Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1's essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the µs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocket knife and for oligomerization. Our results on hGBP1's conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis.

Spatial Distribution of Intracellular Ion Concentrations in Aggregate-Forming HeLa Cells Analyzed by µ-XRF Imaging.

Protein aggregation is a hallmark of several severe neurodegenerative disorders such as Huntington's, Parkinson's, or Alzheimer's disease. Metal ions play a profound role in protein aggregation and altered metal-ion homeostasis is associated with disease progression. Here we utilize µ-X-ray fluorescence imaging in combination with rapid freezing to resolve the elemental distribution of phosphorus, sulfur, potassium, and zinc in huntingtin exon-1-mYFP expressing HeLa cells. Using quantitative XRF analysis, we find a threefold increase in zinc and a 10-fold enrichment of potassium that can be attributed to cellular stress response. While the averaged intracellular ion areal masses are significantly different in aggregate-containing cells, a local intracellular analysis shows no different ion content at the location of intracellular inclusion bodies. The results are compared to corresponding experiments on HeLa cells forming pseudoisocyanine chloride aggregates. As those show similar results, changes in ion concentrations are not exclusively linked to huntingtin exon-1 amyloid formation.

Micro x-ray fluorescence analysis of trace element distribution in frozen hydrated HeLa cells at the P06 beamline at Petra III.

X-ray fluorescence analysis enables the study of trace element distributions in biological specimens. When this analysis is done under cryogenic conditions, cells are cryofixed as closely as possible to their natural physiological state, and the corresponding intracellular elemental densities can be analyzed. Details about the experimental setup used for analysis at the P06 beamline at Petra III, DESY and the used cryo-transfer system are described in this work. The system was applied to analyze the elemental distribution in single HeLa cells, a cell line frequently used in a wide range of biological applications. Cells adhered to silicon nitride substrates were cryoprotected within an amorphous ice matrix. Using a continuous scanning scheme and a KB x-ray focus, the distribution of elements in the cells was studied. We were able to image the intracellular potassium and zinc levels in HeLa cells as two key elements relevant for the physiology of cells.

A biosensor-based framework to measure latent proteostasis capacity.

The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.

A Multiperspective Approach to Solvent Regulation of Enzymatic Activity: HMG-CoA Reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was investigated in different organic cosolvents by means of kinetic and calorimetric measurements, molecular dynamics simulations, and small-angle X-ray scattering. The combined experimental and theoretical techniques were essential to complement each other's limitations in the investigation of the complex interaction pattern between the enzyme, different solvent types, and concentrations. In this way, the underlying mechanisms for the loss of enzyme activity in different water-miscible solvents could be elucidated. These include direct inhibitory effects onto the active center and structural distortions.

Inhibition of Huntingtin Exon-1 Aggregation by the Molecular Tweezer CLR01.

Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. Here we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity.

Conformational dynamics and self-association of intrinsically disordered Huntingtin exon 1 in cells.

Huntington's disease is caused by a CAG trinucleotide expansion mutation in the Huntingtin gene that leads to an artificially long polyglutamine sequence in the Huntingtin protein. A key feature of the disease is the intracellular aggregation of the Huntingtin exon 1 protein (Httex1) into micrometer sized inclusion bodies. The aggregation process of Httex1 has been extensively studied in vitro, however, the crucial early events of nucleation and aggregation in the cell remain elusive. Here, we studied the conformational dynamics and self-association of Httex1 by in-cell experiments using laser-induced temperature jumps and analytical ultracentrifugation. Both short and long polyglutamine variants of Httex1 underwent an apparent temperature-induced conformational collapse. The temperature jumps generated a population of kinetically trapped species selectively for the longer polyglutamine variants of Httex1 proteins. Their occurrence correlated with the formation of inclusion bodies suggesting that such species trigger further self-association.

Simultaneous measurements of photocurrents and H2O2 evolution from solvent exposed photosystem 2 complexes.

In plants, algae, and cyanobacteria, photosystem 2 (PS2) catalyzes the light driven oxidation of water. The main products of this reaction are protons and molecular oxygen. In vitro, however, it was demonstrated that reactive oxygen species like hydrogen peroxide are obtained as partially reduced side products. The transition from oxygen to hydrogen peroxide evolution might be induced by light triggered degradation of PS2's active center. Herein, the authors propose an analytical approach to investigate light induced bioelectrocatalytic processes such as PS2 catalyzed water splitting. By combining chronoamperometry and fluorescence microscopy, the authors can simultaneously monitor the photocurrent and the hydrogen peroxide evolution of light activated, solvent exposed PS2 complexes, which have been immobilized on a functionalized gold electrode. The authors show that under limited electron mediation PS2 displays a lower photostability that correlates with an enhanced H2O2 generation as a side product of the light induced water oxidation.

Infrared laser triggered release of bioactive compounds from single hard shell microcapsules.

Micro composites are commonly characterized in bulk. Here we study the temperature triggered release of a bioactive compound from single isolated microcapsules. We monitor the release process in real-time using a novel thermal microscopy method combining laser-induced heating and fluorescence imaging.

Triphosphate induced dimerization of human guanylate binding protein 1 involves association of the C-terminal helices: a joint double electron-electron resonance and FRET study.

Human guanylate binding protein 1 (hGBP1) is a member of the dynamin superfamily of large GTPases. During GTP hydrolysis, the protein undergoes structural changes leading to self-assembly. Previous studies have suggested dimerization of the protein by means of its large GTPase (LG) domain and significant conformational changes in helical regions near the LG domain and at its C-terminus. We used site-directed labeling and a combination of pulsed electron paramagnetic resonance and time-resolved fluorescence spectroscopy for structural investigations on hGBP1 dimerization and conformational changes of its C-terminal helix α13. Consistent distance measurements by double electron-electron resonance (DEER, also named pulse double electron resonance = PELDOR) spectroscopy and Förster resonance energy transfer (FRET) measurements using model-free analysis approaches revealed a close interaction of the two α13 helices in the hGBP1 dimer formed upon binding of the nonhydrolyzable nucleoside triphosphate derivate GppNHp. In molecular dynamics (MD) simulations, these two helices form a stable dimer in solution. Our data show that dimer formation of hGBP1 involves multiple spatially distant regions of the protein, namely, the N-terminal LG domain and the C-terminal helices α13. The contacts formed between the two α13 helices and the resulting juxtaposition are expected to be a key step for the physiological membrane localization of hGBP1 through the farnesyl groups attached to the end of α13.

Enzyme activity in the crowded milieu.

The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites) and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, K(m), and the catalytic turnover number, k(cat), of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the K(m) values in the presence and absence of crowding agents have been measured.

Mechanism of GTPase-activity-induced self-assembly of human guanylate binding protein 1.

Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of large GTPases (LGs). In the course of GTP hydrolysis, the protein undergoes structural changes leading to self-assembly of the protein, which is a characteristic property of all family members. For self-assembly, the protein employs two distinct interaction sites, one of which is located within the LG domain of the protein located at the N-terminus, and the second is located in the C-terminal alpha-helical domain. Here, we identify intramolecular contacts between the LG domain and the helical part of hGBP1, which relay nucleotide-dependent structural changes from the N-terminus to the C-terminus and thereby mediate tetramer formation of the protein through a second contact site at the C-terminus. Furthermore, we demonstrate the impact of this intramolecular communication on the enzymatic activity of hGBP1 and on its cellular localization.

Human guanylate-binding protein 1 as a model system investigated by several surface techniques.

In medical technologies concerning the surface immobilization of proteins in a defined orientation, maintaining their activity is a critical aspect. Therefore, in this study, the authors have investigated the activity of an elongated protein attached to a self-assembled monolayer supported streptavidin layer for different relative orientations of the protein with regard to the surface. Several mutants of this protein, human guanylate-binding protein 1 (hGBP1) showing GTPase catalytic activity, have been furnished with either one or two biotin anchors. Various independent methods that are based on different biophysical properties such as surface plasmon resonance, atomic force microscopy, and quartz crystal microbalance have been used to determine the orientation of the hGBP1 variants after anchoring them via a streptavidin-linker to a biotinylated surface. The activity of guanosine-triphosphate hydrolysis of hGBP1 monomers bound on the surface is found to depend on their orientation relative to the substrate, relating to their ability to form dimers with other neighboring anchored mutants; the maximum activity is lower than that observed in solutions, as might be expected from diffusion limitations at the solid/liquid interface on the one hand and prevention from homodimer formation due to immobilization on the other hand.

Nucleotide dependent cysteine reactivity of hGBP1 uncovers a domain movement during GTP hydrolysis.

As a member of the dynamin superfamily human guanylate-binding protein 1 (hGBP1) binds and hydrolyses GTP thereby undergoing structural changes which lead to self-assembly of the protein. Here, we employ the reactivity of hGBP1 with a cysteine reactive compound in order to monitor structural changes imposed by GTP binding and hydrolysis. Positions of cysteine residues buried between the C-terminal domain of hGBP1 and the rest of the protein are identified which report a large change of accessibility by the compound after addition of GTP. Our results indicate that nucleotide hydrolysis induces a domain movement in hGBP1, which we suggest enables further assembly of the protein.