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. 2002 Jul;83(1):458–472. doi: 10.1016/S0006-3495(02)75182-5

The effect of core destabilization on the mechanical resistance of I27.

David J Brockwell 1, Godfrey S Beddard 1, John Clarkson 1, Rebecca C Zinober 1, Anthony W Blake 1, John Trinick 1, Peter D Olmsted 1, D Alastair Smith 1, Sheena E Radford 1
PMCID: PMC1302160  PMID: 12080133

Abstract

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.

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Selected References

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  1. Barik S. Site-directed mutagenesis in vitro by megaprimer PCR. Methods Mol Biol. 1996;57:203–215. doi: 10.1385/0-89603-332-5:203. [DOI] [PubMed] [Google Scholar]
  2. Bell G. I. Models for the specific adhesion of cells to cells. Science. 1978 May 12;200(4342):618–627. doi: 10.1126/science.347575. [DOI] [PubMed] [Google Scholar]
  3. Best R. B., Li B., Steward A., Daggett V., Clarke J. Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation. Biophys J. 2001 Oct;81(4):2344–2356. doi: 10.1016/S0006-3495(01)75881-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev Lett. 1986 Mar 3;56(9):930–933. doi: 10.1103/PhysRevLett.56.930. [DOI] [PubMed] [Google Scholar]
  5. Carrion-Vazquez M., Marszalek P. E., Oberhauser A. F., Fernandez J. M. Atomic force microscopy captures length phenotypes in single proteins. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11288–11292. doi: 10.1073/pnas.96.20.11288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carrion-Vazquez M., Oberhauser A. F., Fisher T. E., Marszalek P. E., Li H., Fernandez J. M. Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering. Prog Biophys Mol Biol. 2000;74(1-2):63–91. doi: 10.1016/s0079-6107(00)00017-1. [DOI] [PubMed] [Google Scholar]
  7. Carrion-Vazquez M., Oberhauser A. F., Fowler S. B., Marszalek P. E., Broedel S. E., Clarke J., Fernandez J. M. Mechanical and chemical unfolding of a single protein: a comparison. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3694–3699. doi: 10.1073/pnas.96.7.3694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Evans E. Energy landscapes of biomolecular adhesion and receptor anchoring at interfaces explored with dynamic force spectroscopy. Faraday Discuss. 1998;(111):1–16. doi: 10.1039/a809884k. [DOI] [PubMed] [Google Scholar]
  9. Evans E., Ritchie K. Dynamic strength of molecular adhesion bonds. Biophys J. 1997 Apr;72(4):1541–1555. doi: 10.1016/S0006-3495(97)78802-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ferguson N., Capaldi A. P., James R., Kleanthous C., Radford S. E. Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9. J Mol Biol. 1999 Mar 12;286(5):1597–1608. doi: 10.1006/jmbi.1998.2548. [DOI] [PubMed] [Google Scholar]
  11. Florin E. L., Moy V. T., Gaub H. E. Adhesion forces between individual ligand-receptor pairs. Science. 1994 Apr 15;264(5157):415–417. doi: 10.1126/science.8153628. [DOI] [PubMed] [Google Scholar]
  12. Grandbois M, Beyer M, Rief M, Clausen-Schaumann H, Gaub HE. How strong is a covalent bond? . Science. 1999 Mar 12;283(5408):1727–1730. doi: 10.1126/science.283.5408.1727. [DOI] [PubMed] [Google Scholar]
  13. Hamill S. J., Steward A., Clarke J. The folding of an immunoglobulin-like Greek key protein is defined by a common-core nucleus and regions constrained by topology. J Mol Biol. 2000 Mar 17;297(1):165–178. doi: 10.1006/jmbi.2000.3517. [DOI] [PubMed] [Google Scholar]
  14. Improta S., Krueger J. K., Gautel M., Atkinson R. A., Lefèvre J. F., Moulton S., Trewhella J., Pastore A. The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity. J Mol Biol. 1998 Dec 4;284(3):761–777. doi: 10.1006/jmbi.1998.2028. [DOI] [PubMed] [Google Scholar]
  15. Improta S., Politou A. S., Pastore A. Immunoglobulin-like modules from titin I-band: extensible components of muscle elasticity. Structure. 1996 Mar 15;4(3):323–337. doi: 10.1016/s0969-2126(96)00036-6. [DOI] [PubMed] [Google Scholar]
  16. Kellermayer M. S., Smith S. B., Granzier H. L., Bustamante C. Folding-unfolding transitions in single titin molecules characterized with laser tweezers. Science. 1997 May 16;276(5315):1112–1116. doi: 10.1126/science.276.5315.1112. [DOI] [PubMed] [Google Scholar]
  17. Klimov D. K., Thirumalai D. Native topology determines force-induced unfolding pathways in globular proteins. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7254–7259. doi: 10.1073/pnas.97.13.7254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lee G. U., Chrisey L. A., Colton R. J. Direct measurement of the forces between complementary strands of DNA. Science. 1994 Nov 4;266(5186):771–773. doi: 10.1126/science.7973628. [DOI] [PubMed] [Google Scholar]
  19. Li H., Carrion-Vazquez M., Oberhauser A. F., Marszalek P. E., Fernandez J. M. Point mutations alter the mechanical stability of immunoglobulin modules. Nat Struct Biol. 2000 Dec;7(12):1117–1120. doi: 10.1038/81964. [DOI] [PubMed] [Google Scholar]
  20. Li H., Oberhauser A. F., Fowler S. B., Clarke J., Fernandez J. M. Atomic force microscopy reveals the mechanical design of a modular protein. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6527–6531. doi: 10.1073/pnas.120048697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lu H., Isralewitz B., Krammer A., Vogel V., Schulten K. Unfolding of titin immunoglobulin domains by steered molecular dynamics simulation. Biophys J. 1998 Aug;75(2):662–671. doi: 10.1016/S0006-3495(98)77556-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lu H., Schulten K. Steered molecular dynamics simulations of force-induced protein domain unfolding. Proteins. 1999 Jun 1;35(4):453–463. [PubMed] [Google Scholar]
  23. Lu H., Schulten K. The key event in force-induced unfolding of Titin's immunoglobulin domains. Biophys J. 2000 Jul;79(1):51–65. doi: 10.1016/S0006-3495(00)76273-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Marszalek P. E., Lu H., Li H., Carrion-Vazquez M., Oberhauser A. F., Schulten K., Fernandez J. M. Mechanical unfolding intermediates in titin modules. Nature. 1999 Nov 4;402(6757):100–103. doi: 10.1038/47083. [DOI] [PubMed] [Google Scholar]
  25. Merkel R., Nassoy P., Leung A., Ritchie K., Evans E. Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature. 1999 Jan 7;397(6714):50–53. doi: 10.1038/16219. [DOI] [PubMed] [Google Scholar]
  26. Merritt E. A., Murphy M. E. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994 Nov 1;50(Pt 6):869–873. doi: 10.1107/S0907444994006396. [DOI] [PubMed] [Google Scholar]
  27. Mitsui K., Hara M., Ikai A. Mechanical unfolding of alpha2-macroglobulin molecules with atomic force microscope. FEBS Lett. 1996 Apr 29;385(1-2):29–33. doi: 10.1016/0014-5793(96)00319-5. [DOI] [PubMed] [Google Scholar]
  28. Myers J. K., Pace C. N., Scholtz J. M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 1995 Oct;4(10):2138–2148. doi: 10.1002/pro.5560041020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oberhauser A. F., Marszalek P. E., Erickson H. P., Fernandez J. M. The molecular elasticity of the extracellular matrix protein tenascin. Nature. 1998 May 14;393(6681):181–185. doi: 10.1038/30270. [DOI] [PubMed] [Google Scholar]
  30. Oesterhelt F., Oesterhelt D., Pfeiffer M., Engel A., Gaub H. E., Müller D. J. Unfolding pathways of individual bacteriorhodopsins. Science. 2000 Apr 7;288(5463):143–146. doi: 10.1126/science.288.5463.143. [DOI] [PubMed] [Google Scholar]
  31. Paci E., Karplus M. Unfolding proteins by external forces and temperature: the importance of topology and energetics. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6521–6526. doi: 10.1073/pnas.100124597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rief M., Gautel M., Oesterhelt F., Fernandez J. M., Gaub H. E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science. 1997 May 16;276(5315):1109–1112. doi: 10.1126/science.276.5315.1109. [DOI] [PubMed] [Google Scholar]
  33. Rief M., Pascual J., Saraste M., Gaub H. E. Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles. J Mol Biol. 1999 Feb 19;286(2):553–561. doi: 10.1006/jmbi.1998.2466. [DOI] [PubMed] [Google Scholar]
  34. Rief M, Oesterhelt F, Heymann B, Gaub HE. Single Molecule Force Spectroscopy on Polysaccharides by Atomic Force Microscopy. Science. 1997 Feb 28;275(5304):1295–1297. doi: 10.1126/science.275.5304.1295. [DOI] [PubMed] [Google Scholar]
  35. Santoro M. M., Bolen D. W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry. 1988 Oct 18;27(21):8063–8068. doi: 10.1021/bi00421a014. [DOI] [PubMed] [Google Scholar]
  36. Trinick J. Titin as a scaffold and spring. Cytoskeleton. Curr Biol. 1996 Mar 1;6(3):258–260. doi: 10.1016/s0960-9822(02)00472-4. [DOI] [PubMed] [Google Scholar]
  37. Tskhovrebova L., Trinick J., Sleep J. A., Simmons R. M. Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature. 1997 May 15;387(6630):308–312. doi: 10.1038/387308a0. [DOI] [PubMed] [Google Scholar]
  38. Wymer N., Buchanan L. V., Henderson D., Mehta N., Botting C. H., Pocivavsek L., Fierke C. A., Toone E. J., Naismith J. H. Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli. Structure. 2001 Jan 10;9(1):1–9. doi: 10.1016/s0969-2126(00)00555-4. [DOI] [PubMed] [Google Scholar]
  39. Yang G., Cecconi C., Baase W. A., Vetter I. R., Breyer W. A., Haack J. A., Matthews B. W., Dahlquist F. W., Bustamante C. Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme. Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):139–144. doi: 10.1073/pnas.97.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]

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