A Set of Efficient nD NMR Protocols for Resonance Assignments of Intrinsically Disordered Proteins.

The RF pulse scheme RN[N-CA HEHAHA]NH, which provides a convenient approach to the acquisition of different multidimensional chemical shift correlation NMR spectra leading to backbone resonance assignments, including those of the proline residues of intrinsically disordered proteins (IDPs), is experimentally demonstrated. Depending on the type of correlation data required, the method involves the generation of in-phase ((15) N)(x) magnetisation via different magnetisation transfer pathways such as H→N→CO→N, HA→CA→CO→N, H→N→CA→N and H→CA→N, the subsequent application of (15) N-(13) C(α) heteronuclear Hartmann-Hahn mixing over a period of ≈100 ms, chemical-shift labelling of relevant nuclei before and after the heteronuclear mixing step and amide proton detection in the acquisition dimension. It makes use of the favourable relaxation properties of IDPs and the presence of (1) JCαN and (2) JCαN couplings to achieve efficient correlation of the backbone resonances of each amino acid residue "i" with the backbone amide resonances of residues "i-1" and "i+1". It can be implemented in a straightforward way through simple modifications of the RF pulse schemes commonly employed in protein NMR studies. The efficacy of the approach is demonstrated using a uniformly ((15) N,(13) C) labelled sample of α-synuclein. The different possibilities for obtaining the amino-acid-type information, simultaneously with the connectivity data between the backbone resonances of sequentially neighbouring residues, have also been outlined.

MAS solid state NMR of proteins: simultaneous ¹5N- ¹³CA and ¹5N- ¹³CO dipolar recoupling via low-power symmetry-based RF pulse schemes.

The generation of efficient RN n (ν)s,(ν)k symmetry-based low-power RF pulse schemes for simultaneous (15)N-(13)CA and (15)N-(13)CO dipolar recoupling is demonstrated. The method involves mixing schemes employing phase and amplitude-modulated dual band-selective 180° pulses as basic "R" element and tailoring of the RF field-modulation profile of the 180° pulses so as to obtain efficient magnetisation transfer characteristics over the resonance offset range of the nuclei involved. Mixing schemes leading to simultaneous (15)N-(13)CA and (15)N-(13)CO dipolar recoupling would permit the one-shot acquisition of different chemical shift correlation spectra that are typically utilized for protein backbone resonance assignments and thereby save data acquisition time. At representative MAS frequencies the efficacies of the mixing schemes presented here have been experimentally demonstrated via the simultaneous acquisition of {3D CONH and 3D CANH}, {3D CONH and 3D CO(CA)NH} and {3D CONH, 3D CANH, 3D CO(CA)NH and 3D CA(CO)NH} spectra generated via the magnetisation transfer pathways (1)H → (13)CO → (15)N → (1)H (CONH), (1)H → (13)CA → (15)N → (1)H (CANH) and (1)H → (13)CO → (13)CA → (15)N → (1)H (CO(CA)NH) and (1)H → (13)CA → (13)CO → (15)N → (1)H (CA(CO)NH).

HN-NCA heteronuclear TOCSY-NH experiment for (1)H(N) and (15)N sequential correlations in ((13)C, (15)N) labelled intrinsically disordered proteins.

A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue 'i' with that of residues 'i-1' and 'i+1' in ((13)C, (15)N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploits the favourable relaxation properties of IDPs and the presence of (1) J CαN and (2) J CαN couplings to transfer the (15)N x magnetisation from amino acid residue 'i' to adjacent residues via the application of a band-selective (15)N-(13)C(α) heteronuclear cross-polarisation sequence of ~100 ms duration. Employing non-uniform sampling in the indirect dimensions, the efficacy of the approach has been demonstrated by the acquisition of 3D HNN chemical shift correlation spectra of α-synuclein. The experimental performance of the RF pulse sequence has been compared with that of the conventional INEPT-based HN(CA)NH pulse scheme. As the availability of data from both the HCCNH and HNN experiments will make it possible to use the information extracted from one experiment to simplify the analysis of the data of the other and lead to a robust approach for unambiguous backbone and side-chain resonance assignments, a time-saving strategy for the simultaneous collection of HCCNH and HNN data is also described.

An approach to NMR assignment of intrinsically disordered proteins.

An efficient approach to NMR assignments in intrinsically disordered proteins is presented, making use of the good dispersion of cross peaks observed in [(15) N,(13) C']- and [(13) C',(1) H(N) ]-correlation spectra. The method involves the simultaneous collection of {3D (H)NCO(CAN)H and 3D (HACA)CON(CA)HA} spectra for backbone assignments via sequential H(N) and H(α) correlations and {3D (H)NCO(CACS)HS and 3D (HS)CS(CA)CO(N)H} spectra for side-chain (1) H and (13) C assignments, employing sequential (1) H data acquisitions with direct detection of both the amide and aliphatic protons. The efficacy of the approach for obtaining resonance assignments with complete backbone and side-chain chemical shifts is demonstrated experimentally for the 61-residue [(13) C,(15) N]-labelled peptide of a voltage-gated potassium channel protein of the Kv1.4 channel subunit. The general applicability of the approach for the characterisation of moderately sized globular proteins is also demonstrated.

An approach to sequential NMR assignments of proteins: application to chemical shift restraint-based structure prediction.

A procedure for the simultaneous acquisition of {HNCOCANH & HCCCONH} chemical shift correlation spectra employing sequential [Formula: see text] data acquisition for moderately sized proteins is presented. The suitability of the approach for obtaining sequential resonance assignments, including complete [Formula: see text] and [Formula: see text] chemical shift information, is demonstrated experimentally for a [Formula: see text] and [Formula: see text] labelled sample of the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus. The chemical shift information obtained was used to calculate the global fold of this winged helix domain via CS-Rosetta. This demonstrates that our procedure provides a reliable and straight-forward protocol for a quick global fold determination of moderately-sized proteins.

Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets.

NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23 kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ("scrambling") of NH and CO groups in a standard Escherichia coli expression host is provided.

Structure-function relationship in an archaebacterial methionine sulphoxide reductase B.

Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra.

We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC'C and 3D C'NCA with sequential (13)C acquisitions, 3D NHH and 3D NC'H with sequential (1)H acquisitions and 3D CANH and 3D C'NH with broadband (13)C-(15)N mixing are demonstrated using microcrystalline samples of the ß1 immunoglobulin binding domain of protein G (GB1) and the chicken α-spectrin SH3 domain.

NMR of intrinsically disordered proteins: A note on the application of 15N-13Cα het-TOCSY mixing for 13Cα magnetisation transfers.

Intrinsically disordered proteins (IDPs) or protein regions represent functionally important biomolecules without unique structure. Their inherent flexibility prevents high-resolution structure determination by X-ray or cryo-EM methods. In contrast, NMR spectroscopy provides an extensive and still growing set of experimental approaches to obtain detailed information on structure and dynamics of IDPs. Here, it is experimentally demonstrated that 15N-13Cα band-selective heteronuclear cross-polarisation that has been successfully employed recently to achieve the efficient transfer of 15Nx magnetisation from amino acid residue 'i' to 'i + 1' and 'i - 1' residues in uniformly (15N,13C)-labelled intrinsically disordered proteins can also be applied to transfer, without significant relaxation losses, 13Cαx magnetisation from an amino acid residue to its neighbouring residues. The possibility to obtain in one-shot correlation spectra arising from the simultaneous transfer of 15Nx and 13Cαx magnetisations from an amino acid residue to neighbouring residues is also demonstrated.

Chemical shift correlation at high MAS frequencies employing low-power symmetry-based mixing schemes.

An approach for conveniently implementing low-power CN ( n ) (ν) and RN ( n ) (ν) symmetry-based band-selective mixing sequences for generating homo- and heteronuclear chemical shift correlation NMR spectra of low γ nuclei in biological solids is demonstrated. Efficient magnetisation transfer characteristics are achieved by selecting appropriate symmetries requiring the application of basic RF elements of relatively long duration and numerically tailoring the RF field modulation profile of the basic element. The efficacy of the approach is experimentally shown by the acquisition of (15)N-(13)C dipolar and (13)C-(13)C scalar and dipolar coupling mediated chemical shift correlation spectra at representative MAS frequencies.

Broadband 15N-13C dipolar recoupling via symmetry-based RF pulse schemes at high MAS frequencies.

An approach for generating efficient NR(vS, vk)(n) symmetry-based dual channel RF pulse schemes for gamma-encoded broadband (15)N-(13)C dipolar recoupling at high magic angle spinning frequencies is presented. The method involves the numerical optimisation of the RF phase-modulation profile of the basic "R" element so as to obtain heteronuclear double quantum dipolar recoupling sequences with satisfactory magnetisation transfer characteristics. The basic "R" element was implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by a RF phase and amplitude values. The performance characteristics of the sequences were evaluated via numerical simulations and (15)N-(13)C chemical shift correlation experiments. Employing such (13)C-(15)N double-quantum recoupling sequences and the multiple receiver capabilities available in the current generation of NMR spectrometers, the possibility to simultaneously acquire 3D NCC and CNH chemical shift correlation spectra is also demonstrated.

Solution structure of stem-loop alpha of the hepatitis B virus post-transcriptional regulatory element.

Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regulatory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs, contains a splicing regulatory element and resides in the 3'-region of all viral transcripts. The HPRE consists of three sub-elements alpha (nucleotides 1151-1346), beta1 (nucleotides 1347-1457) and beta2 (nucleotides 1458-1582), which confer together full export competence. Here, we present the NMR solution structure (pdb 2JYM) of the stem-loop alpha (SLalpha, nucleotides 1292-1321) located in the sub-element alpha. The SLalpha contains a CAGGC pentaloop highly conserved in hepatoviruses, which essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLalpha harbours a single bulged G residue flanked by A-helical regions. The structure is highly suggestive of serving two functions in the context of export of unspliced viral RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or preventing the utilization of a 3'-splice site contained within SLalpha.

Numerical design of RN(n)(nu) symmetry-based RF pulse schemes for recoupling and decoupling of nuclear spin interactions at high MAS frequencies.

An approach for the efficient implementation of RN(n)(nu) symmetry-based pulse schemes that are often employed for recoupling and decoupling of nuclear spin interactions in biological solid state NMR investigations is demonstrated at high magic-angle spinning frequencies. RF pulse sequences belonging to the RN(n)(nu) symmetry involve the repeated application of the pulse sandwich {R(phi)R(-phi)}, corresponding to a propagator U(RF) = exp(-i4phiI(z)), where phi = pinu/N and R is typically a pulse that rotates the nuclear spins through 180 degrees about the x-axis. In this study, broadband, phase-modulated 180 degrees pulses of constant amplitude were employed as the initial 'R' element and the phase-modulation profile of this 'R' element was numerically optimised for generating RN(n)(nu) symmetry-based pulse schemes with satisfactory magnetisation transfer characteristics. At representative MAS frequencies, RF pulse sequences were implemented for achieving 13C-13C double-quantum dipolar recoupling and through bond scalar coupling mediated chemical shift correlation and evaluated via numerical simulations and experimental measurements. The results from these investigations are presented here.

Design of high-power, broadband 180 degrees pulses and mixing sequences for fast MAS solid state chemical shift correlation NMR spectroscopy.

An approach for the design of high-power, broadband 180 degrees pulses and mixing sequences for generating dipolar and scalar coupling mediated (13)C-(13)C chemical shift correlation spectra of isotopically labelled biological systems at fast magic-angle spinning frequencies without (1)H decoupling during mixing is presented. Considering RF field strengths in the range of 100-120 kHz, as typically available in MAS probes employed at high spinning speeds, and limited B (1) field inhomogeneities, the Fourier coefficients defining the phase modulation profile of the RF pulses were optimised numerically to obtain broadband inversion and refocussing pulses and mixing sequences. Experimental measurements were carried out to assess the performance characteristics of the mixing sequences reported here.

Recoupling and decoupling of nuclear spin interactions at high MAS frequencies: numerical design of CN(n)(nu) symmetry-based RF pulse schemes.

The CN(n)(nu) class of RF pulse schemes, commonly employed for recoupling and decoupling of nuclear spin interactions in magic angle spinning solid state NMR studies of biological systems, involves the application of a basic "C" element corresponding to an RF cycle with unity propagator. In this study, the design of CN(n)(nu) symmetry-based RF pulse sequences for achieving 13C-13C double-quantum dipolar recoupling and through bond scalar coupling mediated 13C-13C chemical shift correlation has been examined at high MAS frequencies employing broadband, constant-amplitude, phase-modulated basic "C" elements. The basic elements were implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by an RF phase value. The phase-modulation profile of the "C" element was optimised numerically so as to generate efficient RF pulse sequences. The performances of the sequences were evaluated via numerical simulations and experimental measurements and are presented here.

NMR experiments on the transient interaction of the intrinsically disordered N-terminal peptide of cystathionine-ß-synthase with heme.

The N-terminal segment of human cystathionine-ß-synthase (CBS(1-40)) constitutes an intrinsically disordered protein stretch that transiently interacts with heme. We illustrate that the HCBCACON experimental protocol provides an efficient alternative approach for probing transient interactions of intrinsically disordered proteins with heme in situations where the applicability of the conventional [1H, 15N]-HSQC experiment may be limited. This experiment starting with the excitation of protein side chain protons delivers information about the proline residues and thereby makes it possible to use these residues in interaction mapping experiments. Employing this approach in conjunction with site-specific mutation we show that transient heme binding is mediated by the Cys15-Pro16 motif of CBS(1-40).

1H, 13C, and 15N resonance assignments of the cytokine interleukin-36ß isoform-2.

Interleukins are cytokines performing central tasks in the human immune system. Interleukin-36ß (IL-36ß) is a member of the interleukin-1 superfamily as are its homologues IL-36α and IL-36γ. All of them interact with a common receptor composed of IL-36R and IL-1R/acP. IL-36 cytokines can activate IL-36R to proliferation of CD4 + lymphocytes or stimulate M2 macrophages as potently as IL-1ß. Within our efforts to study the structure-function relationship of the three interleukins IL-36α, IL-36ß and IL-36γ by heteronuclear multidimensional NMR, we here report the 1H, 13C, and 15N resonance assignments for the backbone and side chain nuclei of cytokine interleukin-36ß isoform-2.

Structural insights into heme binding to IL-36α proinflammatory cytokine.

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins.

Heme interaction of the intrinsically disordered N-terminal peptide segment of human cystathionine-ß-synthase.

Cystathionine-ß-synthase (CBS) belongs to a large family of pyridoxal 5'-phosphate (PLP)-dependent enzymes, responsible for the sulfur metabolism. The heme-dependent protein CBS is part of regulatory pathways also involving the gasotransmitter hydrogen sulfide. Malfunction of CBS can lead to pathologic conditions like cancer, cardiovascular and neurodegenerative disorders. Truncation of residues 1-40, absent in X-ray structures of CBS, reduces but does not abolish the activity of the enzyme. Here we report the NMR resonance assignment and heme interaction studies for the N-terminal peptide stretch of CBS. We present NMR-spectral evidence that residues 1-40 constitute an intrinsically disordered region in CBS and interact with heme via a cysteine-proline based motif.

A novel cGUUAg tetraloop structure with a conserved yYNMGg-type backbone conformation from cloverleaf 1 of bovine enterovirus 1 RNA.

The 5'-terminal cloverleaf (CL)-like RNA structures are essential for the initiation of positive- and negative-strand RNA synthesis of entero- and rhinoviruses. SLD is the cognate RNA ligand of the viral proteinase 3C (3C(pro)), which is an indispensable component of the viral replication initiation complex. The structure of an 18mer RNA representing the apical stem and the cGUUAg D-loop of SLD from the first 5'-CL of BEV1 was determined in solution to a root-mean-square deviation (r.m.s.d.) (all heavy atoms) of 0.59 A (PDB 1Z30). The first (antiG) and last (synA) nucleotide of the D-loop forms a novel 'pseudo base pair' without direct hydrogen bonds. The backbone conformation and the base-stacking pattern of the cGUUAg-loop, however, are highly similar to that of the coxsackieviral uCACGg D-loop (PDB 1RFR) and of the stable cUUCGg tetraloop (PDB 1F7Y) but surprisingly dissimilar to the structure of a cGUAAg stable tetraloop (PDB 1MSY), even though the cGUUAg BEV D-loop and the cGUAAg tetraloop differ by 1 nt only. Together with the presented binding data, these findings provide independent experimental evidence for our model [O. Ohlenschlager, J. Wohnert, E. Bucci, S. Seitz, S. Hafner, R. Ramachandran, R. Zell and M. Gorlach (2004) Structure, 12, 237-248] that the proteinase 3C(pro) recognizes structure rather than sequence.