Biophysical Techniques Research Articles

The path to the G protein-coupled receptor structural landscape: Major milestones and future directions.

G protein-coupled receptors (GPCRs) play a crucial role in cell function by transducing signals from the extracellular environment to the inside of the cell. They mediate the effects of various stimuli, including hormones, neurotransmitters, ions, photons, food tastants and odorants, and are renowned drug targets. Advancements in structural biology techniques, including X-ray crystallography and cryo-electron microscopy (cryo-EM), have driven the elucidation of an increasing number of GPCR structures. These structures reveal novel features that shed light on receptor activation, dimerization and oligomerization, dichotomy between orthosteric and allosteric modulation, and the intricate interactions underlying signal transduction, providing insights into diverse ligand-binding modes and signalling pathways. However, a substantial portion of the GPCR repertoire and their activation states remain structurally unexplored. Future efforts should prioritize capturing the full structural diversity of GPCRs across multiple dimensions. To do so, the integration of structural biology with biophysical and computational techniques will be essential. We describe in this review the progress of nuclear magnetic resonance (NMR) to examine GPCR plasticity and conformational dynamics, of atomic force microscopy (AFM) to explore the spatial-temporal dynamics and kinetic aspects of GPCRs, and the recent breakthroughs in artificial intelligence for protein structure prediction to characterize the structures of the entire GPCRome. In summary, the journey through GPCR structural biology provided in this review illustrates how far we have come in decoding these essential proteins architecture and function. Looking ahead, integrating cutting-edge biophysics and computational tools offers a path to navigating the GPCR structural landscape, ultimately advancing GPCR-based applications.

Structural modeling and characterization of the Mycobacterium tuberculosis MmpL3 C-terminal domain.

The Mycobacterium tuberculosis (Mtb) cell envelope provides a protective barrier against the immune response and antibiotics. The mycobacterial membrane protein large (MmpL) family of proteins export cell envelope lipids and siderophores; therefore, these proteins are important for the basic biology and pathogenicity of Mtb. In particular, MmpL3 is essential and a known drug target. Despite interest in MmpL3, the structural data in the field are incomplete. Utilizing homology modeling, AlphaFold, and biophysical techniques, we characterized the cytoplasmic C-terminal domain (CTD) of MmpL3 to better understand its structure and function. Our in silico models of the MmpL11TB and MmpL3TB CTD reveal notable features including a long unstructured linker that connects the globular domain to the last transmembrane (TM) in each transporter, charged lysine and arginine residues facing the membrane, and a C-terminal alpha helix. Our predicted overall structure enables a better understanding of these transporters.

How to Find a Fragment: Methods for Screening and Validation in Fragment-Based Drug Dscovery.

Fragment-based drug discovery (FBDD) is a crucial strategy for developing new drugs that have been applied to diverse targets, from neglected infectious diseases to cancer. With at least seven drugs already launched to the market, this approach has gained interest in both academics and industry in the last 20 years. FBDD relies on screening small libraries with about 1000-2000 compounds of low molecular weight (about 300 Da) using several biophysical methods. Because of the reduced size of the compounds, the chemical space and diversity can be better explored than large libraries used in high throughput screenings. This review summarises the most common biophysical techniques used in fragment screening and orthogonal validation. We also explore the advantages and drawbacks of the different biophysical techniques and examples of applications and strategies.

Is there reversible dimerization of albumin in blood plasma? And does it matter?

Most albumin in blood plasma is thought to be monomeric with some 5% covalently dimerized. However, many reports in the recent biophysics literature find that albumin is reversibly dimerized or even oligomerized. We review data on this from X-ray crystallography and diverse biophysical techniques. The number-average molecular weight of albumin would be increased by dimerization, affecting size-dependent filtration processes of albumin such as at the glycocalyx of the capillary endothelium and the podocyte slit-diaphragm of the renal glomerulus. If correct, and depending on characteristics of the process, such as Kd, reversible dimerization of albumin in plasma would have major implications for normal physiology and medicine. We present quantitative models of the impact of dimerization on albumin molecular forms, on the number-average molecular weight of albumin, and estimate the effect on the colloid osmotic pressure of albumin. Dimerization reduces colloid osmotic pressure as total albumin concentration increases below that expected in the absence of dimerization. Current models of albumin filtration by the renal glomerulus would need revision to account for the dynamic size of albumin molecules filtered. More robust biophysical data are needed to give a definitive answer to the questions posed and we suggest possible approaches to this.

Protein - carbohydrate interaction studies using domestic animals as role models support the search of new glycomimetic molecules

The structural dynamics of the interactions between defensins or lysozymes and various saccharide chains that are covalently linked to lipids or proteins were analyzed in relation to the sub-molecular architecture of the carbohydrate binding sites of lectins. Using tissue materials from rare and endangered domestic animals as well as from dogs it was possible to compare these results with data obtained from a human glioblastoma tissue. The binding mechanisms were analyzed on a cellular and a sub-molecular size level using biophysical techniques (e.g. NMR, AFM, MS) which are supported by molecular modeling tools. This leads to characteristic structural patterns being helpful to understand glyco-biochemical pathways in which galectins, defensins or lysozymes are involved. Carbohydrate chains have a distinct impact on cell differentiation, cell migration and immunological processes. The absence or the presence of sialic acids and the conformational dynamics in glycans are often correlated with zoonoses such as influenza- and coronavirus-infections. Receptor-sensitive glycomimetics could be a solution. The new findings concerning the function of galectin-3 in the nucleus in relation to differentiation processes can be understood when the binding specificity of neuroleptic molecules as well as the interactions between proteins and nucleic acids are describable on a sub-molecular size level.

Development of a gold nanoparticle-based novel diagnostic prototype for in vivo detection of Indian red scorpion (Mesobuthus tamulus) venom

Indian red scorpion Mesobuthus tamulus is responsible for substantial mortality in India and Sri Lanka; however, no specific diagnostic method is available to detect the venom of this scorpion in envenomed plasma or body fluid. Therefore, we have proposed a novel, simple, and rapid method for detecting M. tamulus venom (MTV) in the plasma of envenomed animals using polyclonal antibodies (PAb) raised against three modified custom peptides representing the antigenic epitopes of K+ (Tamapin) and Na+ (α-neurotoxin) channel toxins, the two major MTV toxins identified by proteomic analysis. The optimum PAb formulation containing PAb 1, 2, and 3 in proportion (1:1:1, w/w/w) acted synergistically, demonstrating significantly higher immunological recognition of MTV than anti-scorpion antivenom (developed against native toxins) and individual antibodies against peptide immunogens. The PAb formulation could detect MTV optimally in envenomed rat plasma (intravenous and subcutaneous routes) at 30–60 min post-injection. The acetonitrile precipitation method developed in this study to augment the MTV detection sensitivity enriched the low molecular mass peptide toxins in envenomed rat plasma, which was ascertained by mass spectrometry analysis. The gold nanoparticles conjugated PAb formulation, characterised by biophysical techniques such as Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM), demonstrated their interaction with low molecular mass MTV peptide toxins in envenomed rat plasma. This interaction results in the accumulation of the gold nanoparticles, thus leading to signal change in absorbance spectra that can be discerned within 10 min. From a standard curve of MTV spiked plasma, the quantity of MTV in envenomed rat plasma could be determined by gold nanoparticle-PAb formulation conjugate.

Alginate binding enhances the structural stability and potentiates the lytic activity of bacteriophage endolysin's partially folded conformation

Polysaccharide polymers are increasingly being used as chaperon-like macromolecules in assisting protein folding of unfolded protein molecules. They interact with unfolded or partially folded proteins in a charge and conformation specific manner that results in the formation of stable protein-polysaccharide complexes. In most of the cases, the complex formation of protein-polysaccharide is driven via non-covalent interactions that have found to endorse the activity of proteins. T4L (18.7 kDa) and T7L (17 kDa) endolysins belong to the hydrolase and amidase class of peptidoglycan degrading enzymes. Both T4L and T7L exist in partially folded forms and are devoid of lytic activity at low pH conditions. In the current study, we assessed the binding of alginate with T4L and T7L at pH 7 and 3 using variety of biophysical and biochemical techniques. Spectroscopic studies revealed differential structural modulations of partially folded T4L and T7L upon their interaction with alginate. Further, the complex formation of alginate with partially folded T4L/T7L was confirmed by ITC and STEM. Additionally, the formed complexes of alginate with both T4L/T7L PF endolysins were found to be chemically and enzymatically stable. Moreover, such complexes were also marked with differential enhancement in their lytic activities at acidic pH conditions. This implied the potency of alginate as an excellent choice of matrix to preserve the structural and functional integrity of partially folded forms of T4L and T7L at highly acidic conditions.

Insights on cold atmospheric plasma treatment of ethidium bromide and its binding to protein BSA

Ethidium bromide (Et-Br) is a dye prevalently used in microbiology laboratories to visualize its binding to DNA under UV light. After usage, the Et-Br thrown from the lab as waste can reach waterbodies and it is toxic not only to aquatic life but also to humankind. Cold atmospheric plasma (CAP) is a novel technology used to degrade various hazardous chemicals as it produces reactive species (ROS/ RNS) which work synergistically to degrade these hazardous chemicals. Molecular docking studies show that Et-Br has strong binding affinity with BSA. Here, to test the efficacy of CAP treatment, the binding interaction of Et-Br with protein Bovine Serum Albumin (BSA) before and after CAP treatment has been studied. Herein, various biophysical techniques were used to study interaction of Et-Br with BSA before and after CAP treatment. Our investigation provides insight into the applicability of CAP to degrade hazardous chemicals such as Et-Br with potential environmental applications.

Enhanced Recognition of a Herbal Compound Epiberberine by a DNA Quadruplex-Duplex Structure.

The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence with four telomeric repeats (Q4), resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI-binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures under physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is an excellent EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.

Multi-species cryoEM calibration and workflow verification standard.

Cryogenic electron microscopy (cryoEM) is a rapidly growing structural biology modality that has been successful in revealing molecular details of biological systems. However, unlike established biophysical and analytical techniques with calibration standards, cryoEM has lacked comprehensive biological test samples. We introduce a cryoEM calibration sample that is a mixture of compatible macromolecules that can be used not only for resolution optimization but also provides multiple reference points for evaluating instrument performance, data quality, and image processing workflows in a single experiment. This combined test specimen provides researchers a reference point for validating their cryoEM pipeline, benchmarking their methodologies, and testing new algorithms.

Practical Perspectives and Roles of XRD in Drug Discovery: A Review

Abstract: Biophysical techniques include various methodologies applied in studying biological systems at the molecular and cellular level for the drug discovery process. Various methods like isothermal calorimetry, electron microscopy, XRD (X-ray diffraction), electron microscopy, mass spectrometry, atomic force microscopy, differential scanning calorimetry, surface plasmon resonance, and nuclear magnetic resonance are important techniques for drug discovery. Out of these techniques, XRD is widely employed in structure-based drug discovery, whereas FBDD (fragment-based drug discovery) is widely used in the different phases of drug discovery. XRD was considered one of the most important tools for structure determination of biomolecules and peptides. Consistent development and advancement in XRD improved the various aspects of data processing, collection, sample loading, and increased throughput. This advancement is crucial in obtaining highly resolved protein and other biomolecule crystal structures. The structure obtained from XRD forms the core of structure-based drug discovery and FBDD. This review article focuses on the different roles of biophysical techniques with special emphasis on advancement, data collection, and XRD's role in different drug discovery phases.

Chitosan decorated magnetic nanobiocatalyst of Bacillus derived α-amylase as a role model for starchy wastewater treatment, detergent additive and textile desizer

In this study, Bacillus tequilensis TB5 α-amylase from rice-milled by-products (rice bran and de-oiled rice bran) was successfully immobilized onto biologically synthesized magnetic nanoparticles fabricated with chitosan (MNP-Ch) and characterized via different biophysical techniques. Furthermore, the study emphasized incorporating this nanostructure framework (MNP@2mgchitosan_DORB-amy and MNP@3mgchitosan_RB-amy) to offer diverse applications, including enzymatic desizing, cleaning starchy stains, and treating synthetic starchy wastewater. An enzyme loading of > 90 % for both enzymes indicated increased binding sites due to the functional moieties of chitosan on the MNP. The Km was 0.28 and 0.31 mg/mL for the immobilized and free forms of DORB-amy, respectively, and 0.18 and 0.27 mg/mL for the immobilized and free forms of RB-amy, respectively. A low Km indicated an increased affinity of MNP-Ch-immobilized forms of enzymes toward the substrate. The performance of both immobilized enzymes improved at a wide range of pH and temperature, which may be attributed to the covalent binding of the enzyme on to the MNP-Ch. The nanobiocatalysts in the detergent act synergistically to fade the starchy stains. Furthermore, an 8–9 TEGEWA scale rating with > 11 % of starch removal was obtained through the biodesizing of starch-sized cotton fabric. The nanobiocatalyst efficiently decomposed starch and liberated 650–670 mg/L of reducing sugar from the synthetic wastewater, therefore offering promising opportunities for its exploration in a wastewater treatment plant. Thus, the study recommends the potential exploration of sturdy matrices like MNP to offer remarkable applications with maximum operational stability, easier recovery, and higher efficiency.

Characterization of G-quadruplexes in the Helicobacter pylori genome and assessment of therapeutic potential of G4 ligands.

Helicobacter pylori, a leading human pathogen associated with duodenal ulcer and gastric cancer, presents a significant threat to human health due to increasing antibiotic resistance rates. This study investigates G-quadruplexes (G4s), which are non-canonical secondary structures form in G-rich regions within the H. pylori genome. Extensive research on G4s in eukaryotes has revealed their role in epigenetically regulating cellular processes like gene transcription, DNA replication, and oncogene expression. However, understanding of G4-mediated gene regulation in other organisms, especially bacterial pathogens, remains limited. Although G4 motifs have been extensively studied in a few bacterial species such as Mycobacterium, Streptococci, and Helicobacter, research on G4 motifs in other bacterial species is still sparse. Like in other organisms such as archaea, mammals, and viruses, G4s in H. pylori display a non-random distribution primarily situated within open reading frames of various protein-coding genes. The occurrence of G4s in functional regions of the genome and their conservation across different species indicates that their placement is not random, suggesting an evolutionary pressure to maintain these sequences at specific genomic sites. Moreover, G-quadruplexes show enrichment in specific gene classes, suggesting their potential involvement in regulating the expression of genes related to cell wall/membrane/envelope biogenesis, amino acid transport, and metabolism. This indicates a probable regulatory role for G4s in controlling the expression of genes essential for H. pylori survival and virulence. Biophysical techniques such as Circular Dichroism spectroscopy and Nuclear Magnetic Resonance were used to characterize G4 motifs within selected H. pylori genes. The study revealed that G-quadruplex ligand inhibited the growth of H. pylori, with minimal inhibitory concentrations in the low micromolar range. This suggests that targeting G4 structures could offer a promising approach for developing novel anti-H. pylori drugs.

The antiviral drug Ribavirin effectively modulates the amyloid transformation of α-Synuclein protein

α-Synuclein (α-syn) is an intrinsically disordered protein, linked genetically and neuropathologically to Parkinson's disease where this protein aggregates within the brain. Hence, identifying compounds capable of impeding α-syn aggregation puts forward a promising approach for the development of disease-modifying therapies. Herein, we investigated the efficacy of Ribavirin, an FDA-approved compound, in curtailing α-syn amyloid transformation, employing an array of bioinformatic tools and systematic analysis using biophysical techniques. Ribavirin shows a dose dependent anti-aggregation propensity where it effectively subdued the formation of mature fibrillar aggregates of α-syn, where even at the lowest concentration there was a 69 % reduction in the ThT maxima. Ribavirin averts the formation of mature fibrillar aggregates by interacting with the NAC domain of α-syn. Ribavirin redirects the amyloid transformation of α-syn by emanating aggregates of lower order with reduced cross β-sheet signature and revokes the formation of on-pathway amyloids. Collectively, our study puts forward the novel potency of Ribavirin as a promising molecule for therapeutic intervention in Parkinson’s disease.

Protocell Effects on RNA Folding, Function, and Evolution.

ConspectusCreating a living system from nonliving matter is a great challenge in chemistry and biophysics. The early history of life can provide inspiration from the idea of the prebiotic "RNA World" established by ribozymes, in which all genetic and catalytic activities were executed by RNA. Such a system could be much simpler than the interdependent central dogma characterizing life today. At the same time, cooperative systems require a mechanism such as cellular compartmentalization in order to survive and evolve. Minimal cells might therefore consist of simple vesicles enclosing a prebiotic RNA metabolism.The internal volume of a vesicle is a distinctive environment due to its closed boundary, which alters diffusion and available volume for macromolecules and changes effective molecular concentrations, among other considerations. These physical effects are mechanistically distinct from chemical interactions, such as electrostatic repulsion, that might also occur between the membrane boundary and encapsulated contents. Both indirect and direct interactions between the membrane and RNA can give rise to nonintuitive, "emergent" behaviors in the model protocell system. We have been examining how encapsulation inside membrane vesicles would affect the folding and activity of entrapped RNA.Using biophysical techniques such as FRET, we characterized ribozyme folding and activity inside vesicles. Encapsulation inside model protocells generally promoted RNA folding, consistent with an excluded volume effect, independently of chemical interactions. This energetic stabilization translated into increased ribozyme activity in two different systems that were studied (hairpin ribozyme and self-aminoacylating RNAs). A particularly intriguing finding was that encapsulation could rescue the activity of mutant ribozymes, suggesting that encapsulation could affect not only folding and activity but also evolution. To study this further, we developed a high-throughput sequencing assay to measure the aminoacylation kinetics of many thousands of ribozyme variants in parallel. The results revealed an unexpected tendency for encapsulation to improve the better ribozyme variants more than worse variants. During evolution, this effect would create a tilted playing field, so to speak, that would give additional fitness gains to already-high-activity variants. According to Fisher's Fundamental Theorem of Natural Selection, the increased variance in fitness should manifest as faster evolutionary adaptation. This prediction was borne out experimentally during in vitro evolution, where we observed that the initially diverse ribozyme population converged more quickly to the most active sequences when they were encapsulated inside vesicles.The studies in this Account have expanded our understanding of emergent protocell behavior, by showing how simply entrapping an RNA inside a vesicle, which could occur spontaneously during vesicle formation, might profoundly affect the evolutionary landscape of the RNA. Because of the exponential dynamics of replication and selection, even small changes to activity and function could lead to major evolutionary consequences. By closely studying the details of minimal yet surprisingly complex protocells, we might one day trace a pathway from encapsulated RNA to a living system.

Molecular Basis for the Calcium-Dependent Activation of the Ribonuclease EndoU.

Ribonucleases (RNases) are ubiquitous enzymes that process or degrade RNA, essential for cellular functions and immune responses. The EndoU-like superfamily includes endoribonucleases conserved across bacteria, eukaryotes, and certain viruses, with an ancient evolutionary link to the ribonuclease A-like superfamily. Both bacterial EndoU and animal RNase A share a similar fold and function independently of cofactors. In contrast, the eukaryotic EndoU catalytic domain requires divalent metal ions for catalysis, possibly due to an N-terminal extension near the catalytic core. In this study, we used biophysical and computational techniques along with in vitro assays to investigate the calcium-dependent activation of human EndoU. We determined the crystal structure of EndoU bound to calcium and found that calcium binding remote from the catalytic triad triggers water-mediated intramolecular signaling and structural changes, activating the enzyme through allostery. Calcium-binding involves residues from both the catalytic core and the N-terminal extension, indicating that the N-terminal extension interacts with the catalytic core to modulate activity in response to calcium. Our findings suggest that similar mechanisms may be present across all eukaryotic EndoUs, highlighting a unique evolutionary adaptation that connects endoribonuclease activity to cellular signaling in eukaryotes.

Strong heterologous electron sink outcompetes alternative electron transport pathways in photosynthesis.

Improvement of photosynthesis requires a thorough understanding of electron partitioning under both natural and strong electron sink conditions. We applied a wide array of state-of-the-art biophysical and biochemical techniques to thoroughly investigate the fate of photosynthetic electrons in the engineered cyanobacterium Synechocystis sp. PCC 6803, a blueprint for photosynthetic biotechnology, expressing the heterologous gene for ene-reductase, YqjM. This recombinant enzyme catalyses the reduction of an exogenously added substrate into the desired product by utilising photosynthetically produced NAD(P)H, enabling whole-cell biotransformation. Through coupling the biotransformation reaction with biophysical measurements, we demonstrated that the strong artificial electron sink, outcompetes the natural electron valves, the flavodiiron protein-driven Mehler-like reaction and cyclic electron transport. These results show that ferredoxin-NAD(P)H-oxidoreductase is the preferred route for delivering photosynthetic electrons from reduced ferredoxin and the cellular NADPH/NADP+ ratio as a key factor in orchestrating photosynthetic electron flux. These insights are crucial for understanding molecular mechanisms of photosynthetic electron transport and harnessing photosynthesis for sustainable bioproduction by engineering the cellular source/sink balance. Furthermore, we conclude that identifying the bioenergetic bottleneck of a heterologous electron sink is a crucial prerequisite for targeted engineering of photosynthetic biotransformation platforms.

Algal-based biochar and hydrochar: A holistic and sustainable approach to wastewater treatment

Biochar and hydrochar are carbon-rich solid products of algal and terrestrial biomass obtained through various thermochemical processes, mainly hydrothermal liquefaction, pyrolysis, and hydrothermal carbonization. Both are regarded as economical, potent, and environmentally friendly adsorbents for wastewater remediation. The versatile applicability of biochar and hydrochar, which is due to their enhanced surface physicochemical properties, includes efficient metal biosorption, soil fertility enhancement, and carbon sequestration. Consequently, there has been an increase in research for producing and exploiting biochar and hydrochar from algae. Both micro- and macro-algal biomass have strong potential due to their sustainability footprint and distinct properties. This review focuses on a comprehensive account of the synthesis of algal biochar and hydrochar and use in wastewater treatment to develop innovative solutions for efficient mitigation of several aqueous pollutants and heavy metal ions. The review discusses the various thermochemical production routes, biophysical characterization techniques, and modes of mechanism for wastewater bioremediation. Algal-based biochar and hydrochar are reported to have higher porosity and more diverse functional groups such as amines, hydroxyl, and carboxyl compared to their cellulosic and waste-derived counterparts, demonstrating an increased wastewater remediation efficiency. In addition, presence of inorganic metal including sodium, potassium, magnesium, and phosphorus in algal chars facilitates the formation of mesopores and graphite structures, improving their cation exchange capacity. Moreover, algal chars may exhibit pH buffering capacity, which could help stabilize the pH during wastewater treatment processes. Notably, the low O/C content of algal hydrochars enhances the binding and removal of organic pollutants including toxic dyes and antibiotics. The review highlights the development of modified algal chars to enhance the porosity, surface area, structural integrity, and adsorption capacity enabling a higher bioremediation potential.

Fragment-Based Discovery of a Series of Allosteric-Binding Site Modulators of β-Glucocerebrosidase.

β-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.

Allosteric regulation of kinase activity.

The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.