Interplay of Rotational , Relaxational , and Shear Dynamics in Solid 4 He

(2004). 4. R. K. Jha et al., J. Mol. Biol. 400, 257 (2010). 5. P. S. Huang, J. J. Love, S. L. Mayo, Protein Sci. 16, 2770 (2007). 6. J. Karanicolas et al., Mol. Cell 42, 250 (2011). 7. S. Liu et al., Proc. Natl. Acad. Sci. U.S.A. 104, 5330 (2007). 8. Writing Committee of the WHO Consultation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza, N. Engl. J. Med. 362, 1708 (2010). 9. J. Sui et al., Nat. Struct. Mol. Biol. 16, 265 (2009). 10. D. C. Ekiert et al., Science 324, 246 (2009). 11. Group 1 includes 10 of the 16 HA subtypes: H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16. Group 2 includes the remaining six subtypes: H3, H4, H7, H10, H14, and H15. 12. L. Lo Conte, C. Chothia, J. Janin, J. Mol. Biol. 285, 2177 (1999). 13. T. Clackson, J. A. Wells, Science 267, 383 (1995). 14. Materials and methods are available as supporting material on Science Online. 15. M. G. Rossmann, J. Biol. Chem. 264, 14587 (1989). 16. J. Chen, J. J. Skehel, D. C. Wiley, Proc. Natl. Acad. Sci. U.S.A. 96, 8967 (1999). 17. The other hot spot residues (HS1 and HS2) differed from the side chains observed in the crystal structures in their conformation or identity. Each hot spot residue was further diversified by constructing all conformations, the terminal atoms of which coincided with those modeled above. For instance, for HS3, these consisted of all Tyr conformations that matched the position of the aromatic ring and hydrogen bond. This diversification step produced a “fan” of backbone positions for each residue in the hot spot libraries. 18. Proteins in the scaffold set contained no disulfides, were expressed in E. coli, and were predicted to form monomers (14). 19. D. Schneidman-Duhovny, Y. Inbar, R. Nussinov, H. J. Wolfson, Nucleic Acids Res. 33 (Web Server issue), W363 (2005). 20. J. J. Gray et al., J. Mol. Biol. 331, 281 (2003). 21. B. Kuhlman et al., Science 302, 1364 (2003). 22. G. Chao et al., Nat. Protoc. 1, 755 (2006). 23. A third design HB35 bound HA at apparent low-micromolar affinity; however, binding was only partially abolished upon coincubation of HA with the CR6261 Fab, which indicated, at most, partial contact with the target surface on the stem region of HA, and so this design was eliminated from further consideration (fig. S7). A handful of other designs bound HA, albeit weakly and with incomplete reproducibility (14). 24. We recorded dissociation constants using two main methods: by titration of HA against yeast surface-displayed designs and by fitting both kinetic and equilibrium measurements using surface plasmon resonance. As there is a discrepancy in determining Kd values between the methods, measurements derived from yeast surface-display titrations are listed as apparent Kd and should be viewed qualitatively (14). 25. C. E. Stevenson et al., Proteins 65, 1041 (2006). 26. R. Das, D. Baker, Annu. Rev. Biochem. 77, 363 (2008). 27. The alanine-scan mutations were as follows: for HB36.3, Phe, Met, and Trp; for HB80.1 Phe, Phe, and Tyr (table S4 and SOM text). 28. HB36.3 was not able to block the pH-induced conformational changes in the H1 and H5 HAs under identical assay conditions, even though HB36.3 and HB80.3 have very similar dissociation constants and kinetic off-rates at pH 7.5 (fig. S12 and SOM text). 29. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. Acknowledgments. Computational designs were generated on resources generously provided by participants of Rosetta @ Home and the Argonne National Leadership Computing Facility. We thank J. L. Gallaher for protein preparation. S.J.F. was supported by a long-term fellowship from the Human Frontier Science Program, J.E.C. was supported by the Jane Coffin Childs Memorial Fund, and E.M.S. by a career development award from the National Institute of Allergy and Infectious Diseases, NIH, AI057141. Research in the Baker laboratory was supported by grants from the Defense Advanced Research Projects Agency, the NIH yeast resource center, the Defense Threat Reduction Agency, and the Howard Hughes Medical Institute and in the Wilson laboratory by NIH AI058113, predoctoral fellowships from the Achievement Rewards for College Scientists Foundation and the NIH Molecular Evolution Training Program GM080209 (D.C.E.), and the Skaggs Institute for Chemical Biology. X-ray diffraction data sets were collected at the Stanford Synchrotron Radiation Lightsource beamline 9-2 and at the Advanced Photon Source (APS) beamline 23ID-B (GM/CA-CAT). The GM/CA CAT 23-ID-B beamline has been funded in whole or in part with federal funds from National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science, NIH (Y1-GM-1104). Use of the APS was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357. Coordinates and structure factors were deposited in the Protein Data Bank as entry 3R2X.