Six plasmids for NC5 sample expression and 2D [ 1 H, 15 N] HSQC screening Rossmann2x3_58: OR25

1. Red: Design Red: Design Green: NMR, PDB 2KPO Green: NMR, PDB 2KI8 H2 L1 F44 L41 V31 L37 Y3 L5 I7 V4 L2 I14 L28 I6 H1 NMR:green, PDB 2KI8 Design:red L69 H3 I75 A70 A66 I51 V57 L63 I55 V53 V82 L54 L56 F95 A88 H4 I92 • NMR Structures of De Novo Designed “Ideal Structure” Proteins β4 β3 β1 β1 Gaohua Liu1, Nobuyasu Koga2, Rie Koga2, Rong Xiao1, Haleema Janjua1, Keith Hamilton1, Thomas Acton1, John Everett,1David Baker2, Gaetano T. Montelione1 1Department of Molecular Biology and Biochemistry, Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854; 2Department of Biochemistry and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195 II. NMR Screening and Structures • Background i) OR15,designed Ferredoxin-like protein, agree with design 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). RMSD=0.99Å • iii) OR28 and OR36 (not shown), designed Rossmann2x3 fold proteins, disagree with design ii) OR16, designed Rossmann2x2 fold protein, agree with design • Six plasmids for NC5 sample expression and 2D [1H, 15N] HSQC screening • Rossmann2x3_58: OR25 • Rossmann2x3_59: OR26 • Rossmann2x3_61: OR27 • Rossmann2x3_71: OR29 • Rossmann2x3_74: OR30 (no express) • Rossmann2x3_66: OR28 (best) • 134aa, 16kD, better HSQC at 308K • For Structure determination Design CS-Rosetta H2 H1 • Purpose • Establish rational methods to design structures de novo. • Reveal principle of protein folding how amino acid sequence determines native 3D-structure • Can be used as the base to introduce functional site • 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 15N labeled protein samples were prepared for selected protein candidates and were further screened by NESG at Rutgers using 1D NMR or 2D [15N-1H] - HSQC. • Suitable protein candidates were then selected for structure determination by NMR or/and X-ray. H3 β1 & β3 swapped NMR H4 RMSD of C1.06Å Rossmann2x3 (Flavodoxin-like) Ferredoxin-like Rossmann2x2 Rossmann3x3 iv) OR32, designed Rossmann3x3 fold protein, disagree with design • Ten unlabeled samples for 1D 1H NMR screening • NSM5(OR31) and NSM10(OR32) for NC5 label and 2D [1H,15N] HSQC screening • OR32 for structure determination Design OR15, PDB 2kl8 OR16, 2kpo OR28, 2l69 OR36, 2lci OR32, 2l82 CS-Rosetta β1 & β4 swapped NMR • 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 b-strands swapped compared to designed models. These NMR experimental structures provide unique valuable information on how to improve the protein designed 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, 1364-1368.