Calculation of Residual Dipolar Couplings from Disordered State Ensembles Using Local Alignment

Marsh, Joseph A.; Baker, Jennifer M.; Tollinger, Martin; Forman-Kay, Julie D.

doi:10.1021/ja802220c

Cited by 68 publications

(106 citation statements)

References 20 publications

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“…Chemical shifts were predicted using SPARTA (36), SAXS using CRYSOL (73)(74)(75), and PRE as described previously (76). Local alignment windows of 15 amino acids in length were used to calculate averaged RDCs (77). These were then combined with a generic baseline to account for long-range effects (34, 76).…”

Section: Methodsmentioning

confidence: 99%

Functionally specific binding regions of microtubule-associated protein 2c exhibit distinct conformations and dynamics

Melková

Zapletal

Jansen

et al. 2018

Journal of Biological Chemistry

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Microtubule-associated protein 2c (MAP2c)1 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 longrange 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 poly-proline II and α-helices were found in Fyn-and plectin-binding sites of MAP2c, respectively. The region binding 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.Cytoskeletal microtubule associated proteins (MAPs) bind, stabilize, and regulate dynam- Conformation and Dynamics of MAP2cics of microtubules, in a phosphorylation-dependant manner. MAP2 and Tau are neuronal MAPs, MAP2 being expressed mainly in dendrites, whereas Tau is found in axons (1). Both MAP2 and Tau are expressed as different spliced variants. Tau isoforms expressed in human brain differ in the number of near-amino terminal inserts as well as in the number of repeats in the Microtubule Binding Domain (MTBD), whereas MAP2 isoforms differ in the length of the N-terminal projection domain (2, 3). Expression of both MAP2 and Tau isoforms is regulated during development. The high-molecular weight isoforms, MAP2a and MAP2b, contain 1830 amino-acids, and the two low molecular weight variants, MAP2c and MAP2d, consist of 467 and 498 amino acids, respectively. MAP2c is the smallest functional isoform, expressed mainly during embryonal brain development. After birth, its expression is restricted to regions exhibiting post-natal plasticity, such as the olfactory bulb (4), suggesting a role in neuronal development.Tau and MAP2c share a high sequence homology in the C-terminal part, containing MTBD, but differ in the N-terminal part, comprising the acidi...

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Section: Methodsmentioning

confidence: 99%

Functionally specific binding regions of microtubule-associated protein 2c exhibit distinct conformations and dynamics

Melková

Zapletal

Jansen

et al. 2018

Journal of Biological Chemistry

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“…4.8d). In order to overcome this problem, we can divide the protein chain into shorter, uncoupled segments and predict the RDCs for the central amino acid of each segment (Marsh et al 2008), thereby achieving sufficient convergence of the RDC average with only a few hundred structures (Fig. 4.8d).…”

Section: Ensemble Size In Relation To Convergence Properties Of Nmr Pmentioning

confidence: 99%

Ensemble Calculation for Intrinsically Disordered Proteins Using NMR Parameters

Kragelj

Blackledge

Jensen

2015

Advances in Experimental Medicine and Biology

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Intrinsically disordered proteins (IDPs) perform their function despite their lack of well-defined tertiary structure. Residual structure has been observed in IDPs, commonly described as transient/dynamic or expressed in terms of fractional populations. In order to understand how the protein primary sequence dictates the dynamic and structural properties of IDPs and in general to understand how IDPs function, atomic-level descriptions are needed. Nuclear magnetic resonance spectroscopy provides information about local and long-range structure in IDPs at amino acid specific resolution and can be used in combination with ensemble descriptions to represent the dynamic nature of IDPs. In this chapter we describe sample-and-select approaches for ensemble modelling of local structural propensities in IDPs with specific emphasis on validation of these ensembles.

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“…60 The input parameters in TraDES were set as recommended by Forman-Kay and coworkers. 61 It has been demonstrated that this program provides a reasonable sampling of the conformational space sampled by unfolded proteins. 61,62 As a next step, an MTSL tag was attached to the protein molecule in silico.…”

Section: Model 4: Diffusion In Numerically Defined Realistic Potentialmentioning

confidence: 99%

“…61 It has been demonstrated that this program provides a reasonable sampling of the conformational space sampled by unfolded proteins. 61,62 As a next step, an MTSL tag was attached to the protein molecule in silico. The topology of the MTSL, as coded by Battiste,63 was taken from the Xplor-NIH package.…”

Section: Model 4: Diffusion In Numerically Defined Realistic Potentialmentioning

confidence: 99%

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

et al. 2009

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Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as 1 H N is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean-force potential (Smoluchowski equation); (iv) explicit-atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the 1 H N residue number and static magnetic field strength; the results are appreciably different from the Gillespie-Shortle model. At the same time, the Gillespie-Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between 1 H N spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin-labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme-using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates-lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random-coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random-coil protein in good solvent/poor solvent.Additional Supporting Information may be found in the online version of this article.Abbreviations: drkN SH3, N-terminal SH3 domain of the Drosophila adapter protein drk; EDTA, ethylenediaminetetraacetic acid; ESR, electron spin resonance; FRET, fluorescence resonance energy transfer; MTSL, methanethiosulfonate spin label; NOE, nuclear Overhauser effect; NAG, N-acetyl-glycine.

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Calculation of Residual Dipolar Couplings from Disordered State Ensembles Using Local Alignment

Cited by 68 publications

References 20 publications

Functionally specific binding regions of microtubule-associated protein 2c exhibit distinct conformations and dynamics

Functionally specific binding regions of microtubule-associated protein 2c exhibit distinct conformations and dynamics

Ensemble Calculation for Intrinsically Disordered Proteins Using NMR Parameters

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

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