| Abstract
| - A core Y61F mutant of the gene 5 single-stranded DNA-bindingprotein (g5p) of f1 bacterialvirus aggregated when expressed from a plasmid, but, after refolding invitro, it behaved much like wild-type and may be a stability or folding mutant. Circular dichroism(CD) titrations showed the samecooperative polynucleotide binding modes for Y61F and wild-type g5p.There are n = 4 and n ≅ 2.5modes for binding to poly[d(A)] at low ionic strengths, butn = 4, n = 3, and n ≅ 2−2.5modes forbinding to fd single-stranded viral DNA (fd ssDNA), where nis the number of nucleotides occluded byeach bound g5p monomer in a given mode. Y61F g5p has slightlyreduced affinity in the n = 4 mode.Electron microscopy showed that Y61F g5p forms left-handednucleoprotein superhelices indistinguishablefrom wild-type. Progression from binding to fd ssDNA in then = 4 to n = 3 to n ≅ 2−2.5mode isaccompanied by an increase in the number of helical turns, an increasefrom (7.7 ± 0.3) to (9.5 ± 0.3)to (∼10−13) g5p dimers per turn, and a decrease in the number ofDNA nucleotides per turn. From CDspectra for four of five possible Y → F g5p mutants, we infer thatthe fifth tyrosine, Tyr 56, contributesstrongly to the CD. Retention of a strong 229 nm CD band in allmutants indicates that all retain elementsof the native structure. Spectra of Y26F, Y34F, and Y61F g5p implylimited mobility of the replacementPhe. Comparison of measured with calculated CD spectra alsosuggests limited mobility for Tyr 26 andTyr 34 in g5p in solution, and provides new information that the g5pstructure in solution may be dominatedby Tyr 41 rotamers differing from that stabilized in thecrystal.
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