow, solid-state, NMR lines. Finally, we employed MD to predict the behaviour of the salt-bridge network in HET-s(218-289), which cannot be obtained easily by experiment. Simulations at different temperatures indicated that the network is highly dynamic and that it contributes to the thermostability of the HET-s(218-289) amyloids.

Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex.

Transactivation-response element (TAR) is a stable stem-loop structure of HIV RNA, which plays a crucial role during the life cycle of the virus. The apical loop of TAR acts as a binding site for essential cellular cofactors required for the replication of HIV. High-affinity aptamers directed against the apical loop of TAR have been identified by the SELEX approach. The RNA aptamers with the highest affinity for TAR fold as hairpins and form kissing complexes with the targeted RNA through loop-loop interactions. The aptamers with the strongest binding properties all possess a GA base pair combination at the loop-closing position. Using liquid-crystal NMR methodology, we have obtained a structural model in solution of a TAR-aptamer kissing complex with an unprecedented accuracy. This high-resolution structure reveals that the GA base pair is unilaterally shifted toward the 5' strand and is stabilized by a network of intersugar hydrogen bonds. This specific conformation of the GA base pair allows for the formation of two supplementary stable base-pair interactions. By systematic permutations of the loop-closing base pair, we establish that the identified atomic interactions, which form the basis for the high stability of the complex, are maintained in several other kissing complexes. This study rationalizes the stabilizing role of the loop-closing GA base pairs in kissing complexes and may help the development or improvement of drugs against RNA loops of viruses or pathogens as well as the conception of biochemical tools targeting RNA hairpins involved in important biological functions.

Amyloid fibrils of the HET-s(218-289) prion form a beta solenoid with a triangular hydrophobic core.

Prion and nonprion forms of proteins are believed to differ solely in their three-dimensional structure, which is therefore of paramount importance for the prion function. However, no atomic-resolution structure of the fibrillar state that is likely infectious has been reported to date. We present a structural model based on solid-state nuclear magnetic resonance restraints for amyloid fibrils from the prion-forming domain (residues 218 to 289) of the HET-s protein from the filamentous fungus Podospora anserina. On the basis of 134 intra- and intermolecular experimental distance restraints, we find that HET-s(218-289) forms a left-handed beta solenoid, with each molecule forming two helical windings, a compact hydrophobic core, at least 23 hydrogen bonds, three salt bridges, and two asparagine ladders. The structure is likely to have broad implications for understanding the infectious amyloid state.

Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against beta-lactam antibiotics.

In absence of beta-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in beta-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to beta-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

Speeding up three-dimensional protein NMR experiments to a few minutes.

We demonstrate for different protein samples that three-dimensional HNCO and HNCA correlation spectra may be recorded in a few minutes acquisition time using the band-selective excitation short-transient sequences presented here. This opens new perspectives for the NMR structural investigation of unstable protein samples and real-time site-resolved studies of protein kinetics.

Resolution-enhanced base-type-edited HCN experiment for RNA.

New base-type-edited transverse-relaxation optimized CT-HCN(C) experiments are presented that yield intra-base and sugar-to-base correlations for 13C-15N labeled RNA. High spectral resolution in the 13C and 15N dimensions is achieved by constant time (CT) frequency editing. A spectral editing filter applied during the CT 15N labeling period separates the correlation peaks arising from G/U and A/C nucleotide bases. This provides the increased spectral resolution required to unambiguously connect the 1H and 13C resonances in sugar and base moieties of RNA nucleotides. In addition, the experiment allows base type identification for each residue, and therefore presents an attractive spectroscopic alternative to nucleotide-specific isotope labeling. Application to a 33-nucleotide RNA aptamer demonstrates the performance of the new pulse scheme.

Suppression of artifacts induced by homonuclear decoupling in amino-acid-type edited methyl 1H-13C correlation experiments.

A detailed theoretical and experimental analysis of the artifacts induced by homonuclear band-selective decoupling during CT frequency labeling is presented. The effects are discussed in the context of an amino-acid-type editing filter implemented in (1)H-(13)C CT-HSQC experiments of methyl groups in proteins. It is shown that both Bloch-Siegert shifts and modulation sidebands are efficiently suppressed by using additional off-resonance decoupling as proposed by Zhang and Gorenstein [J. Magn. Reson. 132 (1998) 81], and appropriate adjustment of a set of pulse sequence parameters. The theoretical predictions are confirmed by experiments performed on (13)C-labeled protein samples, yielding artifact-free amino-acid-type edited methyl spectra.

Guanidinium chloride denaturation of the dimeric Bacillus licheniformis BlaI repressor highlights an independent domain unfolding pathway.

The Bacillus licheniformis 749/I BlaI repressor is a prokaryotic regulator that, in the absence of a beta-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase. The BlaI repressor is composed of two structural domains. The 82-residue NTD (N-terminal domain) is a DNA-binding domain, and the CTD (C-terminal domain) containing the next 46 residues is a dimerization domain. Recent studies have shown the existence of the monomeric, dimeric and tetrameric forms of BlaI in solution. In the present study, we analyse the equilibrium unfolding of BlaI in the presence of GdmCl (guanidinium chloride) using different techniques: intrinsic and ANS (8-anilinonaphthalene-l-sulphonic acid) fluorescence, far- and near-UV CD spectroscopy, cross-linking, analytical ultracentrifugation, size exclusion chromatography and NMR spectroscopy. In addition, the intact NTD and CTD were purified after proteolysis of BlaI by papain, and their unfolding by GdmCl was also studied. GdmCl-induced equilibrium unfolding was shown to be fully reversible for BlaI and for the two isolated fragments. The results demonstrate that the NTD and CTD of BlaI fold/unfold independently in a four-step process, with no significant co-operative interactions between them. During the first step, the unfolding of the BlaI CTD occurs, followed in the second step by the formation of an 'ANS-bound' intermediate state. Cross-linking and analytical ultracentrifugation experiments suggest that the dissociation of the dimer into two partially unfolded monomers takes place in the third step. Finally, the unfolding of the BlaI NTD occurs at a GdmCl concentration of approx. 4 M. In summary, it is shown that the BlaI CTD is structured, more flexible and less stable than the NTD upon GdmCl denaturation. These results contribute to the characterization of the BlaI dimerization domain (i.e. CTD) involved in the induction process.

Amino acid-type edited NMR experiments for methyl-methyl distance measurement in 13C-labeled proteins.

New NMR experiments are presented for the measurement of methyl-methyl distances in (13)C-labeled proteins from a series of amino acid-type separated 2D or 3D NOESY spectra. Hadamard amino acid-type encoding of the proximal methyl groups provides the high spectral resolution required for unambiguous methyl-methyl NOE assignment, which is particularly important for fast global fold determination of proteins. The experiments can be applied to a wide range of protein systems, as exemplified for two small proteins, ubiquitin and MerAa, and the 30 kDa BRP-Blm complex.