Preparation of neocarzinostatin apoprotein mutants and the randomized library on the chromophore-binding cavity.
W39F, F52Y, S98G, S98A, and S98C mutants of the neocarzinostatin apoprotein (apo-NCS) were newly prepared and investigated their physicochemical properties. The circular dichroism (CD) spectra of F78W, F52Y, S98A, S98G, S98C were superimposable with that of wild type 1R49 protein although the minor spectral change seemed to be in the ellipticity of W39F. The results suggest that position 52, 78, and 98 involving natural chromophore binding do not play a major role in the inducing overall structural changes of the protein. Conversely, the position 39 would be affected slightly. Ethidium bromide (EtdBr) binding to mutants was also evaluated by the monitoring of total fluorescence intensity and fluorescence polarization (FP). The observed dissociation constant in the FP study was 4.4 microM for wild type, 2.2 microM for S98A, 1.3 microM for S98G, 9.7 microM for S98C, respectively. When S98G and F52Y, the calculated maximum change of the total fluorescence intensity was increased, suggesting that the EtdBr binding to S98G or F52Y were slightly improved compared with the wild type. Then, a total of 14 amino acids randomly substituted phage displayed library of apo-NCS was successfully prepared, because substitution of the amino acid structured the chromophore-binding cavity were not change the overall structural features. The phages which bound glycyrrhetic acid conjugated bovine serum albumin were enriched from this library using phage display technique as the pilot experiments. Although more precision investigation still needs, it should be possible to select variants that have new functions not found in nature.
Tomioka
Yoshihisa
Y
Division of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Kisara
Shigeki
S
Yoshizawa
Saki
S
Ozawa
Mika
M
Suzuki
Naoto
N
Yamaguchi
Hiroaki
H
Hishinuma
Takanori
T
Mizugaki
Michinao
M
Goto
Junichi
J
D001059:Apoproteins / Q000096:biosynthesis / Q000235:genetics; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004996:Ethidium / Q000378:metabolism; D005454:Fluorescence Polarization; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D012710:Serum Albumin, Bovine; D009353:Zinostatin / Q000096:biosynthesis*
D001059:Apoproteins; D019151:Peptide Library; D012710:Serum Albumin, Bovine; D009353:Zinostatin; D004996:Ethidium