Skip to main content
SpringerLink
Log in

Engineering heme binding sites in monomeric rop

  • Original Paper
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

Heme ligands were introduced in the hydrophobic core of an engineered monomeric ColE1 repressor of primer (rop-S55) in two different layers of the heptad repeat. Mutants rop-L63M/F121H (layer 1) and rop-L56H/L113H (layer 3) were found to bind heme with a K D of 1.1 ± 0.2 and 0.47 ± 0.07 μM, respectively. The unfolding of heme-bound and heme-free mutants, in the presence of guanidinium hydrochloride, was monitored by both circular dichroism and fluorescence spectroscopy. For the heme-bound rop mutants, the total free energy change was 0.5 kcal/mol higher in the layer 3 mutant compared with that in the layer1 mutant. Heme binding also stabilized these mutants by increasing the \( \Updelta G_{\text{obs}}^{{{\text{H}}_{ 2} {\text{O}}}}, \) by 1.4 and 1.8 kcal/mol in rop-L63M/F121H and rop-L56H/L113H, respectively. The reduction potentials measured by spectroelectrochemical titrations were calculated to be −154 ± 2 mV for rop-56H/113H and −87.5 ± 1.2 mV for rop-L63M/F121H. The mutant designed to bind heme in a more buried environment (layer 3) showed tighter heme binding, a higher stability, and a different reduction potential compared with the mutant designed to bind heme in layer 1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Farinas E, Regan L (1998) Protein Sci 7:1939–1946

    Article  PubMed  CAS  Google Scholar 

  2. Benson DE, Wisz M, Liu W, Hellinga HW (1998) Biochemistry 37:7070–7076

    Article  PubMed  CAS  Google Scholar 

  3. Benson DE, Wisz MS, Hellinga HW (2000) Proc Natl Acad Sci USA 97:6292–6297

    Article  PubMed  CAS  Google Scholar 

  4. DeGrado WF, Summa CM, Pavone V, Nastri F, Lombardi A (1999) Annu Rev Biochem 68:779–819

    Article  PubMed  CAS  Google Scholar 

  5. Cowley AB, Kennedy ML, Silchenko S, Lukat-Rodgers GS, Rodgers KR, Benson DR (2006) Inorg Chem 45:9985–10001

    Article  PubMed  CAS  Google Scholar 

  6. Harris N, Presnell S, Cohen F (1994) J Mol Biol 236:1356–1368

    Article  PubMed  CAS  Google Scholar 

  7. Kamtekar S, Hecht MH (1995) FASEB J 9:1013–1022

    PubMed  CAS  Google Scholar 

  8. Grove A, Mutter M, Rivier JE, Montal M (1993) J Am Chem Soc 115:5919–5924

    Article  CAS  Google Scholar 

  9. Choma CT, Lear JD, Nelson MJ, Dutton PL, Robertson DE, DeGrado WF (1994) J Am Chem Soc 116:856–865

    Article  CAS  Google Scholar 

  10. Betz S, Liebman P, DeGrado WF (1997) Biochemistry 36:2450–2458

    Article  PubMed  CAS  Google Scholar 

  11. Robertson D, Farid R, Moser C, Urbauer J, Mulholland S, Pidikiti R, Lear J, Wand A, DeGrado WF, Dutton P (1994) Nature 368:425–431

    Article  PubMed  CAS  Google Scholar 

  12. Gibney B, Mulholland S, Rabanal F, Dutton P (1996) Proc Natl Acad Sci USA 93:15041–15046

    Article  PubMed  CAS  Google Scholar 

  13. Gibney BR, Johansson JS, Rabanal F, Skalicky JJ, Wand AJ, Dutton PL (1997) Biochemistry 36:2798–2806

    Article  PubMed  CAS  Google Scholar 

  14. Sharp R, Diers J, Bocian D, Dutton P (1998) J Am Chem Soc 120:7103–7104

    Article  CAS  Google Scholar 

  15. Cochran F, Wu S, Wang W, Nanda V, Saven J, Therien M, DeGrado WF (2005) J Am Chem Soc 127:1346–1347

    Article  PubMed  CAS  Google Scholar 

  16. Bender G, Lehmann A, Zou H, Cheng H, Fry H, Engel D, Therien M, Blasie J, Roder H, Saven J, DeGrado WF (2007) J Am Chem Soc 129:10732–10740

    Article  PubMed  CAS  Google Scholar 

  17. Monien B, Drepper F, Sommerhalter M, Lubitz W, Haehnel W (2007) J Mol Biol 371:739–753

    Article  PubMed  CAS  Google Scholar 

  18. Rau H, DeJonge N, Haehnel W (1998) Proc Natl Acad Sci USA 95:11526–11531

    Article  PubMed  CAS  Google Scholar 

  19. Topoglidis E, Discher BM, Moser CC, Dutton PL, Durrant JR (2003) Chembiochem 4:1332–1339

    Article  PubMed  CAS  Google Scholar 

  20. Willner I, Heleg-Shabtai V, Katz E, Rau H, Haehnel W (1999) J Am Chem Soc 121:6455–6468

    Article  CAS  Google Scholar 

  21. Lombardi A, Nastri F, Pavone V (2001) Chem Rev 101:3165–3189

    Google Scholar 

  22. Helmer-Citterich M, Anceschi M, Banner D, Cesareni G (1988) EMBO J 7:557–566

    PubMed  CAS  Google Scholar 

  23. Cesareni G, Helmer-Citterich M, Castagnoli L (1991) Trends Genet 7:230–235

    PubMed  CAS  Google Scholar 

  24. Banner D, Kokkinidis M, Tsernoglou D (1987) J Mol Biol 196:657–675

    Article  PubMed  CAS  Google Scholar 

  25. Hoffmann D, Knapp EW (1997) J Phys Chem B 101:6734–6740

    Article  CAS  Google Scholar 

  26. Kokkinidis M, Vlassi M, Papanikolaou Y, Kotsifaki D, Kingswell A, Tsernoglou D, Hinz HJ (1993) Proteins 16:214–216

    Article  PubMed  CAS  Google Scholar 

  27. Steif C, Hinz H, Cesareni G (1995) Proteins 23:83–96

    Article  PubMed  CAS  Google Scholar 

  28. Ceruso M, Grottesi A, Di Nola A (1999) Proteins 36:436–446

    Article  PubMed  CAS  Google Scholar 

  29. Predki P, Agrawal V, Brünger A, Regan L (1996) Nat Struct Biol 3:54–58

    Article  PubMed  CAS  Google Scholar 

  30. Vlassi M, Cesareni G, Kokkinidis M (1999) J Mol Biol 285:817–827

    Article  PubMed  CAS  Google Scholar 

  31. Predki P, Regan L (1995) Biochemistry 34:9834–9839

    Article  PubMed  CAS  Google Scholar 

  32. Westerlund K, Moran SD, Privett HK, Hay S, Jarvet J, Gibney BR, Tommos C (2008) Protein Eng Des Sel 21:645–652

    Article  PubMed  CAS  Google Scholar 

  33. Wilson J, Caruana D, Gilardi G (2003) Chem Commun 356–357

  34. Munson M, Predki P, Regan L (1994) Gene 144:59–62

    Article  PubMed  CAS  Google Scholar 

  35. Schellman J (1987) Biopolymers 26:549–559

    Article  PubMed  CAS  Google Scholar 

  36. Hagen W (1989) Eur J Biochem 182:523–530

    Article  PubMed  CAS  Google Scholar 

  37. Eberle W, Pastore A, Sander C, Rösch P (1991) J Biomol NMR 1:71–82

    Article  PubMed  CAS  Google Scholar 

  38. Munson M, O’Brien R, Sturtevant J, Regan L (1994) Protein Sci 3:2015–2022

    Article  PubMed  CAS  Google Scholar 

  39. Adar F (1978) In: Dolphin D (ed) The porphyrins. Electronic absorption spectra of hemes and heme proteins. Academic Press, New York, pp 167–209

  40. Yu L, Xu J, Haley P, Yu C (1987) J Biol Chem 262:1137–1143

    PubMed  CAS  Google Scholar 

  41. Shifman J, Gibney B, Sharp R, Dutton P (2000) Biochemistry 39:14813–14821

    Article  PubMed  CAS  Google Scholar 

  42. Peters K, Hinz H, Cesareni G (1997) Biol Chem 378:1141–1152

    Article  PubMed  CAS  Google Scholar 

  43. Pace NC, Shirley BA, Thomson JA (1990) In: Creghton TF (ed) Protein structure: a practical approach. IRL Press, Oxford, pp 311–330

  44. Myers JK, Pace CN, Scholtz JM (1995) Protein Sci 4:2138–2148

    Article  PubMed  CAS  Google Scholar 

  45. Gibney B, Huang S, Skalicky J, Fuentes E, Wand A, Dutton P (2001) Biochemistry 40:10550–10561

    Article  PubMed  CAS  Google Scholar 

  46. Xu ZJ, Farid RS (2001) Protein Sci 10:236–249

    Article  PubMed  CAS  Google Scholar 

  47. Chapman SK, Daff S, Munro AW (1997) Metal Sites Proteins Models 88:39–70

    Google Scholar 

  48. Reedy CJ, Gibney BR (2004) Chem Rev 104:617–49

    Google Scholar 

  49. Reedy CJ, Elvekrog MM, Gibney BR (2008) Nucleic Acids Res 36:D307–D313

    Article  PubMed  CAS  Google Scholar 

  50. Stellwagen E (1978) Nature 275:73–74

    Article  PubMed  CAS  Google Scholar 

  51. Shelnutt JA, Song XZ, Ma JG, Jia SL, Jentzen W, Medforth CJ (1998) Chem Soc Rev 27:31–41

    Article  CAS  Google Scholar 

  52. Gunner MR, Alexov E, Torres E, Lipovaca S (1997) J Biol Inorg Chem 2:126–134

    Article  CAS  Google Scholar 

  53. Gerstein M (1992) Acta Crystallogr A 48:271–276

    Article  Google Scholar 

Download references

Acknowledgments

The monomeric rop construct, pMR103-S55, was kindly provided by Lynne Regan, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, USA. We thank MIUR project (PRIN) and Piedmont Regional Government (CIPE) for financial support.

Author information

Authors and Affiliations

  1. Department of Human and Animal Biology, University of Turin, Via Accademia Albertina 13, 10123, Turin, Italy

    Giovanna Di Nardo, Sheila J. Sadeghi & Gianfranco Gilardi

  2. Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Via Montpellier 1, 00133, Rome, Italy

    Almerinda Di Venere & Giampiero Mei

  3. Division of Molecular Biosciences, Biochemistry Building, Imperial College London, London, SW7 2AY, UK

    Jon R. Wilson & Gianfranco Gilardi

Authors
  1. Giovanna Di Nardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Almerinda Di Venere
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Giampiero Mei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sheila J. Sadeghi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jon R. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gianfranco Gilardi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gianfranco Gilardi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Di Nardo, G., Di Venere, A., Mei, G. et al. Engineering heme binding sites in monomeric rop. J Biol Inorg Chem 14, 497–505 (2009). https://doi.org/10.1007/s00775-009-0465-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-009-0465-0

Keywords

Search

Navigation