Using chimeric immunity proteins to explore the energy landscape for α-helical protein folding

Neil Ferguson, Wei Li, Andrew P. Capaldi, Colin Kleanthous, Sheena E. Radford

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

Abstract

To address the role of sequence in the folding of homologous proteins, the folding and unfolding kinetics of the all-helical bacterial immunity proteins Im2 and Im9 were characterised, together with six chimeric derivatives of these proteins. We show that both Im2 and Im9 fold rapidly (kUNH2O ≈ 2000 s-1 at pH 7.0, 25°C) in apparent two-state transitions, through rate-limiting transition states that are highly compact (βTS 0.93 and 0.96, respectively). Whilst the folding and unfolding properties of three of the chimeras (Im2 (1-44)Im9, Im2 (1-64)Im9 and Im2 (25-44)Im9) are similar to their parental counterparts, in other chimeric proteins the introduced sequence variation results in altered kinetic behaviour. At low urea concentrations, Im2 (1-29)Im9 and Im2 (56-64)Im9 fold in two-state transitions via transition states that are significantly less compact (βTS ≈ 0.7) than those characterised for the other immunity proteins presented here. At higher urea concentrations, however, the rate-limiting transition state for these two chimeras switches or moves to a more compact species (βTS ≈ 0.9). Surprisingly, Im2 (30-64)Im9 populates a highly collapsed species (βI = 0.87) in the dead-time (2.5 ms) of stopped flow measurements. These data indicate that whilst topology may place significant constraints on the folding process, specific inter-residue interactions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape.

Original languageEnglish (US)
Pages (from-to)393-405
Number of pages13
JournalJournal of Molecular Biology
Volume307
Issue number1
DOIs
StatePublished - Mar 16 2001

Keywords

  • Chimera
  • Intermediate
  • Sequence
  • Topology
  • Transition state

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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