six plasmids for nc5 sample expression and 2d [ 1 h, 15 n] hsqc screening rossmann2x3_58: or25 ...

PowerPoint Presentation

Six plasmids for NC5 sample expression and 2D [1H, 15N] HSQC screeningRossmann2x3_58: OR25Rossmann2x3_59: OR26Rossmann2x3_61: OR27Rossmann2x3_71: OR29Rossmann2x3_74: OR30 (no express)Rossmann2x3_66: OR28 (best)134aa, 16kD, better HSQC at 308KFor Structure determination

Gaohua Liu1, Nobuyasu Koga2, Rie Koga2, Rong Xiao1, Haleema Janjua1, Keith Hamilton1, Thomas Acton1, John Everett,1 David Baker2, Gaetano T. Montelione11Department 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

NMR Structures of De Novo Designed Ideal Structure Proteins

BackgroundStructural 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). PurposeEstablish rational methods to design structures de novo.Reveal principle of protein folding how amino acid sequence determines native 3D-structureCan be used as the base to introduce functional site

TargetsFour different folds were targeted

MethodsProtein 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.

Ferredoxin-like

OR15, PDB 2kl8Rossmann2x2

OR16, 2kpoRossmann2x3(Flavodoxin-like)

OR28, 2l69 OR36, 2lciRossmann3x3

OR32, 2l82II. NMR Screening and Structures

ii) OR16, designed Rossmann2x2 fold protein, agree with designRMSD of C1.06

H4H1H2 H3

F44Y3L1V4L5I6I7L2L41L37H1H2V31I14L28

H4H3F95L54L56I92L69I75A70A66V57L63I51V53V82A88I55Red: DesignGreen: NMR, PDB 2KPO

RMSD=0.99

Design:redNMR:green, PDB 2KI8Red: DesignGreen: NMR, PDB 2KI8i) OR15, designed Ferredoxin-like protein, agree with design

iii) OR28 and OR36 (not shown), designed Rossmann2x3 fold proteins, disagree with design

1 & 3 swapped

1 3 Design NMR CS-Rosetta

iv) OR32, designed Rossmann3x3 fold protein, disagree with designTen unlabeled samples for 1D 1H NMR screeningNSM5(OR31) and NSM10(OR32) for NC5 label and 2D [1H,15N] HSQC screeningOR32 for structure determination

1 & 4 swapped

1 4 Design NMR CS-Rosetta

SummaryTo 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.