1. 03/27/09 NIH Roadmap Structure Determination of Membrane Proteins from Orientational Constraints Homayoun Valafar Roadmap Initiative Meeting 3/26/09-3/27/09

2. 03/27/09 NIH Roadmap REDCRAFT REsidual Dipolar Coupling based Residue Assembly and Filtering Tool (REDCRAFT)‏ A method for structure determination from orientational constraints Utilizes ideal peptide plane geometry Structure is determined entirely in rotamer space Can accommodate 6 RDCs /pp and scalar couplings Operates in two distinct stages: Stage-I and Stage-II CC C’ O N H CC O N CC  Plane1 Plane2 H HH

3. 03/27/09 NIH Roadmap Residual Dipolar Couplings Has substantially impacted the field of solution NMR Can be reintroduced by disrupting isotropic tumbling of the molecule Can be formulated in the following way r  Z H N

4. 03/27/09 NIH Roadmap Order Tensor Matrix Describes the global anisotropic alignment of a rigid molecule Has to be determined for study of structure/dynamics Can be decomposed to provide two types of information Orientation of Alignment Can be used to orient domains Strength of Alignment Can be used to probe internal motion

5. 03/27/09 NIH Roadmap Stage-I: Initial Filtering 3 J HN-H ± 2.5Hz Ranking based on RDC -30 -20 0.403711 -60 170 0.421467 -50 160 0.435446 -50 -80 13.7141 -60 -70 21.597 -70 120 23.8277 Takes place for any two adjacent peptide planes

6. 03/27/09 NIH Roadmap Challenges of Experimental RDC List 1 1296 searched 100 accepted List 2 1296 searched 58 accepted List 3 1296 searched 94 accepted

7. 03/27/09 NIH Roadmap REDCRAFT Stage-II: Fragment Extension Uses the results from Stage-I Any seed structure (initially a dipeptide) is extended one plane at a time Depth of search parameter is used to maintain tractability Combined List 5800 searched 200 accepted Search depth of 200 Combined List 18800 searched 200 accepted Search depth of 200 List 1 1296 searched 100 accepted List 2 1296 searched 58 accepted List 3 1296 searched 94 accepted

8. 03/27/09 NIH Roadmap Other Features Fragmented study of protein backbone: – Computationally efficient – Allows separate study of different regions with different properties – Comparison of the principal order parameters (POP) reveals relative motion Ability to handle average observables: – Can be utilized in application to C  -H  RDC or regions with known dynamics – Can be very beneficial in inclusion of CSA data – Can lead to structure calculation and refinement of multimers

9. 03/27/09 NIH Roadmap Example Structures Size: 56 residues. Data acquisition: 1 week. Data analysis: 2 hours. Results: 1.8 Å with X-ray Size: 70 residues. Data acquisition: 1 week. Data analysis: 1 Day. Results: C-terminal motion. Size: ~60 residues. Data acquisition: 1 week. Data analysis: 2 hours. Results: Unstructured Zn- binding region. ~1.5 Å with X-ray.

10. 03/27/09 NIH Roadmap Why Fragmented Study Allows proper treatment of gaps in data Allows study of internal dynamics Appropriate analysis of dynamics yields a more meaningful structure

11. 03/27/09 NIH Roadmap Static Structure Can generate a dynamic profile

12. 03/27/09 NIH Roadmap Dynamic Structure

13. 03/27/09 NIH Roadmap Minimum Data Requirement RDCs provide the benefit of establishing data requirement without the a-priori knowledge of the structure Total degrees of freedom for a fragment of size N peptide planes with RDCs collected from M media:

14. 03/27/09 NIH Roadmap Minimum Data Requirement

15. 03/27/09 NIH Roadmap Pf1 Coat Protein Backbone N-H RDCs from two alignment media Partial a-priori knowledge of structure Will the knowledge of secondary structure reduce minimum data requirement? Is there enough data for meaningful structure?

16. 03/27/09 NIH Roadmap Pf1 Structure from 2D-RDC Analysis 2D-RDC analysis can be used to determine the two order tensors without structure or assignment

17. 03/27/09 NIH Roadmap Pf1 Structure from REDCRAFT

18. 03/27/09 NIH Roadmap Acknowledgments Dr. Fang Tian (Co-PI) Paul Shealy Rishi Mukhodaphyay Mikhail Simin Elizabeth Timko Zach Swearingen Dr. Stanley Opella Dr. Sang Ho Park Funding provided by NIH-1R01GM081793