Tyrosine mutant helps define overlapping CD bands from fd gene 5 protein · nucleic acid complexes

Abstract

We used a mutant gene 5 protein (g5p) to assign and interpret overlapping CD bands of protein · nucleic acid complexes. The analysis of overlapping protein and nucleic acid CD bands is a common challenge for CD spectroscopists, since both components of the complex may change upon binding. We have now been able to more confidently resolve the bands of nucleic acids complexed with the fd gene 5 protein by exploiting a mutant gene 5 protein that has an insignificant change in tyrosine optical activity at 229 nm upon binding to nucleic acids. We have studied the interactions of the mutant Y34F g5p (Tyr‐34 substituted with phenylalanine) with poly[r(A)], poly[d(A)], and fd single‐stranded DNA (ssDNA). Our results showed the following: (1) The 205–300 nm spectrum of poly[r(A)] saturated with the Y34F mutant (P/N = 0.25) was essentially the sum of the spectra of poly[r(A)] at a high temperature plus the spectrum of the free protein, except for a minor negative band at 257 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 secondary structure of the Y34F protein did not change upon binding to any of these nucleic acids, there could be changes in the environment of individual 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. © 1997 John Wiley and Sons, Inc. Biopoly 42: 337–348, 1997

MBNL1–RNA Recognition: Contributions of MBNL1 Sequence and RNA Conformation

“…The decrease in peak intensity implies reduced base stacking in the bound structures of (CUG) 12 and cTNT21. [36][37][38] Upon binding MBNL1, the wavelength maximum was red-shifted by 3 nm for (CUG) 12 , 3 nm for cTNT18, and 5 nm for cTNT21, which is consistent with changes in RNA structure occurring upon MBNL1 binding. A decrease in peak intensity and shifting towards longer wavelengths upon MBNL1 binding was previously observed with (CUG) 4 RNA.…”

“…[18] This type of change is also typical when ssDNA-binding proteins bind DNA and RNA targets. [36,39] These data support a model in which the structures of (CUG) 12 and cTNT21 are altered towards single-stranded upon binding MBNL1, while that of cTNT18 undergoes structural changes. The CD spectra of the bound cTNT18p and cTNT18 RNAs are nearly identical, although those of the free RNA differ, suggesting that the structures of these RNA sequences become similar upon binding MBNL1.…”

The Binding Affinity of Ff Gene 5 Protein Depends on the Nearest-Neighbor Composition of the ssDNA Substrate

“…A tyrosyl CD band at 229 nm increases in magnitude because of the additions of g5p, but the relative increase is not as great as would be predicted from the known amount of added g5p, because of the perturbation of a single tyrosine (Tyr-34) at the interface of cooperatively bound proteins (Day, 1973;Kansy et al, 1986;Mark et al, 1995;Thompson et al, 1998). Making use of a Y34F mutant protein, we found that CD bands of poly[d(A)] and fd ssDNA below 250 nm are perturbed as if the bases were partially unstacked by heating to 70 -80°C (Mark and Gray, 1997). Thus changes in the nucleic acid upon binding g5p are qualitatively similar to the combined effects of dehydration plus heating, with no obvious CD contributions from interactions between the DNA bases and protein chromophores at wavelengths above 210 nm (Mark and Gray, 1997).…”

“…Making use of a Y34F mutant protein, we found that CD bands of poly[d(A)] and fd ssDNA below 250 nm are perturbed as if the bases were partially unstacked by heating to 70 -80°C (Mark and Gray, 1997). Thus changes in the nucleic acid upon binding g5p are qualitatively similar to the combined effects of dehydration plus heating, with no obvious CD contributions from interactions between the DNA bases and protein chromophores at wavelengths above 210 nm (Mark and Gray, 1997).…”

Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein–protein cooperative interactions

“…For plots of protein CD changes at 229 nm during the titrations, the nucleic acid contribution was subtracted from the measured CD. The CD value at 229 nm of poly[d(A)] when saturated with g5p (at a [protein monomer]/[nucleotide] molar ratio, [P]/[N], of 0.25) has been shown to be close to that of free poly[d(A)] at about 76°C (Mark and Gray 1997). Thus, at [P]/[N] ≥ 0.25, this constant value for the poly[d(A)] component was subtracted from the measured CD.…”