Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.
Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the bacteriophage M13 is hindered by a specific protein aggregation effect. Conditions are described for which NMR spectra of the protein can best be recorded. The aromatic part of the spectrum has been reinvestigated by means of two-dimensional total correlation spectroscopy. Sequence-specific assignments were obtained for all of the aromatic amino acid residues with the help of a series of single-site mutant proteins. The solution properties of the mutants of the aromatic amino acid residues have been fully investigated. It has been shown that, for these proteins, either none or only local changes occur compared to the wild-type molecule. Spin-labeled oligonucleotide-binding studies of wild-type and mutant gene V proteins indicate that tyrosine 26 and phenylalanine 73 are the only aromatic residues involved in binding to short stretches of single-stranded DNA. The degree of aggregation of wild-type gene V protein is dependent on both the total protein and salt concentration. The data obtained suggest the occurrence of specific protein-protein interactions between dimeric gene V protein molecules in which the tyrosine residue at position 41 is involved. This hypothesis is further strengthened by the observation that the solubility of tyrosine 41 mutants of gene V protein is significantly higher than that of the wild-type protein. The discovery of the so-called 'solubility' mutants of M13 gene V protein has finally made it possible to study the solution structure of gene V protein and its interaction with single-stranded DNA by means of two-dimensional NMR.
Folkers
P J
PJ
Nijmegen Son Research Center, University of Nijmegen, The Netherlands.
Stassen
A P
AP
van Duynhoven
J P
JP
Harmsen
B J
BJ
Konings
R N
RN
Hilbers
C W
CW
D000596:Amino Acids / Q000737:chemistry; D001435:Bacteriophages / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D009994:Osmolar Concentration; D014764:Viral Proteins / Q000737:chemistry*
D000596:Amino Acids; D004268:DNA-Binding Proteins; D014764:Viral Proteins