Journal of Molecular Evolution

, Volume 87, Issue 1, pp 27–36 | Cite as

Biophysical Spandrels form a Hot-Spot for Kosmotropic Mutations in Bacteriophage Thermal Adaptation

  • A. Carl WhittingtonEmail author
  • Darin R. Rokyta
Original Article


Temperature plays a dominating role in protein structure and function, and life has evolved myriad strategies to adapt proteins to environmental thermal stress. Cellular systems can utilize kosmotropic osmolytes, the products of complex biochemical pathways, to act as chemical chaperones. These extrinsic molecules, e.g., trehalose, alter local water structure to modulate the strength of the hydrophobic effect and increase protein stability. In contrast, simpler genetic systems must rely on intrinsic mutation to affect protein stability. In naturally occurring microvirid bacteriophages of the subfamily Bullavirinae, capsid stability is randomly distributed across the phylogeny, suggesting it is not phylogenetically linked and could be altered through adaptive mutation. We hypothesized that these phages could utilize an adaptive mechanism that mimics the stabilizing effects of the kosmotrope trehalose through mutation. Kinetic stability of wild-type ID8, a relative of ΦX174, displays a saturable response to trehalose. Thermal adaptation mutations in ID8 improve capsid stability and reduce responsiveness to trehalose suggesting the mutations move stability closer to the kosmotropic saturation point, mimicking the kosmotropic effect of trehalose. These mutations localize to and modulate the hydrophobicity of a cavern formation at the interface of phage coat and spike proteins—an evolutionary spandrel. Across a series of genetically distinct phages, responsiveness to trehalose correlates positively with cavern hydrophobicity suggesting that the level of hydrophobicity of the cavern may provide a biophysical gating mechanism constraining or permitting adaptation in a lineage-specific manner. Our results demonstrate that a single mutation can exploit pre-existing, non-adaptive structural features to mimic the adaptive effects of complex biochemical pathways.


Protein adaptation Evolutionary biophysics Hydrophobic effect Historical contingency Microvirus 



Funding for this work was provided by the U.S. National Institutes of Health to D.R.R. (R01 GM-099723).

Supplementary material

239_2018_9882_MOESM1_ESM.txt (93 kb)
Supplementary material 1 (TXT 92 KB)
239_2018_9882_MOESM2_ESM.txt (41 kb)
Supplementary material 2 (TXT 41 KB)
239_2018_9882_MOESM3_ESM.txt (6 kb)
Supplementary material 3 (TXT 6 KB)
239_2018_9882_MOESM4_ESM.xlsx (11 kb)
Supplementary material 4 (XLSX 10 KB)
239_2018_9882_MOESM5_ESM.docx (83 kb)
Supplementary material 5 (DOCX 83 KB)


  1. Bauer DW, Li D, Huffman J, Homa FL, Wilson K, Leavitt JC, Casjens SR, Baines J, Evilevitch A (2015) Exploring the balance between DNA pressure and capsid stability in herpesviruses and phages. J Virol 89(18):9288–9298. Google Scholar
  2. Bell W, Sun W, Hohmann S, Wera S, Reinders A, De Virgilio C, Wiemken A, Thevelein JM (1998) Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex. J Biol Chem 273(50):33311–33319. Google Scholar
  3. Bernal RA, Hafenstein S, Olson NH, Bowman VD, Chipman PR, Baker TS, Fane BA, Rossmann MG (2003) Structural studies of bacteriophage α3 assembly. J Mol Biol 325(1):11–24. Google Scholar
  4. Bharadwaj A, Leelavathi S, Mazumdar-Leighton S, Ghosh A, Ramakumar S, Reddy VS (2008) The critical role of partially exposed N-terminal valine residue in stabilizing GH10 xylanase from Bacillus sp. NG-27 under poly-extreme conditions. PLoS ONE 3(8):e3063. Google Scholar
  5. Blomberg SP, Garland T, Ives AR (2003) Testing for phylogenetic signal in comparative data: behavioral traits are more labile. Evolution 57(4):717–745. Google Scholar
  6. Bolen DW, Baskakov IV (2001) The osmophobic effect: natural selection of a thermodynamic force in protein folding. J Mol Biol 310(5):955–963. Google Scholar
  7. Bruździak P, Panuszko A, Stangret J (2013) Influence of osmolytes on protein and water structure: a step to understanding the mechanism of protein stabilization. J Phys Chem B 117(39):11502–11508. Google Scholar
  8. Cheng YK, Rossky PJ (1998) Surface topography dependence of biomolecular hydrophobic hydration. Nature 392(6677):696–699. Google Scholar
  9. Cheng X, Imai T, Teeka J, Hirose M, Higuchi T, Sekine M (2013) Inactivation of bacteriophages by high levels of dissolved CO2. Environ Technol 34(1–4):539–544. Google Scholar
  10. Crosby T (1994) How to detect and handle outliers, vol 36. ASQC Quality Press,
  11. Darriba D, Taboada GL, Doallo R, Posada D (2012) JModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9(8):772., arXiv:1011.1669v3Google Scholar
  12. Dill KA (1990) Dominant forces in protein folding. Biochemistry 29(31):7133–7155., arXiv:1011.1669v3Google Scholar
  13. Dill KA, Truskett TM (2005) Modeling water, the hydrophobic effect, and ion solvation. Annu Rev Biophys Biomol Struct 34(1):173–199. Google Scholar
  14. Doore SM, Fane BA (2016) The microviridae: diversity, assembly, and experimental evolution. Virology 491:45–55. Google Scholar
  15. Doore SM, Schweers NJ, Fane BA (2017) Elevating fitness after a horizontal gene exchange in bacteriophage φX174. Virology 501:25–34, Google Scholar
  16. Elbein AD, Pan YT, Pastuszak I, Carroll D (2003) New insights on trehalose: a multifunctional molecule. Glycobiology 13(4):17R–27. Google Scholar
  17. Fane B, Brentlinger K, Burch A, Chen M, Hafenstein S, Moore E, Novak C, Uchiyama A (2006) ΦwX174 et al., the Microviridae. In: Calendar R (ed) The Bacteriophages, Oxford University Press, Oxford, pp 129–145Google Scholar
  18. Ferreira LA, Breydo L, Reichardt C, Uversky VN, Zaslavsky BY (2017) Effects of osmolytes on solvent features of water in aqueous solutions. J Biomol Struct Dyn 35(5):1055–1068. Google Scholar
  19. Fields PA, Dong Y, Meng X, Somero GN (2015) Adaptations of protein structure and function to temperature: there is more than one way to ‘skin a cat’. J Exp Biol 218(12):1801–1811. Google Scholar
  20. Francois J, Parrou JL (2001) Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 25(1):125–145. Google Scholar
  21. Funahashi J, Takano K, Yamagata Y, Yutani K (2000) Role of surface hydrophobic residues in the conformational stability of human lysozyme at three different positions. Biochemistry 39(47):14448–14456. 456Google Scholar
  22. Gould SJ (1997) The exaptive excellence of spandrels as a term and prototype. Proc Natl Acad Sci USA 94(20):10750–10755, Google Scholar
  23. Gould SJ, Lewontin RC (1979) The spandrels of San Marco and the Panglossian paradigm: a critique of the adaptationist programme. Proc R Soc London Biol Sci 205(1161):581–598., 9.23Google Scholar
  24. Guan D, Kniel K, Calci K, Hicks D, Pivarnik L, Hoover D (2006) Response of four types of coliphages to high hydrostatic pressure. Food Microbiol 23(6):546–551. Google Scholar
  25. Harmon LJ, Baumes J, Hughes C, Soberon J, Specht CD, Turner W, Lisle C, Thacker RW (2013) Arbor: comparative analysis workflows for the tree of life. PLoS Curr 5,
  26. Hochberg GKA, Thornton JW (2017) Reconstructing ancient proteins to understand the causes of structure and function. Annu Rev Biophys 46(1):247–269. Google Scholar
  27. Hummer G, Garde S, Garcıa AE, Pratt LR (2000) New perspectives on hydrophobic effects. Chem Phys 258(2–3):349–370. Google Scholar
  28. Ilag LL, McKenna R, Yadav MP, BeMiller JN, Incardona NL, Rossmann MG (1994) Calcium ion-induced structural changes in bacteriophage φX174. J Mol Biol 244(3):291–300. Google Scholar
  29. Kaushik JK, Bhat R (1998) Thermal stability of proteins in aqueous polyol solutions: role of the surface tension of water in the stabilizing effect of polyols. J Phys Chem B 102(98):7058–7066. Google Scholar
  30. Kaushik JK, Bhat R (2003) Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose. J Biol Chem 278(29):26458–26465. Google Scholar
  31. Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng Des Sel 13(3):179–191. Google Scholar
  32. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157(1):105–132., arXiv:1407.5140v1Google Scholar
  33. Lee KH, Miller CR, Nagel AC, Wichman HA, Joyce P, Ytreberg FM (2011) First-step mutations for adaptation at elevated temperature increase capsid stability in a virus. PLoS ONE 6(9):e25640, Google Scholar
  34. Levy Y, Onuchic JN (2006) Water mediation in protein folding and molecular recognition. Annu Rev Biophys Biomol Struct 35(1):389–415. Google Scholar
  35. Lumry R, Eyring H (1954) Conformation changes of proteins. J Phys Chem 58(2):110–120. Google Scholar
  36. Ma Y, Nolte RJ, Cornelissen JJ (2012) Virus-based nanocarriers for drug delivery. Adv Drug Deliv Rev 64(9):811–825. Google Scholar
  37. Machius M, Declerck N, Huber R, Wiegand G (2003) Kinetic stabilization of Bacillus licheniformis α-amylase through introduction of hydrophobic residues at the surface. J Biol Chem 278(13):11,546– 11,553. Google Scholar
  38. Mattos C (2002) Protein–water interactions in a dynamic world. Trends Biochem Sci 27(4):203–208. Google Scholar
  39. McGee LW, Aitchison EW, Caudle SB, Morrison AJ, Zheng L, Yang W, Rokyta DR (2014) Payoffs, not tradeoffs, in the adaptation of a virus to ostensibly conflicting selective pressures. PLoS Genet 10(10):e1004611, Google Scholar
  40. McGee LW, Sackman AM, Morrison AJ, Pierce J, Anisman J, Rokyta DR (2016) Synergistic pleiotropy overrides the costs of complexity in viral adaptation. Genetics 202(1):285–295. Google Scholar
  41. McKenna R, Xia D, Willingmann P, Ilag LL, Krishnaswamy S, Rossmann MG, Olson NH, Baker TS, Incardona NL (1992) Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications. Nature 355(6356):137–143. Google Scholar
  42. McKenna R, Bowman BR, Ilag LL, Rossmann MG, Fane BA (1996) Atomic structure of the degraded procapsid particle of the bacteriophage G4: induced structural changes in the presence of calcium ions and functional implications. J Mol Biol 256(4):736–750. Google Scholar
  43. Miyawaki O, Dozen M, Nomura K (2014) Thermodynamic analysis of osmolyte effect on thermal stability of ribonuclease A in terms of water activity. Biophys Chem 185:19–24. Google Scholar
  44. Nguyen V, Wilson C, Hoemberger M, Stiller JB, Agafonov RV, Kutter S, English J, Theobald DL, Kern D (2017) Evolutionary drivers of thermoadaptation in enzyme catalysis. Science 355(6322):289–294,
  45. Nurmemmedov E, Castelnovo M, Catalano CE, Evilevitch A (2007) Biophysics of viral infectivity: matching genome length with capsid size. Q Rev Biophys 40(04):327–356. Google Scholar
  46. Pace CN, Fu H, Fryar KL, Landua J, Trevino SR, Shirley BA, Hendricks MMN, Iimura S, Gajiwala K, Scholtz JM, Grimsley GR (2011) Contribution of hydrophobic interactions to protein stability. J Mol Biol 408(3):514–528. Google Scholar
  47. Pagel M (1999) Inferring the historical patterns of biological evolution. Nature 401(6756):877–884. Google Scholar
  48. Pepin KM, Domsic J, McKenna R (2008) Genomic evolution in a virus under specific selection for host recognition. Infect Genet Evol 8(6):825–834. Google Scholar
  49. Purohit PK, Inamdar MM, Grayson PD, Squires TM, Kondev J, Phillips R (2005) Forces during bacteriophage DNA packaging and ejection. Biophys J 88(2):851–866. Google Scholar
  50. Robinson DF, Foulds LR (1981) Comparison of phylogenetic trees. Math Biosci 53(1–2):131–147., arXiv:0708.3499v1Google Scholar
  51. Robinson O, Dylus D, Dessimoz C (2016) interactive viewing and comparison of large phylogenetic trees on the web. Mol Biol Evol 33(8):2163–2166., 1602.04258Google Scholar
  52. Rohovie MJ, Nagasawa M, Swartz JR (2017) Virus-like particles: next-generation nanoparticles for targeted therapeutic delivery. Bioeng Transl Med 2(1):43–57. Google Scholar
  53. Rokyta DR, Burch CL, Caudle SB, Wichman HA (2006) Horizontal gene transfer and the evolution of microvirid coliphage genomes horizontal gene transfer and the evolution of microvirid coliphage genomes. J Bacteriol 188(3):1134–1142. Google Scholar
  54. Rokyta DR, Abdo Z, Wichman HA (2009) The genetics of adaptation for eight microvirid bacteriophages. J Mol Evol 69(3):229–239. Google Scholar
  55. Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, H¨ohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) Mrbayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3):539–542. Google Scholar
  56. Roos WH, Ivanovska IL, Evilevitch A, Wuite GJL (2007) Viral capsids: mechanical characteristics, genome packaging and delivery mechanisms. Cell Mol Life Sci 64(12):1484–1497. Google Scholar
  57. Sackman AM, Rokyta DR (2013) The adaptive potential of hybridization demonstrated with bacteriophages. J Mol Evol 77(5–6):221–230., (NIHMS150003)Google Scholar
  58. Sackman AM, Rokyta DR (2018) Additive phenotypes underlie epistasis of fitness effects. Genetics 208(1):339–348. Google Scholar
  59. Sackman AM, Reed D, Rokyta DR (2015) Intergenic incompatibilities reduce fitness in hybrids of extremely closely related bacteriophages. PeerJ 3:e1320. Google Scholar
  60. Šali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234(3):779–815. Google Scholar
  61. Sanchez-Ruiz JM (2010) Protein kinetic stability. Biophys Chem 148(1–3):1–15. Google Scholar
  62. Schellman JA (1997) Temperature, stability, and the hydrophobic interaction. Biophys J 73(6):2960–2964. Google Scholar
  63. Schwehm JM, Kristyanne ES, Biggers CC, Stites WE (1998) Stability effects of increasing the hydrophobicity of solvent-exposed side chains in staphylococcal nuclease. Biochemistry 37(19):6939–6948. Google Scholar
  64. Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L (2005) The FoldX web server: an online force field. Nucleic Acids Res 33(SUPPL. 2):382–388. Google Scholar
  65. Sharma M, Shearer AE, Hoover DG, Liu MN, Solomon MB, Kniel KE (2008) Comparison of hydrostatic and hydrodynamic pressure to inactivate foodborne viruses. Innov Food Sci Emerg Technol 9(4):418–422. Google Scholar
  66. Sharp KA, Madan B (1997) Hydrophobic effect, water structure, and heat capacity changes. J Phys Chem B 101(21):4343–4348. Google Scholar
  67. Singer MA, Lindquist S (1998) Thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of trehalose. Trends Biotechnol 16(11):460–468. Google Scholar
  68. Somero GN (2003) Protein adaptations to temperature and pressure: complementary roles of adaptive changes in amino acid sequence and internal milieu. Comp Biochem Physiol B Biochem Mol Biol 136(4):577–591. Google Scholar
  69. Somero GN (2010) The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine ‘winners’ and ‘losers’. J Exp Biol 213(6):912–920. Google Scholar
  70. Starr TN, Thornton JW (2016) Epistasis in protein evolution. Protein Sci 25(7):1204–1218. Google Scholar
  71. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680., arXiv:1011.1669v3Google Scholar
  72. Timasheff SN (1998) Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated. Adv Protein Chem 51:355–432Google Scholar
  73. Tisi LC, Evans PA (1995) Conserved structural features on protein surfaces: Small exterior hydrophobic clusters. J Mol Biol 249(2):251–258. Google Scholar
  74. Van den Burg B, Dijkstra BW, Vriend G, Van der Vinne B, Venema G, Eijsink VG (1994) Protein stabilization by hydrophobic interactions at the surface. Eur J Biochem 220(3):981–985Google Scholar
  75. Viña J (2002) Biochemical adaptation: mechanism and process in physiological evolution, vol 30. Oxford University Press, Oxford. Google Scholar
  76. Vo HT, Imai T, Ho TT, Sekine M, Kanno A, Higuchi T, Yamamoto K, Yamamoto H (2014) Inactivation effect of pressurized carbon dioxide on bacteriophage Qβ and ΦX174 as a novel disinfectant for water treatment. J Environ Sci 26(6):1301–1306. Google Scholar
  77. Wichman HA, Brown CJ (2010) Experimental evolution of viruses: microviridae as a model system. Philos Trans R Soc B Biol Sci 365(1552):2495–2501. Google Scholar
  78. Xie G, Timasheff SN (1997) The thermodynamic mechanism of protein stabilization by trehalose. Biophys Chem 64(1–3):25–43. Google Scholar
  79. Yancey PH (2005) Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J Exp Biol 208(15):2819–2830. Google Scholar
  80. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217(4566):1214–1222. Google Scholar
  81. Yildiz I, Shukla S, Steinmetz NF (2011) Applications of viral nanoparticles in medicine. Curr Opin Biotechnol 22(6):901–908. Google Scholar
  82. Závodszky P, Hajdú I (2013) Evolution of the concept of conformational dynamics of enzyme functions over half of a century: a personal view. Biopolymers 99(4):263–269. Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biological ScienceFlorida State UniversityTallahasseeUSA

Personalised recommendations