Drug retention after intradiscal administration.

Intradiscal drug delivery is a promising strategy for treating intervertebral disk degeneration (IVDD). Local degenerative processes and intrinsically low fluid exchange are likely to influence drug retention. Understanding their connection will enable the optimization of IVDD therapeutics.

Phosphorylation of the HMGN1 Nucleosome Binding Domain Decreases Helicity and Interactions with the Acidic Patch.

Intrinsically disordered proteins are abundant in the nucleus and are prime sites for posttranslational modifications that modulate transcriptional regulation. Lacking a defined three-dimensional structure, intrinsically disordered proteins populate an ensemble of several conformational states, which are dynamic and often altered by posttranslational modifications, or by binding to interaction partners. Although there is growing appreciation for the role that intrinsically disordered regions have in regulating protein-protein interactions, we still have a poor understanding of how to determine conformational population shifts, their causes under various conditions, and how to represent and model conformational ensembles.

Structural dynamics in chromatin unraveling by pioneer transcription factors.

Pioneer transcription factors are proteins with a dual function. First, they regulate transcription by binding to nucleosome-free DNA regulatory elements. Second, they bind to DNA while wrapped around histone proteins in the chromatin and mediate chromatin opening.

Microscale Thermophoresis Analysis of Chromatin Interactions.

Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya.

The Structure and Function of the Bacterial Osmotically Inducible Protein Y.

The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary tract. Environmental stress often provides cross-protection against other challenges and increases antibiotic tolerance of bacteria. Thus, it is critical to understand how E.

Monitoring Anthracycline Cancer Drug-Nucleosome Interaction by NMR Using a Specific Isotope Labeling Approach for Nucleosomal DNA.

Chromatinized DNA is targeted by proteins and small molecules to regulate chromatin function. For example, anthracycline cancer drugs evict nucleosomes in a mechanism that is still poorly understood. We here developed a flexible method for specific isotope labeling of nucleosomal DNA enabling NMR studies of such nucleosome interactions.

A structural and dynamic visualization of the interaction between 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.

H-detected characterization of carbon-carbon networks in highly flexible protonated biomolecules using MAS NMR.

In the last three decades, the scope of solid-state NMR has expanded to exploring complex biomolecules, from large protein assemblies to intact cells at atomic-level resolution. This diversity in macromolecules frequently features highly flexible components whose insoluble environment precludes the use of solution NMR to study their structure and interactions. While High-resolution Magic-Angle Spinning (HR-MAS) probes offer the capacity for gradient-based H-detected spectroscopy in solids, such probes are not commonly used for routine MAS NMR experiments.

Resonance assignments of the microtubule-binding domain of the microtubule-associated protein 7 (MAP7).

The microtubule-associated protein 7 (MAP7) is a protein involved in cargo transport along microtubules (MTs) by interacting with kinesin-1 through the C-terminal kinesin-binding domain. Moreover, the protein is reported to stabilize MT, thereby playing a key role in axonal branch development. An important element for this latter function is the 112 amino-acid long N-terminal microtubule-binding domain (MTBD) of MAP7.

Structural and Functional Characterization of the Newly Designed Antimicrobial Peptide Crabrolin21.

(1) Background: antimicrobial resistance is becoming a dramatic problem for public health, and the design of new antimicrobial agents is an active research area. (2) Methods: based on our previous work, we designed an improved version of the crabrolin peptide and characterized its functional and structural properties with a wide range of techniques. (3) Results: the newly designed peptide, crabrolin21, is much more active than the previous ones and shows specific selectivity towards bacterial cells.

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. Here, we show that the acidic domain of DNA repair factor APLF (APLF) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes.

Mapping the electrostatic potential of the nucleosome acidic patch.

The nucleosome surface contains an area with negative electrostatic potential known as the acidic patch, which functions as a binding platform for various proteins to regulate chromatin biology. The dense clustering of acidic residues may impact their effective pKa and thus the electronegativity of the acidic patch, which in turn could influence nucleosome-protein interactions. We here set out to determine the pKa values of residues in and around the acidic patch in the free H2A-H2B dimer using NMR spectroscopy.

Beyond the Nucleosome: Nucleosome-Protein Interactions and Higher Order Chromatin Structure.

The regulation of chromatin biology ultimately depends on the manipulation of its smallest subunit, the nucleosome. The proteins that bind and operate on the nucleosome do so, while their substrate is part of a polymer embedded in the dense nuclear environment. Their molecular interactions must in some way be tuned to deal with this complexity.

Characterization of nucleosome sediments for protein interaction studies by solid-state NMR spectroscopy.

Regulation of DNA-templated processes such as gene transcription and DNA repair depend on the interaction of a wide range of proteins with the nucleosome, the fundamental building block of chromatin. Both solution and solid-state NMR spectroscopy have become an attractive approach to study the dynamics and interactions of nucleosomes, despite their high molecular weight of ~ 200 kDa. For solid-state NMR (ssNMR) studies, dilute solutions of nucleosomes are converted to a dense phase by sedimentation or precipitation.

Mechanical and structural properties of archaeal hypernucleosomes.

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone-DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an 'endless' histone-protein core.

Mimicking the Nucleosomal Context in Peptide-Based Binders of a H3K36me Reader Increases Binding Affinity While Altering the Binding Mode.

Targeting of proteins in the histone modification machinery has emerged as a promising new direction to fight disease. The search for compounds that inhibit proteins that readout histone modification has led to several new epigenetic drugs, mostly for proteins involved in recognition of acetylated lysines. However, this approach proved to be a challenging task for methyllysine readers, which typically feature shallow binding pockets.

Dynamics of Ligand Binding to a Rigid Glycosidase*.

The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion.

β-Lactamase of Mycobacterium tuberculosis Shows Dynamics in the Active Site That Increase upon Inhibitor Binding.

The β-lactamase BlaC is a broad-spectrum β-lactamase that can convert a range of β-lactam antibiotics. Enzymes with low specificity are expected to exhibit active-site flexibility. To probe the motions in BlaC, we studied the dynamic behavior in solution using nuclear magnetic resonance (NMR) spectroscopy.

Nucleosome binding peptide presents laudable biophysical and in vivo effects.

Chromatin state is highly dependent on the nucleosome binding proteins. Herein, we used a multipronged approach employing biophysical and in vivo experiments to characterize the effects of Nucleosome Binding Peptides (NBPeps) on nucleosome and cell activity. We performed a series of structure-based calculations on the nucleosome surface interaction with GMIP1 (a novel NBPep generated in silico), and HMGN2 (nucleosome binding motif of HMGN2), which contains sites that bind DNA and the acid patch, and also LANA and H4pep (nucleosome binding motif of H4 histone tail) that only bind to the acidic patch.

Ramified rolling circle amplification for 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.

Folding Then Binding vs Folding Through Binding in Macrocyclic Peptide Inhibitors of Human Pancreatic α-Amylase.

macrocyclic peptides, derived using selection technologies such as phage and mRNA display, present unique and unexpected solutions to challenging biological problems. This is due in part to their unusual folds, which are able to present side chains in ways not available to canonical structures such as α-helices and β-sheets. Despite much recent interest in these molecules, their folding and binding behavior remains poorly characterized.

Structural basis of specific H2A K13/K15 ubiquitination by RNF168.

Ubiquitination of chromatin by modification of histone H2A is a critical step in both regulation of DNA repair and regulation of cell fate. These very different outcomes depend on the selective modification of distinct lysine residues in H2A, each by a specific E3 ligase. While polycomb PRC1 complexes modify K119, resulting in gene silencing, the E3 ligase RNF168 modifies K13/15, which is a key event in the response to DNA double-strand breaks.

Unspinning chromatin: Revealing the dynamic nucleosome landscape by NMR.

NMR is an essential technique for obtaining information at atomic resolution on the structure, motions and interactions of biomolecules. Here, we review the contribution of NMR to our understanding of the fundamental unit of chromatin: the nucleosome. Nucleosomes compact the genome by wrapping the DNA around a protein core, the histone octamer, thereby protecting genomic integrity.

Kinetic analysis of multistep USP7 mechanism shows critical role for target protein in activity.

USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7.