NMR Characterization of Long‐Range Contacts in Intrinsically Disordered Proteins from Paramagnetic Relaxation Enhancement in <sup>13</sup>C Direct‐Detection Experiments

Mateos, Borja; Konrat, Robert; Pierattelli, Roberta; Felli, Isabella C.

doi:10.1002/cbic.201800539

Cited by 21 publications

(18 citation statements)

References 54 publications

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“…2B, can be considered the reference spectrum for a series of multidimensional CON-based experiments in which correlations to additional nuclear spins in the indirect dimensions of the experiments provide the needed information to achieve the sequence-specific assignment. These multidimensional spectra include the 13 C-detected 3D (H)CBCACON 23 , (H)CBCANCO 24 , (H) COCON 25,26 , and 5D (HCA)CONCACON 27 spectra. Inspection of the spectra constituted the starting point for the sequence-specific assignment 22,27 .…”

Section: Resultsmentioning

confidence: 99%

Ensemble description of the intrinsically disordered N-terminal domain of the Nipah virus P/V protein from combined NMR and SAXS

Schiavina

Salladini

Murrali

et al. 2020

Sci Rep

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Using SAXS and NMR spectroscopy, we herein provide a high-resolution description of the intrinsically disordered N-terminal domain (PNT, aa 1–406) shared by the Nipah virus (NiV) phosphoprotein (P) and V protein, two key players in viral genome replication and in evasion of the host innate immune response, respectively. The use of multidimensional NMR spectroscopy allowed us to assign as much as 91% of the residues of this intrinsically disordered domain whose size constitutes a technical challenge for NMR studies. Chemical shifts and nuclear relaxation measurements provide the picture of a highly flexible protein. The combination of SAXS and NMR information enabled the description of the conformational ensemble of the protein in solution. The present results, beyond providing an overall description of the conformational behavior of this intrinsically disordered region, also constitute an asset for obtaining atomistic information in future interaction studies with viral and/or cellular partners. The present study can thus be regarded as the starting point towards the design of inhibitors that by targeting crucial protein–protein interactions involving PNT might be instrumental to combat this deadly virus.

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Section: Resultsmentioning

confidence: 99%

Ensemble description of the intrinsically disordered N-terminal domain of the Nipah virus P/V protein from combined NMR and SAXS

Schiavina

Salladini

Murrali

et al. 2020

Sci Rep

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“…In order to probe putative changes in long-range contacts resulting from cis/trans isomerization of proline residues, we carried out a series of paramagnetic relaxation enhancement (PRE) experiments. We exploit 13 C-detected PRE by observing intensities of the paramagnetic and diamagnetic forms [69]. 1 H α -start experiments are more sensitive to paramagnetic effects because protons, with their high gyromagnetic ratio compared to carbon, are reintroduced in the coherence transfer pathway to transfer coherence from protons to heteronuclei ( 13 C or 15 N).…”

Section: On the Role Of Proline Isomerization In Modulating Global Rementioning

confidence: 99%

The Ambivalent Role of Proline Residues in an Intrinsically Disordered Protein: From Disorder Promoters to Compaction Facilitators

Mateos

Conrad-Billroth

Schiavina

et al. 2020

Journal of Molecular Biology

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Intrinsically disordered proteins (IDPs) carry out many biological functions. They lack a stable three-dimensional structure, but rather adopt many different conformations in dynamic equilibrium. The interplay between local dynamics and global rearrangements is key for their function. In IDPs, proline residues are significantly enriched. Given their unique physicochemical and structural properties, a more detailed understanding of their potential role in stabilizing partially folded states in IDPs is highly desirable. Nuclear magnetic resonance (NMR) spectroscopy, and in particular 13 C-detected NMR, is especially suitable to address these questions. We applied a 13 C-detected strategy to study Osteopontin, a largely disordered IDP with a central compact region. By employing the exquisite sensitivity and spectral resolution of these novel techniques we gained unprecedented insight into cis-Pro populations, their local structural dynamics and their role in mediating long-range contacts. Our findings clearly call for a reassessment of the structural and functional role of proline residues in IDPs. The emerging picture shows that proline residues have ambivalent structural roles. They are not simply disorder promoters but rather can, depending on the primary sequence context, act as nucleation sites for structural compaction in IDPs. These unexpected features provide a versatile mechanistic toolbox to enrich the conformational ensembles of IDPs with specific features for adapting to changing molecular and cellular environments.

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“…Different electronic relaxation times, together with other experimental parameters, such as magnetic field strength and protein size will give rise to different radii for the blind sphere and for the sphere where PREs are measurable. Also the replacement of 1 H with 13 C or 15 N nuclei reduces both spheres and provides an alternate route to obtain PREs (Mateos et al 2019 ). To extend the range of application of PREs in 1 H detected experiments it is necessary, on the one hand, to increase the accuracy of experimental methods and observe smaller PRE values and, on the other hand, to measure with reliability R 2 values larger than those achievable with the existing experimental approaches.…”

Section: Resultsmentioning

confidence: 99%

Measuring transverse relaxation in highly paramagnetic systems

et al. 2020

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The enhancement of nuclear relaxation rates due to the interaction with a paramagnetic center (known as Paramagnetic Relaxation Enhancement) is a powerful source of structural and dynamics information, widely used in structural biology. However, many signals affected by the hyperfine interaction relax faster than the evolution periods of common NMR experiments and therefore they are broadened beyond detection. This gives rise to a so-called blind sphere around the paramagnetic center, which is a major limitation in the use of PREs. Reducing the blind sphere is extremely important in paramagnetic metalloproteins. The identification, characterization, and proper structural restraining of the first coordination sphere of the metal ion(s) and its immediate neighboring regions is key to understand their biological function. The novel HSQC scheme we propose here, that we termed R2-weighted, HSQC-AP, achieves this aim by detecting signals that escaped detection in a conventional HSQC experiment and provides fully reliable R2 values in the range of 1H R2 rates ca. 50–400 s−1. Independently on the type of paramagnetic center and on the size of the molecule, this experiment decreases the radius of the blind sphere and increases the number of detectable PREs. Here, we report the validation of this approach for the case of PioC, a small protein containing a high potential 4Fe-4S cluster in the reduced [Fe4S4]2+ form. The blind sphere was contracted to a minimal extent, enabling the measurement of R2 rates for the cluster coordinating residues.

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NMR Characterization of Long‐Range Contacts in Intrinsically Disordered Proteins from Paramagnetic Relaxation Enhancement in ¹³C Direct‐Detection Experiments

Cited by 21 publications

References 54 publications

Ensemble description of the intrinsically disordered N-terminal domain of the Nipah virus P/V protein from combined NMR and SAXS

Ensemble description of the intrinsically disordered N-terminal domain of the Nipah virus P/V protein from combined NMR and SAXS

The Ambivalent Role of Proline Residues in an Intrinsically Disordered Protein: From Disorder Promoters to Compaction Facilitators

Measuring transverse relaxation in highly paramagnetic systems

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NMR Characterization of Long‐Range Contacts in Intrinsically Disordered Proteins from Paramagnetic Relaxation Enhancement in 13C Direct‐Detection Experiments

Cited by 21 publications

References 54 publications

Ensemble description of the intrinsically disordered N-terminal domain of the Nipah virus P/V protein from combined NMR and SAXS

Ensemble description of the intrinsically disordered N-terminal domain of the Nipah virus P/V protein from combined NMR and SAXS

The Ambivalent Role of Proline Residues in an Intrinsically Disordered Protein: From Disorder Promoters to Compaction Facilitators

Measuring transverse relaxation in highly paramagnetic systems

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NMR Characterization of Long‐Range Contacts in Intrinsically Disordered Proteins from Paramagnetic Relaxation Enhancement in ¹³C Direct‐Detection Experiments