Synthesis of the p10 single-stranded nucleic acid binding protein from murine leukemia virus.
The p10 murine leukemia virus (MuLV) protein is a basic single-stranded nucleic acid binding protein encoded by the extreme 3' region of the gag gene of MuLV type C. It contains the Cys-X2-Cys-X4-His-X4-Cys sequence shared by all retroviral gag polyproteins. A similar sequence is found in the gene 32 single-stranded DNA binding protein of bacteriophage T4 and is believed to be the zinc binding region of the protein. Solid phase synthesis of p10 was carried out based on the known primary structure of the native protein, with the exception that the acetamidomethyl (Acm) derivative of cysteine was incorporated at all three cysteine positions. The structure of the synthetic p10 was confirmed by direct amino-acid sequencing, as well as by amino acid analysis and FAB mass spectrometry of endoproteinase Lys-C peptides derived from p10. A Chou and Fasman analysis of the primary sequence predicts that p10 contains 9% beta strand and/or sheet and 36% alpha helix. Circular dichroism experiments carried out on the Acm derivatized peptide gave somewhat different results, in that they suggest that p10 contains approximately 70% random coil, less than 30% beta strand and/or sheet and less than 10% alpha helix. With a Ka of greater than 10(8) M-1 for single-stranded RNA, the synthetic peptide binds as tightly as the p10 protein does when isolated directly from infected HTG-2 cells. The Acm groups can be removed from the synthetic p10 peptide by the use of mercuric acetate, followed by treatment with dithiothreitol to sequester the mercuric ion.(ABSTRACT TRUNCATED AT 250 WORDS)
Roberts
W J
WJ
Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, CT 06510-8024.
Elliott
J I
JI
McMurray
W J
WJ
Williams
K R
KR
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D003545:Cysteine / Q000031:analogs & derivatives; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000138:chemical synthesis*; D015683:Gene Products, gag / Q000138:chemical synthesis*; D015750:Genes, gag / Q000235:genetics*; D009052:Leukemia Virus, Murine / Q000737:chemistry*; D013058:Mass Spectrometry; D011061:Poly A / Q000378:metabolism
D000596:Amino Acids; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D015683:Gene Products, gag; D011061:Poly A; C005163:poly(1,N(6)-ethenoadenylic acid); D003545:Cysteine