Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1998 Sep;7(9):1983–1993. doi: 10.1002/pro.5560070914

A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.

A J Constans 1, M R Mayer 1, S F Sukits 1, J T Lecomte 1
PMCID: PMC2144161  PMID: 9761479

Abstract

The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.

Full Text

The Full Text of this article is available as a PDF (2.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexandrescu A. T., Abeygunawardana C., Shortle D. Structure and dynamics of a denatured 131-residue fragment of staphylococcal nuclease: a heteronuclear NMR study. Biochemistry. 1994 Feb 8;33(5):1063–1072. doi: 10.1021/bi00171a004. [DOI] [PubMed] [Google Scholar]
  2. Anfinsen C. B. Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223–230. doi: 10.1126/science.181.4096.223. [DOI] [PubMed] [Google Scholar]
  3. Attwood T. K., Beck M. E., Bleasby A. J., Parry-Smith D. J. PRINTS--a database of protein motif fingerprints. Nucleic Acids Res. 1994 Sep;22(17):3590–3596. [PMC free article] [PubMed] [Google Scholar]
  4. Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1991 Apr 25;19 (Suppl):2241–2245. doi: 10.1093/nar/19.suppl.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beck von Bodman S., Schuler M. A., Jollie D. R., Sligar S. G. Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9443–9447. doi: 10.1073/pnas.83.24.9443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Behe M. J., Lattman E. E., Rose G. D. The protein-folding problem: the native fold determines packing, but does packing determine the native fold? Proc Natl Acad Sci U S A. 1991 May 15;88(10):4195–4199. doi: 10.1073/pnas.88.10.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bennett M. J., Schlunegger M. P., Eisenberg D. 3D domain swapping: a mechanism for oligomer assembly. Protein Sci. 1995 Dec;4(12):2455–2468. doi: 10.1002/pro.5560041202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bryan P., Wang L., Hoskins J., Ruvinov S., Strausberg S., Alexander P., Almog O., Gilliland G., Gallagher T. Catalysis of a protein folding reaction: mechanistic implications of the 2.0 A structure of the subtilisin-prodomain complex. Biochemistry. 1995 Aug 15;34(32):10310–10318. doi: 10.1021/bi00032a026. [DOI] [PubMed] [Google Scholar]
  9. Dahiyat B. I., Mayo S. L. De novo protein design: fully automated sequence selection. Science. 1997 Oct 3;278(5335):82–87. doi: 10.1126/science.278.5335.82. [DOI] [PubMed] [Google Scholar]
  10. Desjarlais J. R., Handel T. M. De novo design of the hydrophobic cores of proteins. Protein Sci. 1995 Oct;4(10):2006–2018. doi: 10.1002/pro.5560041006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dill K. A. Dominant forces in protein folding. Biochemistry. 1990 Aug 7;29(31):7133–7155. doi: 10.1021/bi00483a001. [DOI] [PubMed] [Google Scholar]
  12. Dion-Schultz A., Howell E. E. Effects of insertions and deletions in a beta-bulge region of Escherichia coli dihydrofolate reductase. Protein Eng. 1997 Mar;10(3):263–272. doi: 10.1093/protein/10.3.263. [DOI] [PubMed] [Google Scholar]
  13. Falzone C. J., Mayer M. R., Whiteman E. L., Moore C. D., Lecomte J. T. Design challenges for hemoproteins: the solution structure of apocytochrome b5. Biochemistry. 1996 May 28;35(21):6519–6526. doi: 10.1021/bi960501q. [DOI] [PubMed] [Google Scholar]
  14. Fetrow J. S., Horner S. R., Oehrl W., Schaak D. L., Boose T. L., Burton R. E. Analysis of the structure and stability of omega loop A replacements in yeast iso-1-cytochrome c. Protein Sci. 1997 Jan;6(1):197–210. doi: 10.1002/pro.5560060122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fetrow J. S. Omega loops: nonregular secondary structures significant in protein function and stability. FASEB J. 1995 Jun;9(9):708–717. [PubMed] [Google Scholar]
  16. Garrett J. B., Mullins L. S., Raushel F. M. Are turns required for the folding of ribonuclease T1? Protein Sci. 1996 Feb;5(2):204–211. doi: 10.1002/pro.5560050203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gekko K., Yamagami K., Kunori Y., Ichihara S., Kodama M., Iwakura M. Effects of point mutation in a flexible loop on the stability and enzymatic function of Escherichia coli dihydrofolate reductase. J Biochem. 1993 Jan;113(1):74–80. doi: 10.1093/oxfordjournals.jbchem.a124007. [DOI] [PubMed] [Google Scholar]
  18. Hamada D., Goto Y. The equilibrium intermediate of beta-lactoglobulin with non-native alpha-helical structure. J Mol Biol. 1997 Jun 20;269(4):479–487. doi: 10.1006/jmbi.1997.1055. [DOI] [PubMed] [Google Scholar]
  19. Hoedemaeker F. J., van Eijsden R. R., Díaz C. L., de Pater B. S., Kijne J. W. Destabilization of pea lectin by substitution of a single amino acid in a surface loop. Plant Mol Biol. 1993 Sep;22(6):1039–1046. doi: 10.1007/BF00028976. [DOI] [PubMed] [Google Scholar]
  20. Holm L., Sander C. Protein structure comparison by alignment of distance matrices. J Mol Biol. 1993 Sep 5;233(1):123–138. doi: 10.1006/jmbi.1993.1489. [DOI] [PubMed] [Google Scholar]
  21. Hooft R. W., Vriend G., Sander C., Abola E. E. Errors in protein structures. Nature. 1996 May 23;381(6580):272–272. doi: 10.1038/381272a0. [DOI] [PubMed] [Google Scholar]
  22. Huntley T. E., Strittmatter P. The effect of heme binding on the tryptophan residue and the protein conformation of cytochrome b 5 . J Biol Chem. 1972 Jul 25;247(14):4641–4647. [PubMed] [Google Scholar]
  23. Kumar A., Ernst R. R., Wüthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980 Jul 16;95(1):1–6. doi: 10.1016/0006-291x(80)90695-6. [DOI] [PubMed] [Google Scholar]
  24. Lattman E. E., Rose G. D. Protein folding--what's the question? Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):439–441. doi: 10.1073/pnas.90.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lazar G. A., Desjarlais J. R., Handel T. M. De novo design of the hydrophobic core of ubiquitin. Protein Sci. 1997 Jun;6(6):1167–1178. doi: 10.1002/pro.5560060605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lee J. C., Timasheff S. N. The stabilization of proteins by sucrose. J Biol Chem. 1981 Jul 25;256(14):7193–7201. [PubMed] [Google Scholar]
  27. Mach H., Middaugh C. R., Lewis R. V. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal Biochem. 1992 Jan;200(1):74–80. doi: 10.1016/0003-2697(92)90279-g. [DOI] [PubMed] [Google Scholar]
  28. Mathews F. S. The structure, function and evolution of cytochromes. Prog Biophys Mol Biol. 1985;45(1):1–56. doi: 10.1016/0079-6107(85)90004-5. [DOI] [PubMed] [Google Scholar]
  29. Matthews C. R. Pathways of protein folding. Annu Rev Biochem. 1993;62:653–683. doi: 10.1146/annurev.bi.62.070193.003253. [DOI] [PubMed] [Google Scholar]
  30. Miranker A. D., Dobson C. M. Collapse and cooperativity in protein folding. Curr Opin Struct Biol. 1996 Feb;6(1):31–42. doi: 10.1016/s0959-440x(96)80092-3. [DOI] [PubMed] [Google Scholar]
  31. Moore C. D., Lecomte J. T. Characterization of an independent structural unit in apocytochrome b5. Biochemistry. 1993 Jan 12;32(1):199–207. doi: 10.1021/bi00052a026. [DOI] [PubMed] [Google Scholar]
  32. Moore C. D., Lecomte J. T. Structural properties of apocytochrome b5: presence of a stable native core. Biochemistry. 1990 Feb 27;29(8):1984–1989. doi: 10.1021/bi00460a004. [DOI] [PubMed] [Google Scholar]
  33. Muñoz V., Cronet P., López-Hernández E., Serrano L. Analysis of the effect of local interactions on protein stability. Fold Des. 1996;1(3):167–178. doi: 10.1016/s1359-0278(96)00029-6. [DOI] [PubMed] [Google Scholar]
  34. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  35. Pfeil W. Thermodynamics of apocytochrome b5 unfolding. Protein Sci. 1993 Sep;2(9):1497–1501. doi: 10.1002/pro.5560020914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Presta L. G., Rose G. D. Helix signals in proteins. Science. 1988 Jun 17;240(4859):1632–1641. doi: 10.1126/science.2837824. [DOI] [PubMed] [Google Scholar]
  37. Prieto J., Wilmans M., Jiménez M. A., Rico M., Serrano L. Non-native local interactions in protein folding and stability: introducing a helical tendency in the all beta-sheet alpha-spectrin SH3 domain. J Mol Biol. 1997 May 16;268(4):760–778. doi: 10.1006/jmbi.1997.0984. [DOI] [PubMed] [Google Scholar]
  38. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  39. Richards F. M. Areas, volumes, packing and protein structure. Annu Rev Biophys Bioeng. 1977;6:151–176. doi: 10.1146/annurev.bb.06.060177.001055. [DOI] [PubMed] [Google Scholar]
  40. Rost B., Sander C., Schneider R. PHD--an automatic mail server for protein secondary structure prediction. Comput Appl Biosci. 1994 Feb;10(1):53–60. doi: 10.1093/bioinformatics/10.1.53. [DOI] [PubMed] [Google Scholar]
  41. Shortle D. The denatured state (the other half of the folding equation) and its role in protein stability. FASEB J. 1996 Jan;10(1):27–34. doi: 10.1096/fasebj.10.1.8566543. [DOI] [PubMed] [Google Scholar]
  42. Tanford C. Protein denaturation. C. Theoretical models for the mechanism of denaturation. Adv Protein Chem. 1970;24:1–95. [PubMed] [Google Scholar]
  43. Tanford C. Protein denaturation. Adv Protein Chem. 1968;23:121–282. doi: 10.1016/s0065-3233(08)60401-5. [DOI] [PubMed] [Google Scholar]
  44. Uversky V. N., Kutyshenko V. P., Protasova NYu, Rogov V. V., Vassilenko K. S., Gudkov A. T. Circularly permuted dihydrofolate reductase possesses all the properties of the molten globule state, but can resume functional tertiary structure by interaction with its ligands. Protein Sci. 1996 Sep;5(9):1844–1851. doi: 10.1002/pro.5560050910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Uversky V. N. Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule. Biochemistry. 1993 Dec 7;32(48):13288–13298. doi: 10.1021/bi00211a042. [DOI] [PubMed] [Google Scholar]
  46. Viguera A. R., Serrano L. Loop length, intramolecular diffusion and protein folding. Nat Struct Biol. 1997 Nov;4(11):939–946. doi: 10.1038/nsb1197-939. [DOI] [PubMed] [Google Scholar]
  47. Viguera A. R., Villegas V., Avilés F. X., Serrano L. Favourable native-like helical local interactions can accelerate protein folding. Fold Des. 1997;2(1):23–33. doi: 10.1016/S1359-0278(97)00003-5. [DOI] [PubMed] [Google Scholar]
  48. Villegas V, V, Viguera AR, Aviles FX, Serrano L. Stabilization of proteins by rational design of alpha-helix stability using helix/coil transition theory. Fold Des. 1995;1(1):29–34. [PubMed] [Google Scholar]
  49. Vriend G., Eijsink V. Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases. J Comput Aided Mol Des. 1993 Aug;7(4):367–396. doi: 10.1007/BF02337558. [DOI] [PubMed] [Google Scholar]
  50. Wrabl J. O., Shortle D. Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics. Protein Sci. 1996 Nov;5(11):2343–2352. doi: 10.1002/pro.5560051121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhang O., Kay L. E., Shortle D., Forman-Kay J. D. Comprehensive NOE characterization of a partially folded large fragment of staphylococcal nuclease Delta131Delta, using NMR methods with improved resolution. J Mol Biol. 1997 Sep 12;272(1):9–20. doi: 10.1006/jmbi.1997.1219. [DOI] [PubMed] [Google Scholar]
  52. Zhang X. J., Baase W. A., Shoichet B. K., Wilson K. P., Matthews B. W. Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive. Protein Eng. 1995 Oct;8(10):1017–1022. doi: 10.1093/protein/8.10.1017. [DOI] [PubMed] [Google Scholar]
  53. el Hawrani A. S., Moreton K. M., Sessions R. B., Clarke A. R., Holbrook J. J. Engineering surface loops of proteins--a preferred strategy for obtaining new enzyme function. Trends Biotechnol. 1994 May;12(5):207–211. doi: 10.1016/0167-7799(94)90084-1. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES