• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of prosciprotein sciencecshl presssubscriptionsetoc alertsthe protein societyjournal home
Protein Sci. Dec 2000; 9(12): 2394–2404.
PMCID: PMC2144517

The effects of disulfide bonds on the denatured state of barnase.

Abstract

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended beta-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.

Full Text

The Full Text of this article is available as a PDF (2.5M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Fardy PS. Cardiac rehabilitation program focuses on total patient. Hospitals. 1978 Nov 1;52(21):101–108. [PubMed]
  • Arcus VL, Vuilleumier S, Freund SM, Bycroft M, Fersht AR. Toward solving the folding pathway of barnase: the complete backbone 13C, 15N, and 1H NMR assignments of its pH-denatured state. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9412–9416. [PMC free article] [PubMed]
  • Arcus VL, Vuilleumier S, Freund SM, Bycroft M, Fersht AR. A comparison of the pH, urea, and temperature-denatured states of barnase by heteronuclear NMR: implications for the initiation of protein folding. J Mol Biol. 1995 Nov 24;254(2):305–321. [PubMed]
  • Betz SF. Disulfide bonds and the stability of globular proteins. Protein Sci. 1993 Oct;2(10):1551–1558. [PMC free article] [PubMed]
  • Betz SF, Marmorino JL, Saunders AJ, Doyle DF, Young GB, Pielak GJ. Unusual effects of an engineered disulfide on global and local protein stability. Biochemistry. 1996 Jun 11;35(23):7422–7428. [PubMed]
  • Bond CJ, Wong KB, Clarke J, Fersht AR, Daggett V. Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: description of the folding pathway. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13409–13413. [PMC free article] [PubMed]
  • Bycroft M, Ludvigsen S, Fersht AR, Poulsen FM. Determination of the three-dimensional solution structure of barnase using nuclear magnetic resonance spectroscopy. Biochemistry. 1991 Sep 3;30(35):8697–8701. [PubMed]
  • Clarke J, Fersht AR. Engineered disulfide bonds as probes of the folding pathway of barnase: increasing the stability of proteins against the rate of denaturation. Biochemistry. 1993 Apr 27;32(16):4322–4329. [PubMed]
  • Clarke J, Henrick K, Fersht AR. Disulfide mutants of barnase. I: Changes in stability and structure assessed by biophysical methods and X-ray crystallography. J Mol Biol. 1995 Oct 27;253(3):493–504. [PubMed]
  • Clarke J, Hounslow AM, Bycroft M, Fersht AR. Local breathing and global unfolding in hydrogen exchange of barnase and its relationship to protein folding pathways. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9837–9841. [PMC free article] [PubMed]
  • Clarke J, Hounslow AM, Fersht AR. Disulfide mutants of barnase. II: Changes in structure and local stability identified by hydrogen exchange. J Mol Biol. 1995 Oct 27;253(3):505–513. [PubMed]
  • CLELAND WW. DITHIOTHREITOL, A NEW PROTECTIVE REAGENT FOR SH GROUPS. Biochemistry. 1964 Apr;3:480–482. [PubMed]
  • Creighton TE. Disulfide bonds as probes of protein folding pathways. Methods Enzymol. 1986;131:83–106. [PubMed]
  • Daggett V, Kollman PA, Kuntz ID. A molecular dynamics simulation of polyalanine: an analysis of equilibrium motions and helix-coil transitions. Biopolymers. 1991 Aug;31(9):1115–1134. [PubMed]
  • Farrow NA, Zhang O, Szabo A, Torchia DA, Kay LE. Spectral density function mapping using 15N relaxation data exclusively. J Biomol NMR. 1995 Sep;6(2):153–162. [PubMed]
  • Freund SM, Wong KB, Fersht AR. Initiation sites of protein folding by NMR analysis. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10600–10603. [PMC free article] [PubMed]
  • Hinck AP, Truckses DM, Markley JL. Engineered disulfide bonds in staphylococcal nuclease: effects on the stability and conformation of the folded protein. Biochemistry. 1996 Aug 13;35(32):10328–10338. [PubMed]
  • Huyghues-Despointes BM, Nelson JW. Stabilities of disulfide bond intermediates in the folding of apamin. Biochemistry. 1992 Feb 11;31(5):1476–1483. [PubMed]
  • Jacobson RH, Matsumura M, Faber HR, Matthews BW. Structure of a stabilizing disulfide bridge mutant that closes the active-site cleft of T4 lysozyme. Protein Sci. 1992 Jan;1(1):46–57. [PMC free article] [PubMed]
  • Johnson CM, Oliveberg M, Clarke J, Fersht AR. Thermodynamics of denaturation of mutants of barnase with disulfide crosslinks. J Mol Biol. 1997 Apr 25;268(1):198–208. [PubMed]
  • Katz BA, Kossiakoff A. The crystallographically determined structures of atypical strained disulfides engineered into subtilisin. J Biol Chem. 1986 Nov 25;261(33):15480–15485. [PubMed]
  • Katz B, Kossiakoff AA. Crystal structures of subtilisin BPN' variants containing disulfide bonds and cavities: concerted structural rearrangements induced by mutagenesis. Proteins. 1990;7(4):343–357. [PubMed]
  • Li A, Daggett V. Molecular dynamics simulation of the unfolding of barnase: characterization of the major intermediate. J Mol Biol. 1998 Jan 30;275(4):677–694. [PubMed]
  • Lin TY, Kim PS. Urea dependence of thiol-disulfide equilibria in thioredoxin: confirmation of the linkage relationship and a sensitive assay for structure. Biochemistry. 1989 Jun 13;28(12):5282–5287. [PubMed]
  • Miller WG, Goebel CV. Dimensions of protein random coils. Biochemistry. 1968 Nov;7(11):3925–3935. [PubMed]
  • Pace CN, Grimsley GR, Thomson JA, Barnett BJ. Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds. J Biol Chem. 1988 Aug 25;263(24):11820–11825. [PubMed]
  • Tidor B, Karplus M. The contribution of vibrational entropy to molecular association. The dimerization of insulin. J Mol Biol. 1994 May 6;238(3):405–414. [PubMed]
  • Villafranca JE, Howell EE, Oatley SJ, Xuong NH, Kraut J. An engineered disulfide bond in dihydrofolate reductase. Biochemistry. 1987 Apr 21;26(8):2182–2189. [PubMed]
  • Weissman JS, Kim PS. Reexamination of the folding of BPTI: predominance of native intermediates. Science. 1991 Sep 20;253(5026):1386–1393. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...