Biophysical characterization of the DNA binding domain of gpNu1, a viral DNA packaging protein.
Terminase enzymes are common to double-stranded DNA viruses. These enzymes "package" the viral genome into a pre-formed capsid. Terminase from bacteriophage lambda is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the expression and biochemical characterization of gpNu1DeltaK100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C. E. (1999) Biochemistry 38, 465-477). Here we present a biophysical characterization of this construct. Thermally induced loss of secondary and tertiary structures is fully reversible. Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative. NMR and limited proteolysis data suggest that approximately 30 amino acids of gpNu1DeltaK100 are solvent-exposed and highly flexible. We therefore constructed gpNu1DeltaE68, a protein consisting of the N-terminal 68 residues of gpNu1. gpNu1DeltaE68 is a dimer with no evidence of dissociation or further aggregation. Thermally induced unfolding of gpNu1DeltaE68 is reversible, with concomitant loss of both secondary and tertiary structure. The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled. The data suggest that gpNu1DeltaE68 represents the minimal DNA binding domain of gpNu1. We further suggest that the C-terminal approximately 30 residues in gpNu1DeltaK100 adopt a pseudo-stable alpha-helix that extends from the folded core of the protein. A model describing the role of this helix in the assembly of the packaging apparatus is discussed.
Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
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Bain
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Berton
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Ortega
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Baran
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Yang
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Catalano
D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D017931:DNA Primers; D006358:Hot Temperature; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D019065:Virus Assembly
D017931:DNA Primers; D014764:Viral Proteins; C050856:lambda phage protein Nu 1