Cooperative deformation of a de novo designed protein.
A de novo protein design has been made to understand the unique packing of natural proteins that have a beta/alpha-barrel fold. A carefully designed 207 amino acid sequence was synthesized using an Escherichia coli expression system and the structural and thermodynamic characteristics of the purified protein were studied. At neutral pH the protein is soluble and monomeric, with large amounts of secondary structure and a hydrophobic core, although the broad resonance peaks of its NMR spectrum suggest that the designed protein does not have a unique structure with tightly packed side chains. In an H-D exchange experiment, no amido protons of the designed protein exchanged slowly with deuterons. At acidic pH, thermal unfolding was observed with a remarkable change in the excess heat capacity measured directly by a differential scanning microcalorimeter. The enthalpy and entropy differences at 110 degrees C, extrapolated from analyzed thermodynamic parameters, are approximately 1/3 of the common values for natural proteins. These measurements indicate that the folding is significantly cooperative as expected, but that the protein is still loosely packed.
Tanaka
T
T
Protein Engineering Research Institute, Osaka, Japan.
Kuroda
Y
Y
Kimura
H
H
Kidokoro
S
S
Nakamura
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics
D011993:Recombinant Fusion Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D014305:Triose-Phosphate Isomerase