NMR Methods

DOI: 10.1002/anie.200602317

Charge-Induced Molecular Alignment ofIntrinsically Disordered Proteins**

Lukasz Skora, Min-Kyu Cho, Hai-Young Kim,Stefan Becker, Claudio O. Fernandez,Martin Blackledge, and Markus Zweckstetter*

Intrinsically disordered or unstructured proteins (IUPs) playa key role in normal and pathological biochemical processes.Despite their importance for function, this category ofproteins remains beyond the reach of classical structuralbiology because of their inherent conformational heteroge-neity. Measurements of global dimensions strongly suggesteda random coil-like behavior of IUPs.[1] In contrast to thesefindings, NMR spectroscopy detected significant amounts oflocal structure in denatured and unfolded states. Recently,these two apparently contradicting behaviors were reconciledwhen it was shown that the local conformational behavior ofIUPs can be described by a simple model based on residue-specific f/f propensities.[2, 3] In particular, residual dipolarcouplings (RDCs), which can be measured for proteins thatare weakly aligned in dilute liquid-crystalline media, could bepredicted from this model by assuming a steric interactionbetween the protein and the alignment medium. Further-more, we showed that RDCs are also sensitive detectors oftransient long-range interactions in IUPs.[4, 5]

[*] L. Skora, M.-K. Cho, H.-Y. Kim, S. Becker, Dr. M. ZweckstetterDepartment of NMR-based Structural BiologyMax Planck Institute for Biophysical ChemistryAm Fassberg 11, 37077 G7ttingen (Germany)Fax: (+49)551-201-2202E-mail: mzwecks@gwdg.deHomepage:http://www.mpibpc.gwdg.de/abteilungen/030/zweckstetter

Dr. C. O. FernandezInstituto de Biolog@a Molecular y Celular de RosarioConsejo Nacional de Investigaciones Cient@ficas y TecnicasUniversidad Nacional de RosarioSuipacha 531, S2002LRK Rosario (Argentina)

Dr. M. BlackledgeInstitute de Biologie Structurale Jean-Pierre EbelCNRS-CEA-UJF, UMR 508541 rue Jules Horowitz38027 Grenoble Cedex (France)

[**] L.S. acknowledges a Marie Curie fellowship (MEST-CT-2004-504193), M.-K.C. a DFG-Graduiertenkolleg (GRK782) scholarship,C.F. an institutional partnership supported by the Alexander vonHumboldt Foundation, and M.Z. a DFG Emmy Noether grant (ZW71/1-5). This work was supported by the CMPB G7ttingen and bythe European Union through UPMAN. We thank Christophe Faresfor help with the CPCl medium and Prof. Dr. Christian Griesinger forstimulating discussions.

Supporting information for this article is available on the WWWunder http://www.angewandte.org or from the author.

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Herein, we demonstrate that the assumption of a stericinteraction between intrinsically disordered proteins and thealignment medium is only valid in the case of unchargedalignment media. Alignment of IUPs in charged mediadepends critically on electrostatic interactions, especially incharged regions of the protein, and is scaled with the ionicstrength of the solution. In this case the RDCs can bepredicted using a simplified electrostatic model.[6]

In our approach we investigated the molecular alignmentof a-synuclein (aS), a natively unstructured protein of140 amino acid residues that is implicated in the onset ofParkinson3s disease, in two charged alignment media withdifferent electrostatic properties: filamentous phage Pf1[7]

and quasiternary surfactant cetylpyridinium chloride/hexa-nol/NaCl (CPCl).[8]

RDCs in aS that was weakly aligned in filamentous phagePf1 are shown in Figure 1. At 300 and 500 mm NaCl the RDCprofile resembles the one that was previously observed in an

uncharged alignment medium.[4] All RDCs were positive, andregions with sizeable RDCs were separated by residues withRDC values close to zero. The C-terminal domain displayedthe largest RDCs. At 100 mm NaCl, the RDCs of residues 23,46, 50, 59, 86, and 103–104 turned from near-zero values tonegative. Furthermore, the magnitude of RDCs for residues15–21 increased, and for residues 124–131 it was nearlydoubled. At even lower salt concentration, the RDCs in theN-terminal domain were further increased, thereby makingseveral residues undetectable owing to strong 1H,1H dipolarcouplings, and in the central part of aS nearly all RDCsbecame negative. This behavior is in agreement with theelectrostatics of the system. The Pf1 alignment medium bearsan overall negative charge on the outer, protein-accessibleside[9] and therefore, at low salt concentrations, stronglyattracts the positively charged N-terminal domain of aS(Figure 1a). At the same time, the negatively charged C-terminal domain of aS is repelled from the Pf1 surface.

Experimental RDCs were com-pared to values that were predictedfrom an ensemble of structures by usingthe software PALES. An ensemblecomprising 50000 structures was gen-erated using the flexible-meccano algo-rithm,[3] which sequentially builds pep-tide chains by random selection of f/fangles from a database of amino acidspecific conformations found in loopregions of high-resolution X-ray struc-tures. From these 50000 structures(30000–50000 structures were neededfor convergence) RDCs were predictedon the basis of a highly simplifiedalignment model that approximatesthe electrostatic interaction between asolute and an ordered liquid-crystalparticle as that between the soluteside-chain charge and the electric fieldof the phage.[6] For Pf1 bacteriophagethe following parameters were used:average surface charge density�0.47 enm�2, order parameter 0.9, con-centration 15 mgmL�1. CPCl was mod-elled as a 26.8-? thick, uniformlycharged wall with a surface chargedensity of + 0.08 enm�2, order parame-ter of 0.8, and a concentration of 5%(w/v).[10]

Reasonably close agreementbetween experimental and charge-shape-predicted RDC patterns wasfound under all investigated conditionsfor Pf1 alignment medium (Figure 1c,dand the Supporting Information). Theprediction of the magnitude of align-ment is better at intermediate ionicstrengths whereas at higher salt con-centrations the RDC magnitude tendsto be underestimated by PALES.[6]

Figure 1. a) Surface charge distribution in aS. Positively and negatively charged residues aremarked blue and red, respectively. Sites of mutations associated with Parkinson’s disease andproline residues are indicated by arrows. b) 1H,15N RDCs in Pf1 phage at 50 mm (black),100 mm (blue), 300 mm (green), and 500 mm (red) NaCl, scaled with respect to the splitting ofthe 2H signal at 50 mm NaCl. All spectra were acquired on a Bruker DRX600 spectrometer with0.2 mm aS in 20 mm Tris-HCl, pH 7.4, at 288 K. c,d) Comparison of experimental (solid line)and predicted (dashed line) 1H,15N RDCs in 15 mgmL�1 Pf1 bacteriophage at 50 mm NaCl (c)and 500 mm NaCl (d). Below 200 mm NaCl the predicted and experimental RDC magnitudestrongly depend on the exact ionic-strength value and the Pf1 batch used.

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Therefore, average RDC values predicted at 100 mm and500 mm NaCl were scaled up by factors of 2.5 and 4,respectively, to maximize agreement with experimentalvalues. At 50 mm NaCl no scaling was required.

CPCl is positively charged and interacts strongly with theacidic C-terminal region of aS, such that severe line broad-ening and disappearance of the corresponding signals wasobserved (Supporting Information). In agreement with thedisappearance of signals, PALES predicted an up to eightfoldincrease in the RDC magnitude of residues 110–140. How-ever, RDCs for residues 10–90 remained small, thus suggest-ing that the different domains of aS align independently.Experimental RDCs in this region fit reasonably well topredicted values. Moreover, a strong influence of the electro-static interaction on the alignment properties of CPCl wasobserved. At 150 mm NaCl, the liquid-crystalline phaseshowed a 2H splitting of 15 Hz, which dropped sixfold uponaddition of aS. Thus, it cannot be excluded that theinteraction with the CPCl medium changes the conforma-tional ensemble of aS, although chemical shifts of observableresidues were not significantly changed.

To further investigate the sign inversion of RDCs in thecentral region of aS at 50 mm NaCl (Figure 1b), we con-structed a 30000-structure ensemble for a polyalanine frag-ment of 10 amino acid residues. Charges of + 5e and �5ewere placed on the N- and C-terminus, respectively, andRDCs were predicted using models of both steric[11] andelectrostatic alignment[6] (Figure 2a). In the steric case, allRDCs were positive and showed the characteristic bell-shaped pattern.[12] In the electrostatic prediction, on the otherhand, average RDC values turned negative, thus indicative ofa preferential alignment perpendicular to the magnetic fieldand in agreement with a dipole-like behavior of the aminoacid fragment. Furthermore, predictions at different salt

concentrations demonstrated that shielding of charges leadsto more steric alignment.

Previously, we showed that the large RDCs in the C-terminal region of aS are a result of long-range interactionsinvolving the C- and N-terminal domains and the NACregion.[4] When steric alignment was assumed, enforcement ofa long-range interaction between residues 1–20 and 120–140in an ensemble of structures generated by flexible-meccanoresulted in an increase in the RDC magnitude predicted forresidues 2–30 and 110–140 and better agreement with RDCsmeasured in both sterically and electrostatically aligningmedia. The situation turns out to be more complicated whenelectrostatic alignment dominates (Figure 2b). AlthoughRDCs for residues 116–121 were slightly increased uponenforcing the long-range contact, average RDCs that werepredicted for the N-terminal region were strongly reduced.Thus, two opposing effects have to be taken into account.Owing to the long-range interaction the protein backbonebecomes more rigid, potentially leading to larger RDCs, butat the same time the proximity of the negatively charged C-terminal region partially compensates the positive charges inthe N-terminus, thus causing a decrease in the RDCmagnitude.

This report demonstrates that molecular alignment ofintrinsically unstructured and other disordered proteins incharged nematic media strongly depends on electrostaticinteractions between the protein and the alignment medium.A simple electrostatic alignment model, however, reliablypredicts RDCs under different sample conditions. Electro-static effects have to be taken into account when using RDCsfor the interpretation of residual structure in disorderedproteins. At the same time, however, charge modulation ofalignment provides an independent set of RDCs in disorderedproteins, potentially improving the structural characterization

of these systems. These findings have importantconsequences for the RDC-based interpretation ofthe structure and dynamics of the unfolded state.

Received: June 9, 2006Published online: September 28, 2006

.Keywords: molecular alignment · NMR spectroscopy ·protein structures · residual dipolar couplings

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Figure 2. a) 1H,15N RDCs in a polyalanine fragment of 10 amino acid residues, predictedusing a steric (red) and an electrostatic alignment model with salt concentrations of50 mm (black), 100 mm (blue), and 500 mm (green) and charges of +5e and �5e at theN- and C-terminus, respectively. Inset: enlarged excerpt. b) 1H,15N RDCs in 15 mgmL�1

Pf1 phage at 100 mm NaCl, predicted with (dashed line) and without (solid line) enforcinga long-range interaction between residues 1–20 and 120–140.

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