Tuning λ6-85 towards downhill folding at its melting temperature

The five-helix bundle lambda(*)(6-85) 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 lambda(*)(6-85) to see if a weaker hydrophobic core can extend the temperature range of downhill folding. The very stable lambda HA is the fastest-folding lambda repressor to date (k(f)(-1)approximate to k(obs)(-1) = 2.3 mu s at 44 degrees C. It folds downhill at low temperature, but transits back to two-state folding at its unfolding midpoint. lambda HG has a weakened hydrophobic core. It is less stable than some slower folding mutants of lambda(*)(6-85), 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 (k(m)(-1)approximate to 2 mu s, k(a)(-1)= 15 mu s at 56 degrees C. We also compare the thermodynamic melting transition of lambda HG with the nominal two-state folding mutant lambda QG, which has a similar melting temperature. Unlike lambda QG, lambda HG 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. (c) 2007 Elsevier Ltd. All rights reserved.