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Matching electrostatic charge between DNA and coat protein in filamentous bacteriophage. Fibre diffraction of
M F Symmons1, L C Welsh, C Nave
1Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, U.K.
Abstract:
The virion of Ff (fd, f1, M13) filamentous bacteriophage consists of a long tube of coat protein subunits in a shingled, helical array, surrounding a genome of circular single-stranded DNA. Modified fd virions have been generated by a mutation (K48A) that removes one positive charge from each coat protein subunit in the C-terminal region of the polypeptide chain facing the DNA. The number of nucleotides in the mutant DNA is unchanged, but the K48A virions are 35% longer than wild-type. We have measured the X-ray diffraction attributable to single virions in hydrated gels of wild-type and K48A bacteriophages. Most of the diffraction pattern shows no significant difference between wild-type and K48A. Since the DNA is only about 12% by weight of the wild-type virion, the diffraction pattern is dominated by the protein contribution, and the absence of significant differences indicates that there are no significant changes in the symmetry or structure of the protein coat. But there is a change in the diffraction pattern in a region where the DNA and protein contributions are comparable. The diffraction pattern of the K48A mutant shows an increase in intensity of one of the weaker equatorial peaks, relative to wild-type, in a region where the protein contribution has negative sign but the DNA contribution has positive sign. This is consistent with a decrease in the ratio of DNA:protein per unit length of the K48A mutant. The results support the view that the protein forms a sheath lined with positive charges interacting electrostatically and non-specifically with a negatively charged DNA core of matching charge density. The lower positive charge density lining the capsid in the K48A mutant means that correspondingly fewer nucleotides can be packaged per coat protein subunit, which in turn requires an elongation of the DNA inside the virion. A longer virion is thus required to package the same amount of DNA. Within the error of measurement, the number of positive charges on the protein interacting with the DNA is the same in K48A as in the wild-type, despite the fact that the mutant is 35% longer than the wild-type.
Insights
A mutation in filamentous bacteriophage (Ff) coat protein causes virions to elongate. This structural change, observed via X-ray diffraction, reveals how DNA packaging is regulated by electrostatic interactions within the phage coat.
Area of Science:
- Structural biology
- Virology
- Biophysics
Background:
- Ff bacteriophages (fd, f1, M13) possess a helical protein coat surrounding a circular single-stranded DNA genome.
- A specific mutation (K48A) alters the C-terminal region of the coat protein, reducing its positive charge.
Purpose of the Study:
- To investigate the structural consequences of the K48A mutation on Ff bacteriophage virions.
- To elucidate the relationship between coat protein charge, DNA packaging, and virion length using X-ray diffraction.
Main Methods:
- X-ray diffraction analysis of hydrated gels containing wild-type and K48A mutant Ff bacteriophages.
- Comparison of diffraction patterns to identify structural and compositional differences between mutant and wild-type virions.
Main Results:
- X-ray diffraction patterns showed no significant changes in the protein coat structure or symmetry between wild-type and K48A mutants.
- A distinct change in diffraction intensity was observed in a region sensitive to both DNA and protein contributions, indicating altered DNA:protein ratio.
- The K48A mutant exhibited increased length (35%) while packaging the same amount of DNA, consistent with reduced positive charge density in the coat.
Conclusions:
- The K48A mutation leads to virion elongation due to decreased electrostatic interaction between the coat protein and the DNA core.
- This suggests that the phage coat's positive charge density is a critical factor in regulating DNA packaging and virion length.
- Despite elongation, the overall positive charge interacting with DNA remains comparable, implying compensatory mechanisms in DNA binding.