1. STRUCTURAL DYNAMICS OF RHODOPSIN: A G-PROTEIN-COUPLED RECEPTOR by Basak Isin

2. OUTLINE  G Protein-Coupled-Receptors (GPCR)  Aim of the project  Gaussian Network Model (GNM)  Anisotropic Network Model (ANM)  Results and Discussion  Conclusion and Summary  A.J.  Simulated protein unfolding by the FIRST software

3. GPCRs  the largest superfamily of cell surface receptors  seven transmembrane helices - their signature motif   involved in a number of clinically important ligand/receptor processes.   couple to heterotrimeric G proteins to convert an extracellular signal into an intracellular signal.   bind ligands from the cell exterior, which induce a conformational change in the cytoplasmic face of the receptor, enabling binding of the G protein.   significant drug targets. 50-60% of approved drugs target members of the GPCR family.

4. RHODOPSIN the first 3-dimensional molecular model for a GPCR  located in the outer segments of rod photoreceptor cells in the retina  responds to environmental signals, i.e., photons  initiates intracellular processes that result in an electrical signal processed by the visual system. 1 2 3 4 6 5 cytoplasmic region extracellular region 7

5. LIGHT ACTIVATION 11-cis retinalAll-trans retinal Metarhodopsin IIRhodopsin

7. AIM OF THE PROJECT understanding the structure-function relationships in rhodopsin understanding the structure-function relationships in rhodopsin derivation of dynamic properties of rhodopsin by analysis of the X-ray crystal structure derivation of dynamic properties of rhodopsin by analysis of the X-ray crystal structure Gaussian network model (GNM) Gaussian network model (GNM) Anisotropic network model (ANM) Anisotropic network model (ANM) APPROACH

8. Gaussian Network Model (GNM) GNM was introduced for estimating the dynamic characteristics of biomolecular structures, based on atomic coordinates in the native conformation. (Bahar, 1999; Bahar et al., 1998; 1997).

9. A NEW REPRESENTATION OF THE STRUCTURE

10.  A biomolecular structure in folded state is represented by a perfectly elastic network.  The virtual bond representation is adopted. The position of each residue is identified by its  -carbon.  The interactions between residues in close proximity are represented by harmonic potentials with a uniform spring constant, .  There is no distinction between bonded and non-bonded neighbors and all residue pairs separated by less than a suitable cutoff distance are assumed to be coupled.

12. CONSTRUCTION OF KIRCHHOFF MATRIX  Rij is the distance between ith and jth residues.  The value of r c = 7 Å includes the neighboring residues located in the first coordination shell near a central residue.

13.  = U  U T   -1 = U  -1 U T The fluctuations associated with k th mode U: orthogonal (NxN) matrix u k : k th eigenvector of Γ (1 ≤ k ≤ N) (shapes of corresponding mode of motion)  : diagonal matrix with eigenvalues ( k ) 1 = 0 <  2 < …< N frequency of modes = (3k B T/  ) ( k -1 [u k ] i [u k ] j )

14.  Provides information on the mechanism of the motion relevant to biological function.  The maxima of the slow mode curves indicate the most flexible regions of the molecule.  Identifies the hinge region that are important for biological function. Slow Modes Global Motions

15. Comparison of theoretically calculated B-factors with those measured by X-ray crystallography RESULTS AND DISCUSSION Science, Palcwezski et. al, 2000

16. First Mode of GNM H1 H2 H3H4 H5H6H7 1 2 7 6 5 3 The color codes are green, cyan, blue, magenta, pink and yellow in the order of increasing mobility.

17. CROSS CORRELATION

18. Fast Modes Critically important ones for the overall stability of the molecule High Frequency Modes Asn55

19.  the extension of the GNM to the 3N-d space of collective modes.  the three components of the inter-residue separation vectors obey Gaussian dynamics.  involves the inversion of a 3N x 3N Hessian matrix H that replaces the N x N Kirchhoff matrix  (Doruker et al., 2000, Atilgan et al., 2001). ANM

20. ANM ANM results Wild type Front ViewBack View ANM resultsWild type ANM results Top View ANM and wildtype Receptor binding site for G protein a b c d

24. Summary and Conclusion The elastic network models (GNM and ANM) are efficient tools to explore the dynamics of proteins and to determine the critically important sites. These are classified in two categories: The first group comprises the residues that are important for coordinating the cooperative motions of the overall molecule. These are identified from the minima of the global mode shapes. Their mutation can impede function. The second group consists of residues experiencing an extremely strong coupling to their close neighbors, and thereby undergoing the highest frequency/smallest amplitude vibrations. Their mutation can impede stability. Both groups of residues are expected to be evolutionarily conserved, the former for function requirements, and the latter for folding and stability.

25. CONCLUSION The opening of helical bundle by torsional rotation of the molecule and exposing various regions possibly for the interaction with the G-protein.

26. Thanks to Dr. Ivet Bahar Dr. Judith Klein-Seetharaman My Advisors: Post-Docs: Dr. A.J. Rader Dr. Dror Tobi