Beier, Ch.

Borovykh, I. V.

Gajula, P.

Gast, P.

Huber, M.

Steinhoff, H.-J.

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John von Neumann Institute for ComputingA Study on the Structure and Dynamics of SpinLabeled Proteins by MD Simulations and EPRSpectroscopyPrasad Gajula, Igor V. Borovykh, Christian Beier,Peter Gast, Martina Huber, Heinz -Ju¨rgen Steinhoffpublished inNIC Workshop 2006,From Computational Biophysics to Systems Biology,Jan Meinke, Olav Zimmermann,Sandipan Mohanty, Ulrich H.E. Hansmann (Editors)John von Neumann Institute for Computing, Ju¨lich,NIC Series, Vol. 34, ISBN-10: 3-9810843-0-6,ISBN-13: 978-3-9810843-0-6, pp. 145-148 , 2006.c© 2006 by John von Neumann Institute for ComputingPermission to make digital or hard copies of portions of this work forpersonal or classroom use is granted provided that the copies are notmade or distributed for profit or commercial advantage and that copiesbear this notice and the full citation on the first page. To copy otherwiserequires prior specific permission by the publisher mentioned above.http://www.fz-juelich.de/nic-series/volume34A Study on the Structure and Dynamics of Spin LabeledProteins by MD Simulations and EPR Spectroscopy.Prasad Gajula1, Igor V. Borovykh1, Christian Beier1,Peter Gast2, Martina Huber2, and Heinz -Ju¨rgen Steinhoff11 Dept.of Physics, University of Osnabrueck,Barbarastr-7, 49076 Osnabru¨ck, Germany2 Dept.of Bio/Mol.physics, Huygens Laboratory, Leiden University, Leiden, The NetherlandsE-mail: prasad.gajula@uos.deInvestigations of conformational changes is a prerequisite to understand a protein’s biologicalfunction. To explore these conformational changes a strategy is developed combining molec-ular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy ofspin labeled proteins. The reaction center (RC) of Rhodobacter sphaeroides, a photosyntheticprotein, is chosen as a model system to study the structure and dynamics that determine electrontransport function. Only in combination EPR experiments and MD simulations positively iden-tify the spin label linker position as Cys156 on subunit H. The distance between the primaryacceptor (QA) and the bound spin label determined by EPR experiments and MD simulationsdeviate by about 7% indicating that the conformations of RC in the crystallized and in thesolubilized states are slightly different.1 IntroductionProteins execute and control essentially all functions in living organisms by changing intodifferent conformational sub-states1. Understanding the underlying mechanisms involvedin these conformational transitions is one of the main challenges of biophysical research.EPR spectroscopy is one of the few techniques available to study the structure and dynam-ics of a protein under physiological conditions. To overcome experimental limitations andto improve the analysis of the experimental results molecular dynamics simulations areapplied. In the present report, the combination of MD simulations with EPR spectroscopyserves as a powerful tool to explore the conformation and conformational dynamics of thereaction center (RC) protein of Rhodobacter sphaeroides, a protein involved in photosyn-thesis. A cysteine specific spin probe, a so called methanethiosulfonate spin label whichcontains an unpaired electron on its nitroxide group, is attached to RC (for a detailed re-view on site-directed spin labeling see2). The EPR spectrum, which is sensitive to the spinlabel reorientational dynamics, reports on the interaction of the spin label side chain withthe protein backbone or neighboring side chains, the conformational dynamics of the pro-tein, and the rotational diffusion of the protein. Inter-residue distances can be determinedby analysis of the dipolar interaction of two unpaired electrons, e.g., a spin label and anative radical center. MD simulations of the spin labeled RC, on the other hand, providetrajectories of the spin label side chain dynamics from which EPR spectra are calculated3.2 Methods and MaterialsThe photosynthetic RC is an integral membrane protein complex, that is composed ofthree polypeptide subunits (L, M and H) and ten non covalently bounded cofactors4. This145complex (Fig.1)with particular spatial configuration plays a central role in the process ofenergy conversion. EPR experiments were performed on spin labeled RC. MD simulationswere performed to identify which of the native cysteines the spin label binds preferen-tially. Simulation of EPR spectra based on MD trajectories allows a direct comparison ofexperimental spectra with the simulated one. For a detailed review on the method, see3.Figure 1. Reaction centre of Rb. Sphaeroides with 5 native cysteines at positions 92, 108, 247 on subunit L andat 156, 234 on subunit H. The spin label can only bind to one of these 5 cysteines.3 Results and Discussion3.1 Identification of the spin label linker positionBiochemical analysis and EPR experiments on the isolated LM complex shows that thespin label is bound to either of the two cysteines on subunit H. Further MD simulationanalysis was performed to identify the exact position of the spin label on subunit H.Figure 2. Orientation distribution of the nitroxide ring(left) and calculated EPR spectra (middle) for spin labelsattached to position 234, C234R1, (top) and 156, C156R1, (bottom). The experimental spectrum is shown ingreen (right). The population distributions projected onto the alpha-beta Euler angle plane reveals the differentrestrictions of the reorientational dynamics of the nitroxides at, two sites. These different nitroxide dynamics leadto distinguishable degrees of motional averaging of the g- and hyperfine tensor anisotropies and are reflected inthe EPR spectral shapes.The conformational space occupied by the nitroxide of spin label C234R1 during a 6 nsMD simulation run shows considerable restriction of the spin label motion. Consequentlythe EPR spectrum calculated for an isotropic distribution of protein orientations and withthe potential determined from the shown orientation distribution(Fig.2) of the nitroxidecoincides with a powder spectrum. In contrast, the space covered by the nitroxide of thespin label side chain at position C156 reveals considerable dynamics. The Euler angleprojection shows a broad distribution of the angle beta with distinct population maxima.146The apparent hyperfine splitting and the spectral shape of C156R1 are similar to that of theexperimental spectrum. Based on this result we conclude that cysteine 156 was labeled inthe present experiment5.3.2 Distance MeasurementsChanges in RC conformation related to electron-transfer events could, in principle, bedetected by measuring the distance between a native cofactor and a spin label. For thatpurpose a two-frequency pulsed electron paramagnetic resonance method (double electronelectron spin resonance - DEER) was used6.Figure 3. Distance between oxygen, O5 of QA and the centre of the N-O bond of the spin label for all framesof the MD run represented as distance distribution and the histogram of results of energy minimization on 30structures (shown in red). (b ) Distance distribution determined from analysis of experimental data (DEER).To facilitate comparision the distance distribution determined by MD simulation shifted by + 0.28 nm is alsoshown(bottom).A distance of 3.05 nm is found in between the light generated semiquinone anion rad-ical of the primary acceptor (QA) and the spin label at position H156 from DEER experi-ments. MD simulations are performed to interpret the distance. A 6 ns MD run comprisingthe entire RC protein yields a distance distribution that is close to the experimental one.However, the average distance value found by the MD simulation is smaller than that ob-tained by DEER by approximately 0.2 nm. Energy minimization on randomly selectedpoints of the MD trajectory confirms this result yielding a slightly broader distributionwidth. The possible reasons for the difference in the distance values determined by twomethods could be(i) incomplete sampling of the nitroxide accessible space during the 6nsMD or (ii) small differences of the conformations of RC in the frozen samples used inexperiments and of the crystal structure used for MD simulations.4 Conclusion and OutlookCombining the molecular dynamics simulations and EPR spectroscopy we identify the spinlabel binding site at position 156 of subunit H of RC protein. The average distance betweenthe native cofactor QA and the spin label bound to position 156 determined by EPR andMD deviate by about 7% indicating that the conformations of RC in the crystallized and inthe solubilized states are slightly different.147References1. Frauenfelder H. and McMahon B. , PNAS 95, 9 (4795-4797)1998.2. Steinhoff, H.-J. Frontiers in Bioscience 7, c97-110 (2002 )3. Steinhoff,H.-J.,Hubbell,W.L. Biophysical Journal 71 (2201)1996.4. Stowell,M.H.B., et.al. Science 276, 812-816 (1997)5. Gajula, P., Borovykh, I.V., Beier, C. , Gast, P., Steinhoff , H.-J. (in preparation)6. Borovykh, I.V., Ceola, S., Gajula, P., Gast, P., Steinhoff , H.-J., Huber, M. JMR 180,178-185 (2006)148

A Study on the Structure and Dynamics of Spin Labeled Proteins by MD Simulations and EPR Spectroscopy

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A Study on the Structure and Dynamics of Spin Labeled Proteins by MD Simulations and EPR Spectroscopy

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