The effect of boundary selection on the stability and folding of the third fibronectin type III domain from human tenascin

被引:77
作者
Hamill, SJ
Meekhof, AE
Clarke, J
机构
[1] Univ Cambridge, Dept Chem, Cambridge CB2 1EW, England
[2] Univ Cambridge, MRC, Ctr Prot Engn, Cambridge CB2 1EW, England
基金
英国惠康基金;
关键词
D O I
10.1021/bi9801659
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Correct selection of domain boundaries is critical for structural analysis of single domains from multimodular proteins. Folding and stability studies of the third fibronectin type III domain from human tenascin (TNfn3(1-90)) have shown that it is moderately stable (Delta G(D-N)(H2O) similar to 5 kcal mol(-1)) and folds with two-state kinetics. In an attempt to stabilize the protein, five domains were constructed with different combinations of extensions to the N- and C-termini. Thermal denaturation studies show that a specific two amino acid (Gly-Leu) extension to the C-terminus is primarily responsible for a significant increase in stability. The Delta Delta G(D-N)(H2O) of the Gly-Leu extension (TNfn3(1-92)) is 2.7 +/- 0.3 kcal mol(-1). Refolding kinetics do not differ significantly, but unfolding is slowed 40-fold. Mutation of leucine 92 to alanine does not affect stability, indicating that the stability of the extension does not come from the packing of the leucine side chain. Hydrogen exchange data suggest that the extension adds new hydrogen bonds and strengthens existing hydrogen bonds in the C-terminal interaction with the A-B and E-F loops. Removal of a very small number of hydrogen bonds substantially increases the unfolding rate, a phenomenon which may be important in stress-relaxation of FNIII-containing muscle proteins such as titin. These experiments demonstrate the importance of a small number of additional long-range interactions in the overall formation of a compact independently folding beta-sheet module.
引用
收藏
页码:8071 / 8079
页数:9
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共 48 条
  • [31] Pace C N, 1986, Methods Enzymol, V131, P266
  • [32] ORGANIZATION OF THE FIBRONECTIN GENE PROVIDES EVIDENCE FOR EXON SHUFFLING DURING EVOLUTION
    PATEL, RS
    ODERMATT, E
    SCHWARZBAUER, JE
    HYNES, RO
    [J]. EMBO JOURNAL, 1987, 6 (09) : 2565 - 2572
  • [33] RELATIONSHIP BETWEEN EQUILIBRIUM AMIDE PROTON-EXCHANGE BEHAVIOR AND THE FOLDING PATHWAY OF BARNASE
    PERRETT, S
    CLARKE, J
    HOUNSLOW, AM
    FERSHT, AR
    [J]. BIOCHEMISTRY, 1995, 34 (29) : 9288 - 9298
  • [34] TERTIARY STRUCTURE OF AN IMMUNOGLOBULIN-LIKE DOMAIN FROM THE GIANT MUSCLE PROTEIN TITIN - A NEW MEMBER OF THE I-SET
    PFUHL, M
    PASTORE, A
    [J]. STRUCTURE, 1995, 3 (04) : 391 - 401
  • [35] When a module is also a domain: The role of the N terminus in the stability and the dynamics of immunoglobulin domains from titin
    Pfuhl, M
    Improta, S
    Politou, AS
    Pastore, A
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1997, 265 (02) : 242 - 256
  • [36] A comparison of the folding kinetics and thermodynamics of two homologous fibronectin type III modules
    Plaxco, KW
    Spitzfaden, C
    Campbell, ID
    Dobson, CM
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1997, 270 (05) : 763 - 770
  • [37] Rapid refolding of a proline-rich all-beta-sheet fibronectin type III module
    Plaxco, KW
    Spitzfaden, C
    Campbell, ID
    Dobson, CM
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (20) : 10703 - 10706
  • [38] The folding and stability of titin immunoglobulin-like modules, with implications for the mechanism of elasticity
    Politou, AS
    Thomas, DJ
    Pastore, A
    [J]. BIOPHYSICAL JOURNAL, 1995, 69 (06) : 2601 - 2610
  • [39] IMMUNOGLOBULIN-TYPE DOMAINS OF TITIN ARE STABILIZED BY AMINO-TERMINAL EXTENSION
    POLITOU, AS
    GAUTEL, M
    JOSEPH, C
    PASTORE, A
    [J]. FEBS LETTERS, 1994, 352 (01) : 27 - 31
  • [40] Reversible unfolding of individual titin immunoglobulin domains by AFM
    Rief, M
    Gautel, M
    Oesterhelt, F
    Fernandez, JM
    Gaub, HE
    [J]. SCIENCE, 1997, 276 (5315) : 1109 - 1112