Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.

The process of mononucleosome assembly mediated by histone chaperone NAP1 was investigated using DNA fragments 146 and 207 bp in length containing the Lytechinus variegatus 5 S rDNA nucleosome positioning sequence. A quantitative description was derived using gel electrophoresis and fluorescent anisotropy data. First, NAP1-bound H3⅐H4 was released forming a DNA-histone tetramer complex with a time constant of k 1 ‫؍‬ (2.5 ؎ 0.7) ⅐ 10 4 M ؊1 s ؊1 .The tetrasome was converted quickly (k 2 ‫؍‬ (4.1 ؎ 3.5) ⅐ 10 5 M ؊1 s ؊1 ), by the addition of a single H2A⅐H2B dimer, into a "hexasome,"i.e. a nucleosome lacking one H2A⅐H2B dimer. From this intermediate a nucleosome was formed by the addition of a second H2A⅐H2B dimer with an average rate constant k 3 ‫؍‬ (6.6 ؎ 1.4) ⅐ 10 3 M ؊1 s ؊1 . For the back-reaction, significant differences were observed between the 146-and 207-bp DNA upon substitution of the canonical H2A histone with H2A.Z. The distinct nucleosome/ hexasome ratios were reflected in the corresponding equilibrium dissociation constants and revealed some differences in nucleosome stability. In a fourth reaction, NAP1 mediated the binding of linker histone H1 to the nucleosome, completing the chromatosome structure with k 4 ‫؍‬ (7.7 ؎ 3.7) ⅐ 10 3 M ؊1 s ؊1 . The activity of the chromatin remodeling complex ACF did not increase the kinetics of the mononucleosome assembly process.The repeating building block of chromatin is the nucleosome, a nucleoprotein complex consisting of two each of histones H2A, H2B, H3, and H4 wrapped around 146/147 bp of DNA (1). Several studies have focused on the mechanism by which these entities are assembled and how a defined chromatin structure is established (2-10). In vivo chromatin assembly is mostly coupled to DNA replication (11), but recent investigations have pointed out the importance of replicationindependent deposition of variant histones such as H2A.Z and H3.3. This process appears to be relevant in the formation of chromatin with differential transcriptional activity (12-16). Assembly of nucleosomes seems to be closely linked to the activity of ATP-dependent chromatin remodeling machineries, required for the formation of evenly spaced nucleosome arrays, which are characteristic for the native chromatin conformation (17).Simple mixing of histones and DNA leads mainly to large, insoluble aggregates (18,19). Accordingly, the transfer of histones to DNA in the cell is carried out by histone chaperones such as the heterotrimeric chromatin assembly factor 1 (CAF1) (20), N1/N2 (21, 22), nucleoplasmin (23-25), HIRA (26,27), and nucleosome assembly protein 1 (NAP1) 2 (28, 29). Per definition, all share the capability of binding histones and releasing them to DNA or other targets, but they differ with respect to in vivo functions and the preferred histone interaction partner. The histone chaperone NAP1 studied here is involved in the shuttling of newly synthesized histone H2A⅐H2B dimers from the cytoplasm to the nucleus and the deposition of histones onto the DNA (3, 17, 30). In a...

NAP1 (nucleosome assembly protein 1) is a histone chaperone that has been described to bind predominantly to the histone H2A⅐H2B dimer in the cell during shuttling of histones into the nucleus, nucleosome assembly/remodeling, and transcription. Here it was examined how NAP1 interacts with chromatin fibers isolated from HeLa cells. NAP1 induced a reversible change toward an extended fiber conformation as demonstrated by sedimentation velocity ultracentrifugation experiments. This transition was due to the removal of the linker histone H1. The H2A⅐H2B dimer remained stably bound to the native fiber fragments and to fibers devoid of linker histone H1. This was in contrast to mononucleosome substrates, which displayed a NAP1-induced removal of a single H2A⅐H2B dimer from the core particle. The effect of NAP1 on the chromatin fiber structure was examined by scanning/atomic force microscopy. A quantitative image analysis of ϳ36,000 nucleosomes revealed an increase of the average internucleosomal distance from 22.3 ؎ 0.4 to 27.6 ؎ 0.6 nm, whereas the overall fiber structure was preserved. This change reflects the disintegration of the chromatosome due to binding of H1 to NAP1 as chromatin fibers stripped from H1 showed an average nucleosome distance of 27.4 ؎ 0.8 nm. The findings suggest a possible role of NAP1 in chromatin remodeling processes involved in transcription and replication by modulating the local linker histone content.The dynamic organization of chromatin in the eukaryotic nucleus is tightly connected to transcription (1-3). Transcribed chromatin is thought to be in a more open conformation with a higher accessibility to DNase I or micrococcal nuclease (4 -7). On the other hand, highly compacted and dense chromatin regions, referred to as heterochromatin, are often transcriptionally inactive and contain a reduced number of genes (3,7,8). Several factors have been identified that promote a transition between a transcriptionally active and a more dense/inactive chromatin conformation. These include linker histones (9 -11), DNA methylation (2, 12), histone modifications (13-15), and the incorporation of histone variants (16 -19). The dynamic nature of chromatin manifests itself by continuous rearrangements of its three-dimensional structure. These changes involve the activity of histone chaperones that mediate the ordered deposition and the removal and exchange of histones (20 -23). The central carrier of the histone H2A⅐H2B dimer in the cell is NAP1 (nucleosome assembly protein 1) (24). NAP1 is involved in the transport of the histone H2A⅐H2B dimer from the cytoplasm to the nucleus and the deposition of histones onto the DNA as described in several reviews (20 -23). NAP1 and other histone chaperones stimulate the binding of transcription factors to chromatin templates (25, 26). In yeast, loss of NAP1 leads to an altered gene expression of about 10% of the genome (27), and several lines of evidence suggest that NAP1 has activities related to transcription. First, it has been shown that NAP1 is present in com...

The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of approximately 1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of DeltaDeltaG approximately 3 kcal mol(-1) as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 +/- 29 degrees and 136 +/- 18 degrees (air-dried samples), and 78 +/- 25 degrees and 137 +/- 25 degrees (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 +/- 34 degrees for complexes in air and 85 +/- 10 degrees in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 +/- 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 +/- 34 basepairs that are wound around the nucleosome.