Sedimentation Velocity Measurements Research Articles

On the detection of nanoparticle cloud migration by a resonant photonic surface signal towards sedimentation velocity measurements

Migration and sedimentation of solid particles in a liquid are physical phenomena involving accumulation of soft matter or decantation of fragmented matter. A thorough understanding together with relevant measurements are prerequisites regarding many fields, including medicine, galenic pharmacology, food processing, and the cosmetics industry. In this paper, we investigate the feasibility of monitoring and detecting the migration of a nanoparticle cloud with a resonant light probe. For this purpose, hybrid silicon/silica/UV210 organic integrated photonic racetrack resonators were patterned by thin film processes to be used as sensors measuring the outcome of the impact of a cloud of nanoparticles, the dynamic migration plus sedimentation phenomenon of the nanoparticle cloud in water. A broadband superluminescent diode has been used for the excitation. Then, the spectral characteristics of the resonant guided modes have been analyzed, considering the observed changes while tracking the free spectral range of the transduced comb spectra as a function of time. The way to operate can be summarized as follows: Solutions based on spherical silica nanoparticles of fixed size are prepared and subjected to rheological measurements to obtain their respective viscosities. Next, a millimeter tank filled with water is conveniently placed on the active surface of the sensing chip, prior to the addition of one of the previously mentioned solutions. The series of spectra are acquired during the whole migration sequence and the transduced optical signal is then directly processed and treated by a specific code operated in real time by way of Lagrange interpolation polynomials. Collected data are then compared to a simple theoretical model describing the sedimentation of a spherical particle in water (Stokes' law). Eventually, the implementation of the device in a characterization platform and the development of a specific protocol allows a global treatment, whose description is followed by discussions on measurements and data. Consequently, after the impact of the drop containing the nanoparticles, the monitoring of a first phase regarding their fast cloud migration into the global study volume (with flow of matter plus vortex) eventually followed by their slow sedimentation, can be detected using such a resonant light probe. The overall duration of the first phase associated with sedimentation velocities is in the order of a few tens of µm/min for particles with submicron diameters (several hundreds of nanometers); a first attempt of comparison of this first phase with the results of the classic Stokes model would give a convergence of the values reaching between 9 and 19% for the sedimentation rates.

Application of holography and automated image processing for laboratory experiments on mass and fall speed of small cloud ice crystals

Abstract. An ice cloud chamber was developed at the Johannes Gutenberg University of Mainz for generating several thousand data points for mass and sedimentation velocity measurements of ice crystals with sizes less than 150 µm. Ice nucleation was initiated from a cloud of supercooled droplets by local cooling using a liquid nitrogen cold finger. Three-dimensional tracks of ice crystals falling through the slightly supersaturated environment were obtained from the reconstruction of sequential holographic images, automated detection of the crystals in the hologram reconstructions, and particle tracking. Through collection of the crystals and investigation under a microscope before and after melting, crystal mass was determined as a function of size. The experimentally obtained mass versus diameter (m(D)) power law relationship resulted in lower masses for small ice crystals than from commonly adopted parameterizations. Thus, they did not support the currently accepted extrapolation of relationships measured for larger crystal sizes. The relationship between Best (X) and Reynolds (Re) numbers for columnar crystals was found to be X=15.3 Re1.2, which is in general agreement with literature parameterizations.

Hesperidin interaction to collagen detected by physico-chemical techniques

ObjectiveDentin collagen can be modified by some plant-derived flavonoids to improve properties of dentin organic matrix. Hesperidin (HPN), a hesperetin-7-O-rutinoside flavonoid, has a potential of dentin modification for being based on evidence that a treatment with HPN may resist collagenase degradation and arrest demineralization of human dentin. In this study, biophysical and molecular-level information on the interaction of HPN and collagen was investigated. MethodsHPN is extracted from citrus fruits. Sample collagenous solution was prepared using atelocollagen (ATCL) as a triple-helical peptide model. We have performed circular dichroism spectroscopic analysis, sedimentation velocity measurement by ultracentrifuge and saturation transfer difference measurement (STD) by NMR on HPN-collagen in solution state. ResultsThe circular dichroism and sedimentation velocity measurement showed the evidence for the molecular interaction between ATCL and HPN, while HPN did not induce any conformational change of ATCL. The STD-NMR study further confirmed this interaction and suggested that HPN interacted with ATCL through its aromatic part, not through its disaccharide moiety. SignificanceThese findings indicated that HPN is weakly bound to ATCL not causing structural modification of collagen. This interaction may contribute to the preservation of collagen by protecting from collagenase degradation.

Laboratory measurements of sedimentation velocity of columnar ice crystals

The terminal velocity of cloud particles may be particularly important to climate studies, because they may have a non‐negligible effect on the radiation budget of the planet, and on the lifetime of clouds. In the present study, the terminal velocity of individual columnar ice crystals was measured under controlled laboratory conditions. The size and fall orientation were determined for ice crystals with maximum dimensions less than 160 µm. The results, which are in agreement with the fall velocity presented in previous studies, show a random orientation of the columnar ice crystals during free fall with a fall velocity which is best represented by their capacitance. A linear relationship between fall velocity and ice crystal capacitance is presented that could be used in cloud modelling. Although the mass of ice crystals was not measured, an estimation of this parameter was made and an empirical Best–Reynolds power‐law relationship was found.

Fractal Models for Floc Density, Sedimentation Velocity, and Floc Volume Fraction for High Peclet Number Reactors

Abstract Analytical models of floc characteristics are developed as components of a comprehensive flocculation model. The models are derived for reactors with a high ratio of advective fluid transport relative to diffusion (i.e., high Peclet number) and batch reactors where the floc size distribution is expected to be relatively narrow compared with completely mixed flow reactors. High Peclet number flows are obtained in gravity-driven hydraulic flocculators without mechanical agitation. Hydraulic flocculators are an ideal technology for a low carbon footprint and where robust, low maintenance operation is required. The models, combined with measurements of floc sedimentation velocity and floc volume fraction, suggest that the fractal dimension of flocs is approximately 2.3 and that 0.65–1 μm is approximately the particle size beyond which fractal geometry is needed for description of floc properties.

Crystal structure of l-sorbose dehydrogenase, a pyrroloquinoline quinone-dependent enzyme with homodimeric assembly, from Ketogulonicigenium vulgare

The crystal structure of the L-sorbose dehydrogenase (SDH) from Ketogulonicigenium vulgare Y25 has been determined at 2.7 Å resolution using the molecular replacement method. The overall structure of SDH is similar to that of other quinoprotein dehydrogenases; consisting of an eight bladed β-propeller PQQ domain and protrusion loops. We identified a stable homodimer in crystal and demonstrated its existence in solution by sedimentation velocity measurement. By biochemical characterization of the SDH in vitro, using L-sorbose as substrate and cytochrome c551 as electron acceptor, we revealed cytochrome c551 acting as physiological primary electron acceptor for SDH.

Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (Kd) of ~2 μM and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.

Size and density measurement of core–shell Si nanoparticles by analytical ultracentrifugation

Si nanocrystals, terminated with cyclohexane and allylamine, were analyzed using analytical ultracentrifugation (AUC), dynamic light scattering (DLS) and transmission electron microscopy. We found that AUC yielded equivalent particle size distribution data to other nanoparticle characterization techniques, while also providing important information not probed by techniques such as DLS regarding the relative size of the particle core and shell components and the overall effective particle density. Estimates of particle core and shell dimensions are consistent with the particle’s optical properties within the quantum confinement representation and available theoretical Si nanocrystal models. Measurement of sedimentation velocity in several solvents with different densities presents a way to circumvent the ambiguity of simultaneous fitting of particle density and sedimentation coefficient and allows us to significantly reduce the uncertainty in the estimates of particle hydrodynamic diameter by finding the effective particle density value.

Are fluorescence-detected sedimentation velocity data reliable?

Sedimentation velocity analytical ultracentrifugation is a classical biophysical technique that is commonly used to analyze the size, shape, and interactions of biological macromolecules in solution. Fluorescence detection provides enhanced sensitivity and selectivity relative to the standard absorption and refractrometric detectors, but data acquisition is more complex and can be subject to interference from several photophysical effects. Here, we describe methods to configure sedimentation velocity measurements using fluorescence detection and evaluate the performance of the fluorescence optical system. The fluorescence detector output is linear over a concentration range of at least 1 to 500nM fluorescein and Alexa Fluor 488. At high concentrations, deviations from linearity can be attributed to the inner filter effect. A duplex DNA labeled with Alexa Fluor 488 was used as a standard to compare sedimentation coefficients obtained using fluorescence and absorbance detectors. Within error, the sedimentation coefficients agree. Thus, the fluorescence detector is capable of providing precise and accurate sedimentation velocity results that are consistent with measurements performed using conventional absorption optics, provided the data are collected at appropriate sample concentrations and the optics are configured correctly.

Chromatin structure outside and inside the nucleus

The structure of the 30-nm chromatin fiber has provided, over the years, an important reference in chromatin studies. Originally derived from electron microscopic studies of soluble chromatin fibers released by restriction digestion, the gross structural features of such fragments have been supported by biophysical methods such as low angle X-ray and neutron scattering, sedimentation, light scattering, and electric dichroism. Electron microscopy and sedimentation velocity measurements demonstrated that reconstituted chromatin fibers, prepared from repeating arrays of high affinity nucleosome positioning sequences, retain the same overall features as observed for native chromatin fibers. It had been suggested that the 30 nm fiber might be the form assumed in vivo by transcriptionally silent chromatin, but individual gene or genome-wide studies of chromatin released from nuclei do not reveal any such simple correlation. Furthermore, even though the 30 nm fiber has been thought to represent an intermediate in the hierarchical folding of DNA into chromosomes, most analyses of chromatin folding within the nucleus do not detect any regular extended compact structures. However, there are important exceptions in chicken erythroid cell nuclei as well as in transcribed regions that form extended loops. Localized domains within the nucleus, either at the surface of chromosome domains or constrained as a specialized kind of constitutive heterochromatin by specific DNA binding proteins, may adopt 30 nm fiber-like structures.

The Stoichiometry of an SK Channel Peptide and Calmodulin

SK potassium channels modulate the cellular membrane potential in response to intracellular calcium. SK requires that calcium binds to the calcium sensor, calmodulin (CaM), to open. Comprised of four identical subunits, SK has at least four putative CaM binding sites; at least one site is encoded per carboxyl tail. Understanding how SK channels operate requires knowing how many CaMs bind to SK. Previous work has shown that in a crystal the SK peptide (SKp) forms a ‘2:2 complex' with CaM in saturating calcium. We have used a recombinant CaM binding SK peptide (SKp) without affinity tags to measure stoichiometry. Orthogonal approaches were used to measure the molecular mass of particles in solution and in the presence of 1 mM calcium. Light scattering results show that SKp and CaM form a complex with a molecular mass close to 29 kDa, which is consistent with a 1:1 SKp:CaM complex. Sedimentation equilibrium studies show an intermediate molecular weight consistent with alternate stoichiometries, but not likely present as a 2:2 complex. In sedimentation velocity measurements, the sedimentation rate of the complex that forms varies with the molar ratio of SKp:CaM. For example, when SKp and CaM are equimolar, van Holde-Weischet analyses yields a sedimentation constant of 2.50 s (s=1x10−13s). If CaM is in two fold molar excess over SKp, the sedimentation constant increases to 2.96 s. If SKp is in two fold molar excess over CaM, a distinct species forms with a sedimentation constant of 2.75 s. Finally, 2D spectrum analyses combined with Monte Carlo statistics support alternative stoichiometric ratios, but also exclude the formation of a ‘2:2 complex'. The data suggest these proteins have different modes of interaction in a dilute solution than occurs in a crystal.

Mg2+-Linked Self-Assembly of FtsZ in the Presence of GTP or a GTP Analogue Involves the Concerted Formation of a Narrow Size Distribution of Oligomeric Species

The assembly of the bacterial cell division FtsZ protein in the presence of constantly replenished GTP was studied as a function of Mg(2+) concentration (at neutral pH and 0.5 M potassium) under steady-state conditions by sedimentation velocity, concentration-gradient light scattering, fluorescence correlation spectroscopy, and dynamic light scattering. Sedimentation velocity measurements confirmed previous results indicating cooperative appearance of a narrow size distribution of finite oligomers with increasing protein concentration. The concentration dependence of light scattering and diffusion coefficients independently verified the cooperative appearance of a narrow distribution of high molecular weight oligomers, and in addition provided a measurement of the average size of these species, which corresponds to 100 ± 20 FtsZ protomers at millimolar Mg(2+) concentration. Parallel experiments on solutions containing guanosine-5'-[(α,β)-methyleno]triphosphate, sodium salt (GMPCPP), a slowly hydrolyzable analogue of GTP, in place of GTP, likewise indicated the concerted formation of a narrow size distribution of fibrillar oligomers with a larger average mass (corresponding to 160 ± 20 FtsZ monomers). The closely similar behavior of FtsZ in the presence of both GTP and GMPCPP suggests that the observations reflect equilibrium rather than nonequilibrium steady-state properties of both solutions and exhibit parallel manifestations of a common association scheme.

Small Amphipathic Molecules Modulate Secondary Structure and Amyloid Fibril-forming Kinetics of Alzheimer Disease Peptide Aβ1–42

Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation.

HspB2/Myotonic Dystrophy Protein Kinase Binding Protein (MKBP) as a Novel Molecular Chaperone: Structural and Functional Aspects

The small heat shock protein, human HspB2, also known as Myotonic Dystrophy Kinase Binding Protein (MKBP), specifically associates with and activates Myotonic Dystrophy Protein Kinase (DMPK), a serine/threonine protein kinase that plays an important role in maintaining muscle structure and function. The structure and function of HspB2 are not well understood. We have cloned and expressed the protein in E.coli and purified it to homogeneity. Far-UV circular dichroic spectrum of the recombinant HspB2 shows a β-sheet structure. Fluorescence spectroscopic studies show that the sole tryptophan residue at the 130th position is almost completely solvent-exposed. Bis-ANS binding shows that though HspB2 exhibits accessible hydrophobic surfaces, it is significantly less than that exhibited by another well characterized small HSP, αB-crystallin. Sedimentation velocity measurements show that the protein exhibits concentration-dependent oligomerization. Fluorescence resonance energy transfer study shows that HspB2 oligomers exchange subunits. Interestingly, HspB2 exhibits target protein-dependent chaperone-like activity: it exhibits significant chaperone-like activity towards dithiothreitol (DTT)-induced aggregation of insulin and heat-induced aggregation of alcohol dehydrogenase, but only partially prevents the heat-induced aggregation of citrate synthase, co-precipitating with the target protein. It also significantly prevents the ordered amyloid fibril formation of α-synuclein. Thus, our study, for the first time, provides biophysical characterization on the structural aspects of HspB2, and shows that it exhibits target protein-dependent chaperone-like activity.

Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.

Probing the Fluctuation-Dissipation Theorem in a Perrin-like Experiment

In this Letter, we present a new experimental approach to investigate the effective temperature concept as a generalization of the fluctuation-dissipation theorem (FDT) for nonequilibrium systems. Simultaneous measurements of diffusion coefficient and sedimentation velocity of heavy colloids, embedded in a Laponite clay suspension, are performed with a fluorescence-recovery-based setup. This nonperturbative dual measurement, performed at a single time in a single sample, allows for a direct application of the FDT to the tracer velocity observable. It thus provides a well-defined derivation of the effective temperature in this ageing colloidal gel. For a wide range of concentrations and ageing times, we report no violation of the FDT, with effective temperature agreeing with bath temperature. This result is consistent with recent theoretical predictions on the coupling between the velocity observable and nonequilibrium gels dynamics.

Inhibition of Cu2+-mediated generation of reactive oxygen species by the small heat shock protein αB-crystallin: The relative contributions of the N- and C-terminal domains

Oxidative stress, Cu2+ homeostasis, and small heat shock proteins (sHsp's) have important implications in several neurodegenerative diseases. The ubiquitous sHsp αB-crystallin is an oligomeric protein that binds Cu2+. We have investigated the relative contributions of the N- and C-terminal (C-TDαB-crystallin) domains of αB-crystallin to its Cu2+-binding and redox-attenuation properties and mapped the Cu2+-binding regions. C-TDαB-crystallin binds Cu2+ with slightly less affinity and inhibits Cu2+-catalyzed, ascorbate-mediated generation of ROS to a lesser extent than αB-crystallin. [Cu2+]/[subunit] stoichiometries for redox attenuation by αB-crystallin and C-TDαB-crystallin are 5 and 2, respectively. Both αB-crystallin and C-TDαB-crystallin also inhibit the Fenton reaction of hydroxyl radical formation. Trypsinization of αB-crystallin bound to a Cu2+–NTA column and MALDI-TOF analysis of column-bound peptides yielded three peptides located in the N-terminal domain, and in-solution trypsinization of αB-crystallin followed by Cu2+–NTA column chromatography identified four additional Cu2+-binding peptides located in the C-terminal domain. Thus, Cu2+-binding regions are distributed in the N- and C-terminal domains. Small-angle X-ray scattering and sedimentation-velocity measurements indicate quaternary structural changes in αB-crystallin upon Cu2+ binding. Our study indicates that an oligomer of αB-crystallin can sequester a large number (~150) of Cu2+ ions. It acts like a “Cu2+ sponge,” exhibits redox attenuation of Cu2+, and has potential roles in Cu2+ homeostasis and in preventing oxidative stress.

Interactions of ionic and non-ionic surfactants with plasma low density lipoprotein.

Surfactants might be useful in facilitating the replacement of the interior of low density lipoprotein (LDL2) (p = 1.02-1.063 g ml-1) with drug molecules. We have used photon correlation spectroscopy, supported by sedimentation velocity measurements to study the effects of surfactants on LDL2. Sodium dodecyl sulphate, cetrimide, and all non-ionic surfactants studied caused rapid increases of ca 50% in the Stokes' radius up to surfactant/LDL2 molar ratios of ca 1000:1. This was interpreted as due to partial unfolding of the LDL2 protein and intercalation of surfactant with the LDL2 surface layer. At higher concentrations, ionic surfactants and non-ionics with HLB values less than 14.6 decreased the Stokes' radius due to delipidation of LDL2. These interactions are similar to those between surfactants and biological membranes, thus LDL2 might be a useful model system to study surfactant structure-activity relationships.

Activation of PKR by Stem Loop RNAs Requires Flanking ssRNA Arms

Protein kinase R (PKR) is a key component of the interferon-induced viral response pathway. PKR contains an N-terminal double-stranded RNA (dsRNA) binding domain and a C-terminal kinase domain connected by a ∼90-residue linker. Upon binding dsRNA, PKR undergoes autophosphorylation and kinase activation. Activated PKR then phosphorylates the alpha subunit of the eukaryotic initiation growth factor 2 and thus inhibits protein synthesis in virally infected cells. A minimum of 30 bp of dsRNA is required to bind two PKRs, leading to dimerization and activation. Stem-loops are common RNA structural motifs. Recently, it was reported that short stem-loops containing 16 bp and ssRNA flanking arms are also able to activate PKR and that activation requires a 5’-triphosphate. However, the activation mechanism is not known. We have prepared RNAs containing a 15 bp stem-loop and various ssRNA flanking arms and characterized activation and binding of PKR. Activation by these RNAs requires ssRNA arms on both the 5’ and 3’ sides but it is independent of the presence of a 5’-triphosphate. Sedimentation velocity measurements indicate that a single PKR binds to the activating stem-loop RNA at 200 mM NaCl with Kd = 309 nM. PKR binding affinity is not strongly affected by removal of either of the ssRNA arms or the 5’-triphosphate. At lower [NaCl] (75 mM), each of the stem-loop RNAs can bind two monomers of PKR. However, only the activating RNA exhibits a positive binding cooperativity. We propose that activation by this RNA is due to enhanced population of the species containing two bound PKRs as a result of cooperative binding of the second PKR monomer.