Protein Design pp 161-182 | Cite as

Symmetric Protein Architecture in Protein Design: Top-Down Symmetric Deconstruction

  • Liam M. Longo
  • Michael Blaber
Part of the Methods in Molecular Biology book series (MIMB, volume 1216)


Top-down symmetric deconstruction (TDSD) is a joint experimental and computational approach to generate a highly stable, functionally benign protein scaffold for intended application in subsequent functional design studies. By focusing on symmetric protein folds, TDSD can leverage the dramatic reduction in sequence space achieved by applying a primary structure symmetric constraint to the design process. Fundamentally, TDSD is an iterative symmetrization process, in which the goal is to maintain or improve properties of thermodynamic stability and folding cooperativity inherent to a starting sequence (the “proxy”). As such, TDSD does not attempt to solve the inverse protein folding problem directly, which is computationally intractable. The present chapter will take the reader through all of the primary steps of TDSD—selecting a proxy, identifying potential mutations, establishing a stability/folding cooperativity screen—relying heavily on a successful TDSD solution for the common β-trefoil fold.

Key words

Symmetric protein design Protein folding Protein engineering Phi-value analysis β-trefoil Protein evolution 


  1. 1.
    Longo LMB, Blaber M (2012) Protein design—a vast unexploited resource. J Protein Proteonomics 3:78–83Google Scholar
  2. 2.
    Yue K, Dill KA (1992) Inverse protein folding problem: designing polymer sequences. Proc Natl Acad Sci U S A 89:4163–4167PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Koga N, Tatsumi-Koga R, Liu G, Xiao R, Acton TB, Montelione GT et al (2012) Principles for designing ideal protein structures. Nature 491:222–227PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Blaber M, Lee J (2012) Designing proteins from simple motifs: opportunities in top-down symmetric deconstruction. Curr Opin Struct Biol 22:442–450PubMedCrossRefGoogle Scholar
  5. 5.
    Lee J, Blaber SI, Dubey VK, Blaber M (2011) A polypeptide “building block” for the ß-trefoil fold identified by “top-down symmetric deconstruction”. J Mol Biol 407:744–763PubMedCrossRefGoogle Scholar
  6. 6.
    Fleishman SJ, Whitehead TA, Ekiert DC, Dreyfus C, Corn JE, Strauch E-M et al (2011) Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science 332:816–821PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Jung J, Lee B (2001) Circularly permuted proteins in the protein structure database. Protein Sci 10:1881–1886PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Levy Y, Cho SS, Shen T, Onuchic JN, Wolynes PG (2005) Symmetry and frustration in protein energy landscapes: a near degeneracy resolves the Rop dimer-folding mystery. Proc Natl Acad Sci U S A 102:2373–2378PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Seitz T, Bocola M, Claren J, Sterner R (2007) Stabilization of a (beta-alpha)8-barrel protein designed from identical half barrels. J Mol Biol 372:114–129PubMedCrossRefGoogle Scholar
  10. 10.
    Fortenberry C, Bowman EA, Proffitt W, Dorr B, Combs S, Harp J et al (2011) Exploring symmetry as an avenue to the computational design of large protein domains. J Am Chem Soc 133:18026–18029PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Yadid I, Tawfik DS (2011) Functional β-propeller lectins by tandem duplications of repetitive units. Protein Eng Des Sel 24:185–195PubMedCrossRefGoogle Scholar
  12. 12.
    Lee J, Blaber M (2011) Experimental support for the evolution of symmetric protein architecture from a simple peptide motif. Proc Natl Acad Sci U S A 108:126–130PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Brych SR, Dubey VK, Bienkieicz E, Lee J, Logan TM, Blaber M (2004) Symmetric primary and tertiary structure mutations within a symmetric superfold: a solution, not a constraint, to achieve a foldable polypeptide. J Mol Biol 344:769–780PubMedCrossRefGoogle Scholar
  14. 14.
    Broom A, Doxey AC, Lobsanov YD, Berthin LG, Rose DR, Howell PL et al (2012) Modular evolution and the origins of symmetry: reconstruction of a three-fold symmetric globular protein. Structure 20:161–171PubMedCrossRefGoogle Scholar
  15. 15.
    Fukuchi S, Nishikawa K (2001) Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria. J Mol Biol 309:835–843PubMedCrossRefGoogle Scholar
  16. 16.
    Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng 13:179–191PubMedCrossRefGoogle Scholar
  17. 17.
    Fukuchi S, Yoshimune K, Wakayama M, Moriguchi M, Nishikawa K (2003) Unique amino acid composition of proteins in halophilic bacteria. J Mol Biol 327:347–357PubMedCrossRefGoogle Scholar
  18. 18.
    Oren A, Larimer F, Richardson P, Lapidus A, Csonka LN (2005) How to be moderately halophilic with broad salt tolerance: clues from the genome of Chromohalobacter salexigens. Extremophiles 9:275–279PubMedCrossRefGoogle Scholar
  19. 19.
    Kennedy SP, Ng WV, Salzberg SL, Hood L, DasSarma S (2001) Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence. Genome Res 11:1641–1650PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Schreiber G, Buckle AM, Fersht AR (1994) Stability and function: two constraints in the evolution of barstar and other proteins. Structure 2:945–951PubMedCrossRefGoogle Scholar
  21. 21.
    Shoichet BK, Baase WA, Kuroki R, Matthews BW (1995) A relationship between protein stability and protein function. Proc Natl Acad Sci U S A 92:452–456PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Longo L, Lee J, Blaber M (2012) Experimental support for the foldability-function tradeoff hypothesis: segregation of the folding nucleus and functional regions in FGF-1. Protein Sci 21:1911–1920PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Capraro DT, Gosavi S, Roy M, Onuchic JN, Jennings PA (2012) Folding circular permutants of IL-1beta: route selection driven by functional frustration. PLoS One 7:e38512PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Gosavi S, Chavez LL, Jennings PA, Onuchic JN (2006) Topological frustration and the folding of interleukin-1β. J Mol Biol 357: 986–996PubMedCrossRefGoogle Scholar
  25. 25.
    Gosavi S, Whitford PC, Jennings PA, Onuchic JN (2008) Extracting function from a beta-trefoil folding motif. Proc Natl Acad Sci U S A 105:10384–10389PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Serrano L, Matouschek A, Fersht AR (1992) The folding of an enzyme. III. Structure of the transition state for unfolding of barnase analysed by a protein engineering procedure. J Mol Biol 224:805–818PubMedCrossRefGoogle Scholar
  27. 27.
    Lowe AR, Itzhaki LS (2007) Rational redesign of the folding pathway of a modular protein. Proc Natl Acad Sci U S A 104(8):2679–2684PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Liu C, Gaspar JA, Wong HJ, Meiering EM (2002) Conserved and nonconserved features of the folding pathway of hisactophilin, a β-trefoil protein. Protein Sci 11:669–679PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Richter M, Bosnali M, Carstensen L, Seitz T, Durchschlag H, Blanquart S et al (2010) Computational and experimental evidence for the evolution of a (βα)8-barrel protein from an ancestral quarter-barrel stabilized by disulfide bonds. J Mol Biol 398:763–773PubMedCrossRefGoogle Scholar
  30. 30.
    Carstensen L, Sperl JM, Bocola M, List F, Schmid FX, Sterner R (2012) Conservation of the folding mechanism between designed primordial (βα)8-barrel proteins and their modern descendant. J Am Chem Soc 134:12786–12791PubMedCrossRefGoogle Scholar
  31. 31.
    Yadid I, Tawfik DS (2007) Reconstruction of functional β-propeller lectins via homo-oligomeric assembly of shorter fragments. J Mol Biol 365:10–17PubMedCrossRefGoogle Scholar
  32. 32.
    Pace CN, Trevino S, Prabhakaran E, Scholtz JM (2004) Protein structure, stability and solubility in water and other solvents. Philos Trans R Soc Lond B Biol Sci 359:1225–1234, discussion 1234–1225PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Barrick D (2009) What have we learned from the studies of two-state folders, and what are the unanswered questions about two-state protein folding? Phys Biol 6:015001PubMedCrossRefGoogle Scholar
  34. 34.
    Lee J, Blaber M (2009) The interaction between thermostability and buried free cysteines in regulating the functional half-life of fibroblast growth factor-1. J Mol Biol 393: 113–127PubMedCrossRefGoogle Scholar
  35. 35.
    Blaber SI, Culajay JF, Khurana A, Blaber M (1999) Reversible thermal denaturation of human FGF-1 induced by low concentrations of guanidine hydrochloride. Biophys J 77:470–477PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Copeland RA, Halfpenny AJ, Williams RW, Thompson KC, Herber WK et al (1991) The structure of human acidic fibroblast growth factor and its interaction with heparin. Arch Biochem Biophys 289:53–61PubMedCrossRefGoogle Scholar
  37. 37.
    Larson SM, Ruczinski I, Davidson AR, Baker D, Plaxco KW (2002) Residues participating in the protein folding nucleus do not exhibit preferential evolutionary conservation. J Mol Biol 316:225–233PubMedCrossRefGoogle Scholar
  38. 38.
    Nickson AA, Clarke J (2010) What lessons can be learned from studying the folding of homologous proteins? Methods 52:38–50PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Beadle BM, Shoichet BK (2002) Structural basis of stability–function tradeoffs in enzymes. J Mol Biol 321:285–296PubMedCrossRefGoogle Scholar
  40. 40.
    Rubini M, Lepthie S, Golbik R, Budisa N (2006) Aminotryptophan-containing barstar: structure-function tradeoff in protein design and engineering with an expanded genetic code. Biochim Biophys Acta 1764:1147–1158PubMedCrossRefGoogle Scholar
  41. 41.
    Steipe B, Schiller B, Pluckthun A, Steinbacher S (1994) Sequence statistics reliably predict stabilizing mutations in a protein domain. J Mol Biol 240:188–192PubMedCrossRefGoogle Scholar
  42. 42.
    Lehmann M, Kostrewa D, Wyss M, Brugger R, D’Arcy A, Pasamontes L et al (2000) From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Protein Eng 13:49–57PubMedCrossRefGoogle Scholar
  43. 43.
    Sullivan BJ, Nguyen T, Durani V, Mathur D, Rojas S, Thomas M et al (2012) Stabilizing proteins from sequence statistics: the interplay of conservation and correlation in triosephosphate isomerase stability. J Mol Biol 420:384–399PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Lee J, Dubey VK, Longo LM, Blaber M (2008) A logical OR redundancy with the Asx-Pro-Asx-Gly type I β-turn motif. J Mol Biol 377:1251–1264PubMedCrossRefGoogle Scholar
  45. 45.
    Karpusas M, Baase WA, Matsumura M, Matthews BW (1989) Hydrophobic packing in T4 lysozyme probed by cavity-filling mutants. Proc Natl Acad Sci U S A 86:8237–8241PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Lassalle MW, Yamada H, Morii H, Ogata K, Sarai A, Akasaka K (2001) Filling a cavity dramatically increases pressure stability of the c-Myb R2 subdomain. Proteins 45:96–101PubMedCrossRefGoogle Scholar
  47. 47.
    Bernett MJ, Somasundaram T, Blaber M (2004) An atomic resolution structure for human fibroblast growth factor 1. Proteins 57:626–634PubMedCrossRefGoogle Scholar
  48. 48.
    Brych SR, Blaber SI, Logan TM, Blaber M (2001) Structure and stability effects of mutations designed to increase the primary sequence symmetry within the core region of a β-trefoil. Protein Sci 10:2587–2599PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    Ponder JW, Richards FM (1987) Tertiary templates for proteins—use of packing criteria in the enumeration of allowed sequences for different structural classes. J Mol Biol 193: 775–791PubMedCrossRefGoogle Scholar
  50. 50.
    Lesk AM, Branden CL, Chothia C (1989) Structural principles of alpha/beta barrel proteins: the packing of the interior of the sheet. Proteins 5:139–148PubMedCrossRefGoogle Scholar
  51. 51.
    Sandberg WS, Terwilliger TC (1991) Energetics of repacking a protein interior. Proc Natl Acad Sci U S A 88:1706–1710PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Ross SA, Sarisky CA, Su A, Mayo SL (2001) Designed protein g core variants fold to native-like structures: sequence selection by orbit tolerates variation in backbone specification. Protein Sci 10:450–454PubMedCrossRefPubMedCentralGoogle Scholar
  53. 53.
    Zou J, Saven JG (2000) Statistical theory of combinatorial libraries of folding proteins: energetic discrimination of a target structure. J Mol Biol 296:281–294PubMedCrossRefGoogle Scholar
  54. 54.
    Dantas G, Corrent C, Reichow SL, Havranek JJ, Eletr ZM, Isern NG et al (2007) High-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design. J Mol Biol 366:1209–1221PubMedCrossRefPubMedCentralGoogle Scholar
  55. 55.
    Pokala N, Handel TM (2004) Energy functions for protein design i: efficient and accurate continuum electrostatics and solvation. Protein Sci 13:925–936PubMedCrossRefPubMedCentralGoogle Scholar
  56. 56.
    Wisz MS, Hellinga HW (2003) An empirical model for electrostatic interactions in proteins incorporating multiple geometry-dependent dielectric constants. Proteins 51:360–377PubMedCrossRefGoogle Scholar
  57. 57.
    Jain T, Cerutti DS, McCammon JA (2006) Configurational-bias sampling technique for predicting side-chain conformations in proteins. Protein Sci 15:2029–2039PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Shaw KL, Scholtz JM, Pace CN, Grimsley GR (2009) Determining the conformational stability of a protein using urea denaturation curves. Methods Mol Biol 490:41–55PubMedCrossRefGoogle Scholar
  59. 59.
    Myers JK, Pace CN, Scholtz JM (1995) Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci 4: 2138–2148PubMedCrossRefPubMedCentralGoogle Scholar
  60. 60.
    Sanchez IE, Kiefhaber T (2003) Evidence of sequential barriers and obligatory intermediates in apparent two-state protein folding. J Mol Biol 325:367–376PubMedCrossRefGoogle Scholar
  61. 61.
    Sanchez IE, Kiefhaber T (2003) Hammond behavior versus ground state effects in protein folding: evidence for narrow free energy barriers and residual structure in unfolded states. J Mol Biol 327:867–884PubMedCrossRefGoogle Scholar
  62. 62.
    Aksel T, Majumdar A, Barrick D (2011) The contribution of entropy, enthalpy, and hydrophobic desolvation to cooperativity in repeat-protein folding. Structure 19:349–360PubMedCrossRefPubMedCentralGoogle Scholar
  63. 63.
    Chowdhry BZ, Cole SC (1989) Differential scanning calorimetry: applications in biotechnology. Trends Biotechnol 7:11–18CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Biomedical Sciences, College of MedicineFlorida State UniversityTallahasseeUSA

Personalised recommendations