Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS

2011 ◽  
Vol 436 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Masanori Noda ◽  
Susumu Uchiyama ◽  
Adam R. McKay ◽  
Akihiro Morimoto ◽  
Shigeki Misawa ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Electrostatic Interactions ◽  
Analytical Ultracentrifugation ◽  
Three Dimensional ◽  
Sedimentation Velocity ◽  
Dimensional Structure ◽  
Histone H2a ◽  
Nucleosome Assembly ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1

Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A–H2B dimer or H3–H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein–protein interactions in solution.

scholarly journals Nucleosome Assembly Protein 1 Exchanges Histone H2A-H2B Dimers and Assists Nucleosome Sliding

2004 ◽  
Vol 280 (3) ◽  
pp. 1817-1825 ◽  
Author(s):  
Young-Jun Park ◽  
Jayanth V. Chodaparambil ◽  
Yunhe Bao ◽  
Steven J. McBryant ◽  
Karolin Luger
Keyword(s):  
Histone H2a ◽  
Nucleosome Assembly ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1 ◽  
Nucleosome Sliding

scholarly journals Histone Chaperone Nap1 Is a Major Regulator of Histone H2A-H2B Dynamics at the InducibleGALLocus

2016 ◽  
Vol 36 (8) ◽  
pp. 1287-1296 ◽  
Author(s):  
Xu Chen ◽  
Sheena D'Arcy ◽  
Catherine A. Radebaugh ◽  
Daniel D. Krzizike ◽  
Holli A. Giebler ◽  
...  
Keyword(s):  
Critical Role ◽  
Histone Chaperones ◽  
Histone H2a ◽  
Nucleosome Assembly ◽  
Content Type ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1 ◽  
Histone Exchange

Histone chaperones, like nucleosome assembly protein 1 (Nap1), play a critical role in the maintenance of chromatin architecture. Here, we use theGALlocus inSaccharomyces cerevisiaeto investigate the influence of Nap1 on chromatin structure and histone dynamics during distinct transcriptional states. When theGALlocus is not expressed, cells lacking Nap1 show an accumulation of histone H2A-H2B but not histone H3-H4 at this locus. Excess H2A-H2B interacts with the linker DNA between nucleosomes, and the interaction is independent of the inherent DNA-binding affinity of H2A-H2B for these particular sequences as measuredin vitro. When theGALlocus is transcribed, excess H2A-H2B is reversed, and levels of all chromatin-bound histones are depleted in cells lacking Nap1. We developed anin vivosystem to measure histone exchange at theGALlocus and observed considerable variability in the rate of exchange across the locus in wild-type cells. We recapitulate this variability within vitronucleosome reconstitutions, which suggests a contribution of DNA sequence to histone dynamics. We also find that Nap1 is required for transcription-dependent H2A-H2B exchange. Altogether, these results indicate that Nap1 is essential for maintaining proper chromatin composition and modulating the exchange of H2A-H2Bin vivo.

scholarly journals Dual Roles of p300 in Chromatin Assembly and Transcriptional Activation in Cooperation with Nucleosome Assembly Protein 1 In Vitro

2002 ◽  
Vol 22 (9) ◽  
pp. 2974-2983 ◽  
Author(s):  
Hiroshi Asahara ◽  
Sophie Tartare-Deckert ◽  
Takeya Nakagawa ◽  
Tsuyoshi Ikehara ◽  
Fumiko Hirose ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Chromatin Assembly ◽  
Micrococcal Nuclease ◽  
Histone H2a ◽  
Nucleosome Assembly ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1 ◽  
Core Histones ◽  
Kix Domain

ABSTRACT In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.

scholarly journals Association States of Nucleosome Assembly Protein 1 and Its Complexes with Histones

2005 ◽  
Vol 280 (16) ◽  
pp. 15690-15699 ◽  
Author(s):  
Katalin Fejes Tóth ◽  
Jacek Mazurkiewicz ◽  
Karsten Rippe
Keyword(s):  
Nuclear Import ◽  
Analytical Ultracentrifugation ◽  
Nucleosome Assembly ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1 ◽  
Core Histones ◽  
A Cell ◽  
Cycle Dependent ◽  
Equilibrium Dissociation ◽  
Prevalent Species

The histone chaperone NAP1 is a carrier of histones during nuclear import, nucleosome assembly, and chromatin remodeling. Analytical ultracentrifugation was used to determine the association states of NAP1 alone and in complexes with core histones. In addition, the concentration dependence of the association was quantified by determining the equilibrium dissociation constant between different NAP1 species. At physiological protein and salt concentrations the prevalent species were the NAP1 dimer and octamer. These were also the association states found to interact with histones in a stoichiometry of one NAP1 monomer per histone. Based on these results a model for a cell cycle-dependent shift of the NAP1 dimer-octamer equilibrium is proposed that reflects different biological functions of NAP1.

Understanding Bacterial Ice Nucleation

2021 ◽  
Author(s):  
Ralph Schwidetzky ◽  
Max Lukas ◽  
Anna T. Kunert ◽  
Ulrich Pöschl ◽  
Janine Fröhlich-Nowoisky ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Affinity Purification ◽  
Hydration Shell ◽  
Three Dimensional ◽  
American Chemical Society ◽  
Chemical Society ◽  
Ice Nucleation ◽  
Dimensional Structure ◽  
Protein Activity ◽  
Ice Nucleators

<p>Bacterial ice-nucleating proteins (INPs) promote heterogeneous ice nucleation better than any known material. On the molecular scale, bacterial INPs are believed to function by organizing water into ice‑like patterns to enable the formation of embryonic crystals. However, the details of their working mechanism remains largely elusive. Here, we report the results of comprehensive evaluations of environmentally relevant effects such as changes in pH, the presence of ions and temperature on the activity, three-dimensional structure and hydration shell of bacterial ice nucleators using ice affinity purification, high-throughput ice nucleation assays and surface-specific sum-frequency generation spectroscopy.</p><p> </p><p>[1] Lukas, Max, et al. "Electrostatic Interactions Control the Functionality of Bacterial Ice Nucleators." Journal of the American Chemical Society 142.15 (2020): 6842-6846.</p><p>[2] Lukas, Max, et al. "Interfacial Water Ordering Is Insufficient to Explain Ice-Nucleating Protein Activity." The Journal of Physical Chemistry Letters 12 (2020): 218-223.</p>

Synthesis of Dendridic NLO Chromophores for the Improvement of Order in Electro-optics

2004 ◽  
Vol 846 ◽  
Author(s):  
Jessica Sinness ◽  
Olivier Clot ◽  
Scott R. Hammond ◽  
Nishant Bhatambrekar ◽  
Harrison L. Rommel ◽  
...  
Keyword(s):  
Aspect Ratio ◽  
Electrostatic Interactions ◽  
Dipole Moments ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Quantum Calculations ◽  
Electro Optic ◽  
The Matrix ◽  
Macroscopic Domain ◽  
Nlo Chromophores

ABSTRACTPrevious research in organic electro-optics has shown dramatic increases in the hyperpolarizablity of NLO chromophores. However, this large microscopic activity has not been translated to the macroscopic domain. The polymeric electro-optic (E-O) materials continue to lack the high noncentrosymmetric order of the poled chromophores within the matrix necessary for high E-O response (r33). This deficiency of order represents one major obstacle that must be overcome before E-O device commercialization can be achieved. This lack of order is partially due to the large dipole moments of high μβ chromophores, which cause the chromophores to align in a centrosymmetric fashion through intermolecular electrostatic interactions. However, quantum calculations show that when the aspect ratio between the width and length of the chromophore system is adjusted to be greater than 1.4:1 by adding bulky side groups around the center of the chromophore, it would prevent side on pairing of the chromophores. This would cause a decrease in the large areas of centrosymmetric aggregation and thus allow for easier poling of the system. Here we report the synthesis of a nanoscale NLO architecture in which dendritic moieties have been incorporated around the center of the chromophore to give a three dimensional structure in order to achieve the 1.4:1 aspect ratio and maximize the macroscopic order of the system.

Organization and expression of the Paramecium caudatum gene encoding nucleosome assembly protein 1

Gene ◽  
2001 ◽  
Vol 280 (1-2) ◽  
pp. 107-114 ◽  
Author(s):  
Norihito Nishiyama ◽  
Shun Sawatsubashi ◽  
Masaki Ishida ◽  
Kiyoshi Yamauchi
Keyword(s):  
Paramecium Caudatum ◽  
Nucleosome Assembly ◽  
Gene Encoding ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1
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