TYROSINE MUTANT HELPS DEFINE OVERLAPPING CD BANDS FROM FD GENE-5-PROTEIN NUCLEIC-ACID COMPLEXES

We used a mutant gene 5 protein (g5p) to assign and interpret overlapp ing CD bands of protein nucleic acid complexes. The analysis of overla pping protein and nucleic acid CD bands is a common challenge for CD s pectroscopists, since both components of the complex may change upon b inding. We have now been able to more confidently resolve the bands of nucleic acids complexed with the fd gene 5 protein by exploiting a mu tant gene 5 protein that has an insignificant change in tyrosine optic al activity at 229 nm upon binding to nucleic acids. We have studied t he interactions of the mutant Y34F g5p (Tyr-34 substituted with phenyl alanine) with poly [r(A) ], poly [d(A)], and fd single-stranded DNA (s sDNA). Our results showed the following: (1) The 205-300 nm spectrum o f poly [r(A)] saturated with the Y34F mutant (P/N = 0.25) was essentia lly the sum of the spectra of poly [r(A)] at a high temperature plus t he spectrum of the free protein, except for a minor negative band at 2 57 nm. (2) The spectra of poly [d(A)] and fd ssDNA saturated with the mutant protein at a P/N = 0.25, minus the spectra of the free nucleic acids at a high temperature, also essentially equaled the spectrum of the free protein in the 205-245 nm region. (3) While the overall secon dary structure of the Y34F protein did not change upon binding to any of these nucleic acids, there could be changes in the environment of i ndividual aromatic residues. (4) Nucleic acids complexed with the g5p are unstacked (as if heated) and (in the cases of the DNAs) perturbed as if part of a dehydrated double-stranded DNA. (5) Difference spectra revealed regions of the spectrum specific for the particular nucleic acid, the protein, and whether g5p was bound to DNA or RNA. (C) 1997 J ohn Wiley di Sons, Inc.