Poly(ADP-ribosyl)ation enhances nucleosome dynamics and organizes DNA damage repair components within biomolecular condensates.

Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3.

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins.

A FRET-Based Assay and Computational Tools to Quantify Enzymatic Rates and Explore the Mechanisms of RNA Deadenylases in Heterogeneous Environments.

We developed a medium-throughput assay that can measure the time-dependent distribution of RNA products generated as a deadenylase degrades a polyadenosine (poly(A)) RNA tract, thereby providing insight into the mechanism of deadenylation. Importantly, this assay can be performed in both homogeneous and heterogeneous environments without relying on gel electrophoresis of RNA products or coupled enzymatic reactions that indirectly report on the RNA distribution through the detection of freed adenosine monophosphate. In parallel, we have established an open-source, Python-based command-line software package, deadenylationkinetics, that can be used to numerically simulate and/or fit the datasets afforded by our assay with different deadenylation mechanisms to determine the most likely case and estimate the associated rate constants.

Flexible Client-Dependent Cages in the Assembly Landscape of the Periplasmic Protease-Chaperone DegP.

The periplasmic protein DegP, which is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that helps to maintain protein homeostasis in Gram-negative bacteria and is essential to bacterial survival under stress conditions. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming ensembles that can include extremely large cage particles, but how this occurs remains an open question.

Correlating histone acetylation with nucleosome core particle dynamics and function.

Epigenetic modifications of chromatin play a critical role in regulating the fidelity of the genetic code and in controlling the translation of genetic information into the protein components of the cell. One key posttranslational modification is acetylation of histone lysine residues. Molecular dynamics simulations, and to a smaller extent experiment, have established that lysine acetylation increases the dynamics of histone tails.

Dissecting the role of interprotomer cooperativity in the activation of oligomeric high-temperature requirement A2 protein.

The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established.

Global multi-method analysis of interaction parameters for reversibly self-associating macromolecules at high concentrations.

Weak macromolecular interactions assume a dominant role in the behavior of highly concentrated solutions, and are at the center of a variety of fields ranging from colloidal chemistry to cell biology, neurodegenerative diseases, and manufacturing of protein drugs. They are frequently measured in different biophysical techniques in the form of second virial coefficients, and nonideality coefficients of sedimentation and diffusion, which may be related mechanistically to macromolecular distance distributions in solution and interparticle potentials. A problem arises for proteins where reversible self-association often complicates the concentration-dependent behavior, such that grossly inconsistent coefficients are measured in experiments based on different techniques, confounding quantitative conclusions.

Oligomeric assembly regulating mitochondrial HtrA2 function as examined by methyl-TROSY NMR.

Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood.

Parallel reaction pathways accelerate folding of a guanine quadruplex.

G-quadruplexes (G4s) are four-stranded, guanine-rich nucleic acid structures that can influence a variety of biological processes such as the transcription and translation of genes and DNA replication. In many cases, a single G4-forming nucleic acid sequence can adopt multiple different folded conformations that interconvert on biologically relevant timescales, entropically stabilizing the folded state. The coexistence of different folded conformations also suggests that there are multiple pathways leading from the unfolded to the folded state ensembles, potentially modulating the folding rate and biological activity.

Analyzing multi-step ligand binding reactions for oligomeric proteins by NMR: Theoretical and computational considerations.

Solution NMR spectroscopy is widely used to investigate the thermodynamics and kinetics of the binding of ligands to their biological receptors, as it provides detailed, atomistic information, potentially leading to microscopic affinities for each binding event, and, to the development of allosteric pathways describing how the binding at one site affects distal sites in the molecule. Importantly, weak interactions that are often invisible to other biophysical methods can also be probed. Methodological advancements in NMR have enabled the investigation of high molecular weight, homo-oligomeric complexes that bind multiple ligand molecules, with increasing numbers of studies of the structural dynamics and binding properties of these systems.

Conformational Dynamics of Strand Register Shifts in DNA G-Quadruplexes.

The complex folding energy landscape of DNA G-quadruplexes leads to numerous conformations for this functionally important class of noncanonical DNA structures. A new layer of conformational heterogeneity comes from sequences with different numbers of G-nucleotides in each of the DNA G-strands that form the four-stranded G-quartet core. The mechanisms by which G-quadruplexes transition from one folded conformation to another are currently unknown.

G-quadruplex dynamics.

G-quadruplexes (GQs) are four-stranded nucleic acid secondary structures formed by guanosine (G)-rich DNA and RNA sequences. It is becoming increasingly clear that cellular processes including gene expression and mRNA translation are regulated by GQs. GQ structures have been extensively characterized, however little attention to date has been paid to their conformational dynamics, despite the fact that many biological GQ sequences populate multiple structures of similar free energies, leading to an ensemble of exchanging conformations.

Stabilization of i-motif structures by 2'-β-fluorination of DNA.

i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH(+)). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands.

G-register exchange dynamics in guanine quadruplexes.

G-quadruplexes (GQs) are 4-stranded DNA structures formed by tracts of stacked, Hoogsteen-hydrogen bonded guanosines. GQs are found in gene promoters and telomeres where they regulate gene transcription and telomere elongation. Though GQ structures are well-characterized, many aspects of their conformational dynamics are poorly understood.