An Approach to NMR Assignment of Intrinsically Disordered Proteins

Goradia, Nishit; Wiedemann, Christoph; Herbst, Christian T.; Görlach, Matthias; Heinemann, Stefan H.; Ohlenschläger, Oliver; Ramachandran, Ramadurai

doi:10.1002/cphc.201402872

Cited by 16 publications

(12 citation statements)

References 39 publications

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“…One possibility would be to adapt the RN[N‐CA HEHAHA]NH protocols to achieve the simultaneous collection of RN[N‐CA HEHAHA]CACSHS correlation spectra. This could be achieved by subjecting the residual 13 C α ( i / i −1) magnetisation present at the end of the HEHAHA mixing period to a period of 13 C‐ 13 C TOCSY mixing first and then transferring the resultant 13 C sc magnetisation to the attached side chain protons through a 13 C‐ 1 H cross polarization step for detection as in our earlier studies . Even the availability of the additional RN[N‐CA HEHAHA]CAHA data set would not only provide further confirmation of the sequential connectivities established via the RN[N‐CA HEHAHA]NH protocol but also be potentially useful in establishing reliable sequential connectivities in regions where the amide proton signal intensities are inherently weak due to exchange of the labile protons with water.…”

Section: Figurementioning

confidence: 97%

“…Even the availability of the additional RN[N‐CA HEHAHA]CAHA data set would not only provide further confirmation of the sequential connectivities established via the RN[N‐CA HEHAHA]NH protocol but also be potentially useful in establishing reliable sequential connectivities in regions where the amide proton signal intensities are inherently weak due to exchange of the labile protons with water. Representative spectral cross‐sections taken from 3D HN[N‐CA HEHAHA](CACS)HS and HN[N‐CA HEHAHA](N)H data sets collected using the sequential data acquisition procedure, without sparse sampling in the indirect dimensions and without and with the application of 13 C‐ 13 C TOCSY mixing are given in the SI. It is worth pointing out that, unlike the INEPT procedure, the application of band‐selective 15 N‐ 13 C α HEHAHA mixing sequence for transferring the 15 N i magnetisation to the adjacent “ i +1” and “ i −1” residues makes it possible to conveniently extract the side chain chemical shift information simultaneously.…”

Section: Figurementioning

confidence: 99%

“…Another approach to simultaneously extract the side chain chemical shifts, using only a single data acquisition, would be to implement RF pulse schemes starting with the excitation of side chain protons such as the HSCSCACON[N‐CA HEHAHA]NH experiment involving the 1 H sc → 13 C sc → 13 C α magnetisation transfer pathway and chemical shift labelling of the side chain resonances in the indirect dimensions (see SI). Starting with the excitation of the initial transverse magnetisation from both the 15 N and 13 C attached protons and employing sequential detection of side chain and amide protons in the direct dimension, as in our recent studies, it is also possible to implement RF pulse schemes leading to the simultaneous acquisition of correlation data of interest, for example, HNCOCACSHS and HACACON[N‐CA HEHAHA]NH. Preliminary studies clearly indicate that these different protocols hold considerable promise and it is planned to carry out further investigations on these and other related issues such as the incorporation of sparse sampling in the sequential acquisition of multi‐dimensional data sets and the performance of different sparse sampling schemes.…”

Section: Figurementioning

confidence: 99%

“…Some of the possibilities to extract these,s imultaneously with the connectivity data, are outlined below.O ne possibility would be to adapt the RN[N-CAH EHAHA]NH protocols to achieve the simultaneous collection of RN[N-CAH EHAHA]-CACSHSc orrelation spectra.T his could be achieved by subjecting the residual 13 C a (i/iÀ1) magnetisation presenta tt he end of the HEHAHA mixing period to ap eriod of 13 C- 13 CT OCSY mixing first and then transferring the resultant 13 C sc magnetisation to the attached side chain protons througha 13 C-1 Hc ross polarization step for detection as in our earlier studies. [5] Even the availability of the additional RN[N-CAH EHAHA]CAHA data set would not only providef urtherconfirmation of the sequential connectivities established via the RN[N-CAH EHAHA]NH protocolb ut also be potentially useful in establishing reliable sequential connectivitiesi nr egions where the amide proton signal intensities are inherently weak due to exchange of the labile protons with water.R epresentative spectral cross-sections taken from 3D HN[N-CA HEHAHA](CACS)HS and HN[N-CA HEHAHA](N)H data sets collected using the sequential dataa cquisitionprocedure, [5,60] withoutsparse sampling in the indirect dimensions and without andw ith the application of 13 C-13 C TOCSY mixinga re given in the SI. It is worth pointingo ut that, unlike the INEPT procedure, the application of band-selective 15 N-13 C a HEHAHA mixing sequence for transferring the 15 N i magnetisation to the adjacent "i + 1" and" iÀ1" residues makes it possible to conveniently extract the side chain chemical shift information simultaneously.T he pulse sequences can also be adapted, using dual receiversa si ne arlier studies [61][62][63][64] to acquire simultaneously RN[N-CAH EHAHA]NCO and RN[N-CA HE-HAHA]CAHA correlation data sets.…”

Section: Dedicatedtoprof Drp Eter Schuster( Vienna) On the Occasionmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Figurementioning

confidence: 97%

Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Dedicatedtoprof Drp Eter Schuster( Vienna) On the Occasionmentioning

confidence: 99%

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

et al. 2016

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“…Since the assignment of an NMR spectrum to each of the atoms along the sequence depends on the tracing of the magnetization transfer, that is the transfer from one atom to the next as in a network, overlap has the consequence of limiting the possibility of uniquely tracing the chain. Several strategies have been suggested to circumvent this problem (1)(2)(3). Yet, no definite answer has been given to the question and new ideas must be considered to eventually find, if possible, a unique and universal strategy.…”

Section: Introductionmentioning

confidence: 99%

A strategy to study intrinsically mixed folded proteins: The structure in solution of ataxin-3

Sicorello

Kelly

Oregioni

et al. 2018

Preprint

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It has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions which both can contribute to function.While equally interesting, the disordered regions are difficult to study because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. Nuclear magnetic resonance spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe assignment of the spectrum of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We used a 42 kDa construct containing a globular N-terminal josephin domain and a C-terminal tail which comprises thirteen polyglutamine repeats within a low-complexity region. We developed a strategy which allowed us to achieve 87% assignment of the spectrum. We show that the C-terminal tail is flexible with extended helical regions and interacts only marginally with the rest of the protein. We could also, for the first time, deduce the structure of the polyglutamine repeats within the context of the full-length protein and show that it has a strong helical propensity stabilized by the preceding region.

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Multiple Acquisition Strategies

Traaseth

2023

Two‐Dimensional (2D) NMR Methods

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An Approach to NMR Assignment of Intrinsically Disordered Proteins

Cited by 16 publications

References 39 publications

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

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

A strategy to study intrinsically mixed folded proteins: The structure in solution of ataxin-3

Multiple Acquisition Strategies

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