title: Thermodynamics of replacing an alpha-helical Pro residue in the P40S mutant of Escherichia coli thioredoxin.
abstract: Escherichia coli thioredoxin is a 108 amino acid oxidoreductase and contains a single Met residue at position 37. The protein contains a long alpha-helical stretch between residues 32 and 49. The central residue of this helix, Pro40, has been replaced by Ser. The stabilities of the oxidized states of two proteins, the single mutant M37L and the double mutant M37L,P40S, have been characterized by differential scanning calorimetry (DSC) and also by a series of isothermal guanidine hydrochloride (GuHCl) melts in the temperature range of 277 to 333 K. The P40S mutation was found to stabilize the protein at all temperatures upto 340 K though both proteins had similar Tm values of about 356 K. At 298 K, the M37L,P40S mutant was found to be more stable than M37L by 1.5 kcal/mol. A combined analysis of GuHCl and calorimetric data was carried out to determine the enthalpy, entropy, and heat capacity change upon unfolding. At 298 K there was a large, stabilizing enthalpic effect in P40S though significant enthalpy-entropy compensation was observed and the two proteins had similar values of deltaCp. Thus, replacement of a Pro in the interior of an alpha helix can have substantial effects on protein stability.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: A
authors.initials: A
authors.lastname: Chakrabarti
authors.forename: S
authors.initials: S
authors.lastname: Srivastava
authors.forename: C P
authors.initials: CP
authors.lastname: Swaminathan
authors.forename: A
authors.initials: A
authors.lastname: Surolia
authors.forename: R
authors.initials: R
authors.lastname: Varadarajan
mesh_terms: D019943:Amino Acid Substitution; D002151:Calorimetry; D004926:Escherichia coli / Q000378:metabolism*; D019791:Guanidine; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011392:Proline; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; D013879:Thioredoxins; D011392:Proline; D019791:Guanidine