超遠心分析と非変性条件下でのMSによるNAP-1の会合状態の解析

抄録

Nucleosome assembly is essential for chromatin structure formation during transcription, translation and for the repair of genomic information. During these processes histones are delivered to naked DNA by proteins known as histone chaperones. NAP-1 (nucleosome assembly protein 1), one of the best characterized protein among 10 or more 15 known histone chaperones, is regarded as a multi-functional protein in interphase. During mitosis, NAP-1 is localized within the cytoplasm and segregated from histones required for the next cell cycle. Previous reports on yeast nucleosome assembly protein 1 (yNAP-1) suggested that yNAP-1 forms dimers and higher oligomers, specifically, octamer, hexadecamer under the physiological salt concentrations (150 mM NaCl), while at higher ionic strength (>750 mM NaCl) only a homogeneous dimer was evident. However, association of the human NAP-1 is still unknown. Here, we performed mass spectrometry (MS) analysis under the denaturing condition, and analytical ultracentrifugation (AUC) to investigate association states of human NAP-1 in solution. MS result clearly indicates that the primary assembly unit of hNAP-1 core is the dimer and that higher oligomers (tetramer, hexamer, octamer, decamer and dodecamer) are formed at physiological ionic strength. Sedimentation velocity measurement by AUC showed that they form a heterogeneous series of large oligomers at physiological ionic strength with their sedimentation coefficients ranging from 4 S to 22 S. At high ionic strength such as 750 mM sodium chloride, higher oligomers are disrupted. These results clearly shows hNAP-1 have different distribution pattern from that of yeast NAP-1 in solution. We here demonstrate the combination of non-denaturing MS and AUC provides is powerful approach for the investigation of non-covalent protein-protein interactions in solution.