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Tyrosine mutant helps define overlapping CD bands from fd gene 5 protein.nucleic acid complexes
1Program in Molecular and Cell Biology, University of Texas at Dallas, Richardson 75083-0688, USA.
Biopolymers
|September 1, 1997
Summary
A mutant gene 5 protein (g5p) with altered tyrosine activity aids in resolving overlapping CD bands in protein-nucleic acid complexes. This method clarifies nucleic acid stacking and perturbation upon binding to fd gene 5 protein.
Area of Science:
- Biophysical Chemistry
- Molecular Biology
- Spectroscopy
Background:
- Analyzing circular dichroism (CD) bands in protein-nucleic acid complexes is challenging due to overlapping signals.
- Changes in both protein and nucleic acid CD spectra occur upon complex formation, complicating interpretation.
Purpose of the Study:
- To develop a method for confidently resolving overlapping CD bands of nucleic acids complexed with fd gene 5 protein (g5p).
- To utilize a mutant g5p with minimal tyrosine optical activity change upon nucleic acid binding for improved spectral analysis.
Main Methods:
- Employed a Y34F mutant of fd gene 5 protein (g5p), where tyrosine at position 34 is replaced by phenylalanine.
- Studied the interactions of the Y34F mutant g5p with polyriboadenylic acid (poly[r(A)]), polydeoxyadenylic acid (poly[d(A)]), and fd single-stranded DNA (ssDNA).
- Utilized circular dichroism (CD) spectroscopy to analyze spectral changes upon complex formation.
Main Results:
- The CD spectrum of poly[r(A)] saturated with Y34F mutant g5p was nearly the sum of free poly[r(A)] and free protein spectra.
- Spectra of poly[d(A)] and fd ssDNA saturated with Y34F mutant g5p, after subtracting free nucleic acid spectra, matched the free protein spectrum.
- The Y34F mutant protein's overall secondary structure remained unchanged upon binding, but individual aromatic residues' environments were altered.
- Nucleic acids complexed with g5p showed unstacking and perturbation, resembling dehydrated double-stranded DNA.
- Difference spectra distinguished specific spectral regions for RNA vs. DNA and protein-nucleic acid interactions.
Conclusions:
- The Y34F mutant g5p effectively resolves overlapping CD bands in protein-nucleic acid complexes.
- Nucleic acids bound to g5p are unstacked and perturbed, with distinct spectral signatures for DNA and RNA interactions.
- This approach enhances the interpretation of CD spectra for protein-nucleic acid complexes.