Tuning lambda6-85 towards downhill folding at its melting temperature

J Mol Biol. 2007 Jul 13;370(3):574-84. doi: 10.1016/j.jmb.2007.04.036. Epub 2007 Apr 19.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacteriophage lambda
  • Circular Dichroism
  • DNA-Binding Proteins* / chemistry
  • DNA-Binding Proteins* / genetics
  • DNA-Binding Proteins* / metabolism
  • Molecular Sequence Data
  • Mutation
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Denaturation*
  • Protein Folding*
  • Repressor Proteins* / chemistry
  • Repressor Proteins* / genetics
  • Repressor Proteins* / metabolism
  • Temperature*
  • Thermodynamics
  • Viral Proteins* / chemistry
  • Viral Proteins* / genetics
  • Viral Proteins* / metabolism
  • Viral Regulatory and Accessory Proteins

Substances

  • DNA-Binding Proteins
  • Repressor Proteins
  • Viral Proteins
  • Viral Regulatory and Accessory Proteins
  • phage repressor proteins