Topological Mutation of Escherichia coli Dihydrofolate Reductase

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Abstract

Proteins appear to contain structural elements which determine the folded structure. If such elements are present, the order of structural elements in the primary structure, <i>i.e.</i> the chain topology, can be disregarded for building of the folded tertiary structure, when they are properly connected to each other by proper linkers. To experimentally examine this, “topological” mutants (designated as GHF33 and GHF34) of <i>Escherichia coli</i> dihydrofolate reductase (DHFR) were designed and constructed by switching two amino acid sequence parts containing the βF strand and βG-βH strands in the primary sequence. In this way, the chain topology of wild-type DHFR, βA→αB→βB→αC→βC→βD→αE→βE→αF→βF<sub>-</sub>→βG<sub>-</sub>→βH<sub>-</sub>, was changed to βA→αB→βB→αC→βC→βD→αE→βE→αF→βG<sub>-</sub>→βH<sub>-</sub>→βF<sub>-</sub>. Such topological mutant proteins were stably expressed and accumulated in <i>E. coli</i> cells, and highly purified. Molecular mass measurements of the purified proteins and their proteolytic fragments confirmed that GHF33 and GHF34 had the designed sequence. In terms of <i>k</i><sub>cat</sub>, the GHF33 and GHF34 proteins showed about 10 and 20% of the DHFR activity of the wild-type with <i>K</i><sub>m</sub> values of 3.3 μM (GHF33) and 2.1 μM (GHF34), respectively. The topological mutants showed a cooperative two-state transition in urea-induced unfolding experiments with <i>Δ</i>G<sup>H<sub>2</sub>O</sup> values of 4.0 and 4.8 kcal/mol. Whereas, the <i>K</i><sub>m</sub> and <i>Δ</i>G<sup>H<sub>2</sub>O</sup> values for wild-type DHFR were 0.9 μM and 6.1 kcal/mol, respectively. The significance of the topological mutations was discussed with respect to protein folding and protein evolution.

Journal

  • The Journal of Biochemistry

    The Journal of Biochemistry 124(4), 769-777, 1998-10-01

    The Japanese Biochemical Society

References:  40

Cited by:  2

Codes

  • NII Article ID (NAID)
    10005850396
  • NII NACSIS-CAT ID (NCID)
    AA00694073
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    0021924X
  • NDL Article ID
    4576623
  • NDL Source Classification
    ZR2(科学技術--生物学--生化学)
  • NDL Call No.
    Z53-B472
  • Data Source
    CJP  CJPref  NDL  J-STAGE 
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