Biology subjects

van Ingen, H.

Publications and source records attributed to van Ingen, H..

5 recordsLinked to original sources

bioRxiv2026

Methyl-TROSY NMR at 1.2 GHz: exploiting resolution and magnetic field alignment for structure and dynamics of large biomolecular assemblies

The range of protein assemblies that can be studied by NMR has greatly expanded both by increases in magnetic field strength and by development of new NMR approaches, such as methyl-group specific transverse relaxation optimized spectroscopy (methyl-TROSY). Recently, a new generation of NMR magnets operating at 1.2 GHz have become available, offering the highest resolution for biomolecular NMR. Here, we investigated the performance of methyl-TROSY NMR at 1.2 GHz in the characterization of the structure and dynamics of large biomolecular assemblies, using the nucleosome, a 200 kDa protein-DNA complex, as a test sample. We find that the increased resolution of the 1.2 GHz system allowed to resolve small asymmetries in sidechain conformations that remained hidden at lower magnetic field strengths. Increased CSA relaxation effectively increased 13C transverse relaxation rates by 20% at 1200 compared to 900 MHz, negatively impacting sensitivity. We further observed significant magnetic field alignment of the nucleosome at 1.2 GHz, giving rise to methyl 1H-13C residual dipolar couplings (RDCs). We show that these histone methyl group RDCs can be used to aid stereo-specific assignment and to determine the overall conformation of the nucleosomal DNA, revealing a significant unwrapping of the DNA from the histone core in solution. We expect that 1.2 GHz NMR will be very advantageous for studies of large biomolecular assemblies, and in particular for protein-DNA complexes.

biochemistry↗

bioRxiv2023

A structural and dynamic visualization of the interaction between the microtubule-associated protein 7 (MAP7) and microtubules

Microtubules (MTs) are key components of the eukaryotic cytoskeleton and are essential for intracellular organization, organelle trafficking and mitosis. MT tasks depend on binding and interactions with MT-associated proteins (MAPs). MT-associated protein 7 (MAP7) has the unusual ability of both MT binding and activating kinesin-1-mediated cargo transport along MTs. Additionally, the protein is reported to stabilize MTs with its 112 amino-acid long MT-binding domain (MTBD). Here we investigate the structural basis of the interaction of MAP7 MTBD with the MT lattice. Using a combination of solid and solution-state nuclear magnetic resonance (NMR) spectroscopy with electron microscopy, fluorescence anisotropy and isothermal titration calorimetry, we shed light on the binding mode of MAP7 to MTs at an atomic level. Our results show that a combination of interactions between MAP7 and MT lattice extending beyond a single tubulin dimer and including tubulin C-terminal tails contribute to formation of the MAP7-MT complex.

biophysics↗

bioRxiv2021

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B(1-5). Here, we show that the acidic domain of DNA repair factor APLF (APLFAD) can assemble the histone octamer in a single step, and deposit it on DNA to form nucleosomes. The crystal structure of the APLFAD-histone octamer complex shows that APLFAD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLFAD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted. one sentence summaryHistone chaperone APLF assembles histones H2A-H2B/H3-H4 into histone octamers to deposit them onto DNA and form nucleosomes.

biochemistry↗

bioRxiv2019

Catalysis by a rigid enzyme

Many enzymes are dynamic entities, sampling conformational states that are relevant for catalytic activity. Crystal structures of catalytic intermediates suggest, however, that not all enzymes require structural changes for activity. The single-domain enzyme xylanase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose. We demonstrate that BCX in solution undergoes minimal structural changes during catalysis. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow, enzyme induced substrate distortion. Therefore, we propose a model in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates catalysis.\n\nOne Sentence SummaryThe rigid matrix of BCX uses substrate flexibility in Michaelis complex formation.

biochemistry↗

bioRxiv2019

Ramified Rolling Circle Amplification for efficient and flexible synthesis of nucleosomal DNA sequences

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that using ramified rolling circle amplification (RCA) milligram amounts of a genomic nucleosomal DNA fragment can be produced in a scalable, one-pot reaction overnight. The ramified RCA reaction is more efficient than the existing methods, is flexible in DNA sequence and shows a 10-fold increase in yield compared to PCR, rivalling the production using plasmids. We demonstrate the method by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.

biochemistry↗