Nejvíce citovaný článek - PubMed ID 29071460
Triple resonance ¹⁵Ν NMR relaxation experiments for studies of intrinsically disordered proteins
Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) is a class of biologically important proteins exhibiting specific biophysical characteristics. They lack a hydrophobic core, and their conformational behavior is strongly influenced by electrostatic interactions. IDPs and IDRs are highly dynamic, and a characterization of the motions of IDPs and IDRs is essential for their physically correct description. NMR together with molecular dynamics simulations are the methods best suited to such a task because they provide information about dynamics of proteins with atomistic resolution. Here, we present a study of motions of a disordered C-terminal domain of the delta subunit of RNA polymerase from Bacillus subtilis. Positively and negatively charged residues in the studied domain form transient electrostatic contacts critical for the biological function. Our study is focused on investigation of ps-ns dynamics of backbone of the delta subunit based on analysis of amide 15N NMR relaxation data and molecular dynamics simulations. In order to extend an informational content of NMR data to lower frequencies, which are more sensitive to slower motions, we combined standard (high-field) NMR relaxation experiments with high-resolution relaxometry. Altogether, we collected data reporting the relaxation at 12 different magnetic fields, resulting in an unprecedented data set. Our results document that the analysis of such data provides a consistent description of dynamics and confirms the validity of so far used protocols of the analysis of dynamics of IDPs also for a partially folded protein. In addition, the potential to access detailed description of motions at the timescale of tens of ns with the help of relaxometry data is discussed. Interestingly, in our case, it appears to be mostly relevant for a region involved in the formation of temporary contacts within the disordered region, which was previously proven to be biologically important.
- MeSH
- amidy MeSH
- DNA řízené RNA-polymerasy chemie MeSH
- konformace proteinů MeSH
- magnetická rezonanční spektroskopie MeSH
- simulace molekulární dynamiky MeSH
- vnitřně neuspořádané proteiny * chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- amidy MeSH
- DNA řízené RNA-polymerasy MeSH
- vnitřně neuspořádané proteiny * MeSH
Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.
Improving our understanding of nanosecond motions in disordered proteins requires the enhanced sampling of the spectral density function obtained from relaxation at low magnetic fields. High-resolution relaxometry and two-field NMR measurements of relaxation have, so far, only been based on the recording of one- or two-dimensional spectra, which provide insufficient resolution for challenging disordered proteins. Here, we introduce a 3D-HNCO-based two-field NMR experiment for measurements of protein backbone 15 N amide longitudinal relaxation rates. The experiment provides accurate longitudinal relaxation rates at low field (0.33 T in our case) preserving the resolution and sensitivity typical for high-field NMR spectroscopy. Radiofrequency pulses applied on six different radiofrequency channels are used to manipulate the spin system at both fields. The experiment was demonstrated on the C-terminal domain of δ subunit of RNA polymerase from Bacillus subtilis, a protein with highly repetitive amino-acid sequence and very low dispersion of backbone chemical shifts.
- Klíčová slova
- Dynamics, High-resolution relaxometry, Intrinsically disordered proteins, Non-uniform sampling, Nuclear magnetic resonance, Relaxation,
- MeSH
- Bacillus subtilis enzymologie MeSH
- bakteriální proteiny chemie MeSH
- DNA řízené RNA-polymerasy chemie MeSH
- nukleární magnetická rezonance biomolekulární * MeSH
- rekombinantní proteiny chemie MeSH
- vnitřně neuspořádané proteiny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
- DNA řízené RNA-polymerasy MeSH
- rekombinantní proteiny MeSH
- vnitřně neuspořádané proteiny MeSH
Microtubule-associated protein 2c (MAP2c) is a 49-kDa intrinsically disordered protein regulating the dynamics of microtubules in developing neurons. MAP2c differs from its sequence homologue Tau in the pattern and kinetics of phosphorylation by cAMP-dependent protein kinase (PKA). Moreover, the mechanisms through which MAP2c interacts with its binding partners and the conformational changes and dynamics associated with these interactions remain unclear. Here, we used NMR relaxation and paramagnetic relaxation enhancement techniques to determine the dynamics and long-range interactions within MAP2c. The relaxation rates revealed large differences in flexibility of individual regions of MAP2c, with the lowest flexibility observed in the known and proposed binding sites. Quantitative conformational analyses of chemical shifts, small-angle X-ray scattering (SAXS), and paramagnetic relaxation enhancement measurements disclosed that MAP2c regions interacting with important protein partners, including Fyn tyrosine kinase, plectin, and PKA, adopt specific conformations. High populations of polyproline II and α-helices were found in Fyn- and plectin-binding sites of MAP2c, respectively. The region binding the regulatory subunit of PKA consists of two helical motifs bridged by a more extended conformation. Of note, although MAP2c and Tau did not differ substantially in their conformations in regions of high sequence identity, we found that they differ significantly in long-range interactions, dynamics, and local conformation motifs in their N-terminal domains. These results highlight that the N-terminal regions of MAP2c provide important specificity to its regulatory roles and indicate a close relationship between MAP2c's biological functions and conformational behavior.
- Klíčová slova
- NMR relaxation, Tau protein (Tau), microtubule-associated protein (MAP), nuclear magnetic resonance (NMR), paramagnetic relaxation enhancement (PRE), protein conformation, small-angle X-ray scattering (SAXS),
- MeSH
- difrakce rentgenového záření MeSH
- fosforylace MeSH
- konformace proteinů * MeSH
- lidé MeSH
- maloúhlový rozptyl MeSH
- plektin chemie metabolismus MeSH
- proteiny asociované s mikrotubuly chemie metabolismus MeSH
- src homologní domény MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- MAP2 protein, human MeSH Prohlížeč
- PLEC protein, human MeSH Prohlížeč
- plektin MeSH
- proteiny asociované s mikrotubuly MeSH
