Discovery of a stable dimeric mutant of cyanovirin-N (CV-N) from a T7 phage-displayed CV-N mutant library.
Mutant proteins with altered properties can be useful probes for investigating structure, ligand binding sites, mechanisms of action, and physicochemical attributes of the corresponding wild-type proteins of interest. In this report, we illuminate properties of mutants of the potent HIV-inactivating protein, cyanovirin-N (CV-N), selected by construction of a mutant library by error-prone polymerase chain reaction and affinity-based screening using T7 phage display technology. After three rounds of biopanning, two phage-displayed, one-point mutants of CV-N, Ser52Pro and Ala77Thr, were isolated. After the elucidation of biological activities of the mutants displayed on phage as well as the Escherichia coli-expressed, purified mutant proteins, we subsequently subjected the mutants to analyses by native PAGE and size-exclusion chromatography. We found that the Ser52Pro mutant not only was active against HIV but also existed exclusively as a dimer in solution. This was in marked contrast to the wild-type CV-N, which exists in solution predominantly as the monomer. The Ser52Pro mutant provides a novel model for further investigations of the folding mechanism as well as structure-activity requirements for CV-N's antiviral properties.
Han
Zhaozhong
Z
Molecular Targets Drug Discovery Program, NCI-Frederick, Frederick, Maryland 21702-1201, USA.
Xiong
Changyun
C
Mori
Toshiyuki
T
Boyd
Michael R
MR
D019943:Amino Acid Substitution; D019380:Anti-HIV Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins; D017123:Bacteriophage T7 / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D002850:Chromatography, Gel; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D017510:Protein Folding; D013329:Structure-Activity Relationship
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D015699:HIV Envelope Protein gp120; D019151:Peptide Library; C107733:cyanovirin N