Tuning lambda6-85 towards downhill folding at its melting temperature.
The five-helix bundle lambda6-85* 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 lambda6-85* to see if a weaker hydrophobic core can extend the temperature range of downhill folding. The very stable lambdaHA is the fastest-folding lambda repressor to date (k(f)(-1) approximately k(obs)(-1)=2.3 micros at 44 degrees C). It folds downhill at low temperature, but transits back to two-state folding at its unfolding midpoint. lambdaHG has a weakened hydrophobic core. It is less stable than some slower folding mutants of lambda6-85*, 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) approximately 2 micros, k(a)(-1)=15 micros at 56 degrees C). We also compare the thermodynamic melting transition of lambdaHG with the nominal two-state folding mutant lambdaQG, which has a similar melting temperature. Unlike lambdaQG, lambdaHG 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.
Liu
Feng
F
Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801, USA. fengliu2@uiuc.edu
Gruebele
Martin
M
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins