An intrinsic-tryptophan-fluorescence study of phage phi 29 connector/nucleic acid interactions.
The protein p10 of bacteriophage phi 29 assembled into connectors exhibit an intrinsic fluorescence with an emission peak centered at 335 nm, which suggests a hydrophobic environment of the three tryptohan residues that the protein contains. Upon incubation with linear DNA (but not with circular DNA), a decrease in the connector intrinsic fluorescence is measured which does not show any sequence specificity. The decrease in fluorescence is not observed when DNA is incubated with proteolyzed connectors, which lack the DNA-binding domain, suggesting that the fluorescence quenching is related to the binding of DNA to the phi 29 connectors. Acrylamide quenching studies reveal a higher accessibility of tryptophan residues to the quencher when the connector is bound to DNA. Protein denaturation by guanidine hydrochloride occurs at lower denaturant concentrations in the presence of linear DNA (but not circular DNA) than in its absence, suggesting a conformational change of phi 29 connector upon binding to linear DNA. This hypothesis is supported by the fact that the proteolyzed connectors, which do not bind DNA, are denatured at the same denaturant concentration, regardless of the presence of DNA. phi 29 connectors also bind RNA, but this interaction does not exert any effect on acrylamide quenching or guanidine hydrochloride denaturation. This result, together with that showing that proteolyzed connectors are able to interact with RNA, reinforces the idea that phi 29 connectors have two independent domains for interaction with DNA and RNA.
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad del País Vasco, Bilbao, Spain.
M A
MA
Urbaneja
S
S
Rivas
J L
JL
Carrascosa
J M
JM
Valpuesta
D017103:Bacillus Phages / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014764:Viral Proteins / Q000737:chemistry*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D014764:Viral Proteins; D014364:Tryptophan