Tuning λ6-85 Towards Downhill Folding at its Melting Temperature

Feng Liu, Martin Gruebele

Research output: Contribution to journalArticlepeer-review


The five-helix bundle λ6-85* is a fast two-state folder. Several stabilized mutants have been reported to fold kinetically near-downhill or downhill. These mutants undergo a transition to two-state folding kinetics when heated. It has been suggested that this transition is caused by increased hydrophobicity at higher temperature. Here we investigate two histidine-containing mutants of λ6-85* to see if a weaker hydrophobic core can extend the temperature range of downhill folding. The very stable λHA is the fastest-folding lambda repressor to date (kf- 1 ≈ kobs- 1 = 2.3 μs at 44 °C). It folds downhill at low temperature, but transits back to two-state folding at its unfolding midpoint. λHG has a weakened hydrophobic core. It is less stable than some slower folding mutants of λ6-85*, and it has more exposed hydrophobic surface area in the folded state. This mutant nonetheless folds very rapidly, and has the non-exponential folding kinetics of an incipient downhill folder even at the unfolding midpoint (km- 1 ≈ 2 μs, ka- 1 = 15 μs at 56 °C). We also compare the thermodynamic melting transition of λHG with the nominal two-state folding mutant λQG, which has a similar melting temperature. Unlike λQG, λHG yields fluorescence wavelength-dependent cooperativities and probe-dependent melting temperatures. This result combined with previous work shows that the energy landscapes of lambda repressor mutants support all standard folding mechanisms.

Original languageEnglish (US)
Pages (from-to)574-584
Number of pages11
JournalJournal of Molecular Biology
Issue number3
StatePublished - Jul 13 2007


  • fluorescence
  • free energy landscape
  • intermediate
  • lambda repressor
  • temperature jump

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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