THE AMINO-ACID-SEQUENCE AND INTERACTION WITH THE NUCLEOSOME CORE DNA OF TRANSITION PROTEIN 4 FROM BOAR LATE SPERMATID NUCLEI

The primary structure of transition protein 4 (TP4) from boar late spermatid nuclei was determined by automated Edman degradation of S-pyridylethylated protein and of peptides generated by cleavage with Staphylococcus aureus V8 protease, lysyl endopeptidase and CNBr. Boar TP4 is a basic protein consisting of a highly basic amino-terminal half (residues 1-73) and a less basic carboxy-terminal half (residues 74-138). The latter half includes a highly hydrophobic segment, a four-times tandemly repeated sequence, N(G)QNKR(K)X, and a carboxy-terminal segment containing Trp126. Ultraviolet absorption and CD spectra of TP4-rat-liver-nucleosome-core-DNA (double-stranded DNA) complexes suggest a TP4-induced local melting of DNA. Although at 1 mM NaCl TP4 brought about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From the results of quenching of tryptophan (Trp126) fluorescence of TP4 upon its binding to double-stranded and single-stranded boar liver nucleosome-core DNA at 50 mM NaCl, the apparent association constants for the binding of TP4 to double-stranded and single-stranded DNA were calculated to be 7.3x10(3) M(-1) and 4.1x10(3) M(-1), respectively. These results suggest that TP4, having different domain structures from TP1-3 and a higher affinity for double-stranded DNA, induces a local destabilization of DNA probably through the stacking of Trp126 with nucleic acid bases.