Protein stabilization through phage display.
RNase S consists of two proteolytic fragments of RNase A, residues 1-20 (S20) and residues 21-124 (S pro). A 15-mer peptide (S15p) with high affinity for S pro was selected from a phage display library. Peptide residues that are buried in the structure of the wild type complex are conserved in S15p though there are several changes at other positions. Isothermal titration calorimetry studies show that the affinity of S15p is comparable to that of the wild type peptide at 25 degrees C. However, the magnitudes of DeltaH(o) and DeltaC(p) are lower for S15p, suggesting that the thermal stability of the complex is enhanced. In agreement with this prediction, at pH 6, the T(m) of the S15p complex was found to be 10 degrees C higher than that of the wild type complex. This suggests that for proteins where fragment complementation systems exist, phage display can be used to find mutations that increase protein thermal stability.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
S
S
Chakravarty
N
N
Mitra
I
I
Queitsch
A
A
Surolia
R
R
Varadarajan
S
S
Dübel
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002151:Calorimetry; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D017931:DNA Primers; D010446:Peptide Fragments; D019151:Peptide Library; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; C022542:ribonuclease S