 Six designs (OR25, OR26, OR27, OR28, OR29, OR30) for 2D [ 1 H, 15 N] HSQC screening  OR28 for structure determination Gaohua Liu 1, Nobuyasu Koga 2, Rie Koga 2, Rong Xiao 1, Haleema Janjua 1, Keith Hamilton 1, Thomas Acton 1, John Everett 1, David Baker 2, Gaetano T. Montelione 1 1 Department of Molecular Biology and Biochemistry, Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854; 2 Department of Biochemistry and Howard Hughes Medical Institute, University of Washington, Seattle, WA NMR Structures of De Novo Designed “Ideal Structure” Proteins I.Background Structural characterization of designed proteins is a critical (and often neglected) step in validating computational design methodology. Many of the groups involved in computational protein design have limited resources for 3D structure determination, and structural genomics platforms are ideally suited for collaborative projects aimed at accelerating the field. Designed proteins are often relatively small, making them especially well suited to NMR structure determination. Computational design also often yields imperfect core packing (and marginal thermal stability), which may prevent crystallization, and also rendering NMR structure determination challenging due to exchange broadening effects. None the less, NMR has traditionally been invaluable for characterizing the structures of designed proteins (Kuhlman et al., 2003). Purpose  Establish rational methods to design structures de novo.  Reveal principles of how amino acid sequence determines native 3D-structure  Utilize designed structural scaffolds to introduce functional sites Targets  Four different folds were targeted Methods  Protein candidates with different primary sequences were computational designed and pre- selected based on computational energy at University of Washington.  Unlabeled or 15 N labeled protein samples were prepared for selected protein candidates and were further screened by NESG at Rutgers using 1D NMR or 2D [ 15 N- 1 H] - HSQC.  Suitable protein candidates were then selected for structure determination by NMR or/and X-ray. Ferredoxin-like OR15, PDB 2kl8 Rossmann2x2 OR16, 2kpo Rossmann2x3 (Flavodoxin-like) OR28, 2l69 OR36, 2lci Rossmann3x3 OR32, 2l82 II. NMR Screening and Structures ii) OR16, designed Rossmann2x2 fold protein, agrees with design H4 H1 H2 H3 F44 Y3 L1 V4 L5 I6 I7 L2 L41 L37 H1 H2 V31 I14 L28 H4 H3 F95 L54 L56 I92 L69 I75 A70 A66 V57 L63 I51 V53 V82 A88 I55 Red: Design Green: NMR, PDB 2KPO RMSD of C  1.06 Å Design:red NMR:green, PDB 2KI8 Red: Design Green: NMR, PDB 2KI8 RMSD of C  0.99 Å i) OR15, designed Ferredoxin-like protein, agrees with design iii) OR28 and OR36 (not shown), designed Rossmann2x3 fold proteins, disagree with design β1 & β3 swapped β1β1 β3β3 Design NMR CS-Rosetta iv) OR32, designed Rossmann3x3 fold protein, disagrees with design Ten unlabeled samples (NSM1-10) for 1D 1 H NMR screening NSM5(OR31) and NSM10(OR32) for 2D [ 1 H, 15 N] HSQC screening OR32 for structure determination β1 & β4 swapped β1β1 β4β4 Design RPF Precision Map III. Summary To date, solution NMR structures have been determined for five targets of four folds. The Ferredoxin-like protein (NESG ID OR15); Rossmann 2x2 fold protein OR16; Flavodoxin-like proteins OR28 and OR36, Rossmann 3x3 fold protein OR32. The experimental NMR structures of OR15 and OR16 are in excellent agreement with their designed models. However, structures of the three proteins OR28, OR36 and OR32 turn out to be P-loop NTPase fold structures that have two  -strands swapped compared to designed models. These NMR experimental structures provide unique valuable information on how to improve the protein design strategies. Reference: Kuhlman, B., Dantas, G., Ireton, G.C., Varani, G., Stoddard, B.L., and Baker, D. (2003). Design of a novel globular protein fold with atomic-level accuracy. Science 302, OR25 OR26 OR27 OR28 OR29 OR30, no expression NMR Red: Design Green: NMR, PDB 2L69 RMSD of C  ~3.4 Å Red: Design Green: NMR, PDB 2L82 RMSD of C  ~2.9 Å NMR OR31 OR32 NSM1NSM2NSM3NSM4 NSM6NSM5 NSM9 NSM10 NSM7NSM8 CS-Rosetta NMR RPF Precision Map  OR28 NMR structure turns out to be P-loop NTPase fold structure that has two  strands swapped compared to designed model.  OR32 NMR structure turns out to be P-loop NTPase fold structure that has two  strands swapped compared to designed model.