Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Exploring Host-Guest Interactions within a 600 kDa DegP Protease Cage Complex Using Hydrodynamics Measurements and Methyl-TROSY NMR.

The DegP protease-chaperone operates within the periplasm of Gram-negative bacteria, where it assists in the regulation of protein homeostasis, promotes virulence, and is essential to survival under stress. To carry out these tasks, DegP forms a network of preorganized apo oligomers that facilitate the capture of substrates within distributions of cage-like complexes which expand to encapsulate clients of various sizes. Although the architectures of DegP cage complexes are well understood, little is known about the structures, dynamics, and interactions of client proteins within DegP cages and the relationship between client structural dynamics and function. Here, we probe host-guest interactions within a 600 kDa DegP cage complex throughout the DegP activation cycle using a model α-helical client protein through a combination of hydrodynamics measurements, methyl-transverse relaxation optimized spectroscopy-based solution nuclear magnetic resonance studies, and proteolytic activity assays. We find that in the presence of the client, DegP cages assemble cooperatively with few intermediates. Our data further show that the N-terminal half of the bound client, which projects into the interior of the cages, is predominantly unfolded and flexible, and exchanges between multiple conformational states over a wide range of time scales. Finally, we show that a concerted structural transition of the protease domains of DegP occurs upon client engagement, leading to activation. Together, our findings support a model of DegP as a highly cooperative and dynamic molecular machine that stabilizes unfolded states of clients, primarily via interactions with their C-termini, giving rise to efficient cleavage.

An automated interface for sedimentation velocity analysis in SEDFIT.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is an indispensable tool for the study of particle size distributions in biopharmaceutical industry, for example, to characterize protein therapeutics and vaccine products. In particular, the diffusion-deconvoluted sedimentation coefficient distribution analysis, in the software SEDFIT, has found widespread applications due to its relatively high resolution and sensitivity. However, a lack of suitable software compatible with Good Manufacturing Practices (GMP) has hampered the use of SV-AUC in this regulatory environment. To address this, we have created an interface for SEDFIT so that it can serve as an automatically spawned module with controlled data input through command line parameters and output of key results in files. The interface can be integrated in custom GMP compatible software, and in scripts that provide documentation and meta-analyses for replicate or related samples, for example, to streamline analysis of large families of experimental data, such as binding isotherm analyses in the study of protein interactions. To test and demonstrate this approach we provide a MATLAB script mlSEDFIT.

Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP.

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a "resting" hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP's substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.

Characterization of DNA-protein complexes by nanoparticle tracking analysis and their association with systemic lupus erythematosus.

Nanotechnology enables investigations of single biomacromolecules, but technical challenges have limited the application in liquid biopsies, for example, blood plasma. Nonetheless, tools to characterize single molecular species in such samples represent a significant unmet need with the increasing appreciation of the physiological importance of protein structural changes at nanometer scale. Mannose-binding lectin (MBL) is an oligomeric plasma protein and part of the innate immune system through its ability to activate complement. MBL also serves a role as a scavenger for cellular debris, especially DNA. This may link functions of MBL with several inflammatory diseases in which cell-free DNA now appears to play a role, but mechanistic insight has been lacking. By making nanoparticle tracking analysis possible in human plasma, we now show that superoligomeric structures of MBL form nanoparticles with DNA. These oligomers correlate with disease activity in systemic lupus erythematosus patients. With the direct quantification of the hydrodynamic radius, calculations following the principles of Taylor dispersion in the blood stream connect the size of these complexes to endothelial inflammation, which is among the most important morbidities in lupus. Mechanistic insight from an animal model of lupus supported that DNA-stabilized superoligomers stimulate the formation of germinal center B cells and drive loss of immunological tolerance. The formation involves an inverse relationship between the concentration of MBL superoligomers and antibodies to double-stranded DNA. Our approach implicates the structure of DNA-protein nanoparticulates in the pathobiology of autoimmune diseases.

Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms.

BACKGROUND: C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood. METHODS: We combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors. RESULTS: CrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. CONCLUSION: Our study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstract.

An intrinsically disordered motif regulates the interaction between the p47 adaptor and the p97 AAA+ ATPase.

VCP/p97, an enzyme critical to proteostasis, is regulated through interactions with protein adaptors targeting it to specific cellular tasks. One such adaptor, p47, forms a complex with p97 to direct lipid membrane remodeling. Here, we use NMR and other biophysical methods to study the structural dynamics of p47 and p47-p97 complexes. Disordered regions in p47 are shown to be critical in directing intra-p47 and p47-p97 interactions via a pair of previously unidentified linear motifs. One of these, an SHP domain, regulates p47 binding to p97 in a manner that depends on the nucleotide state of p97. NMR and electron cryomicroscopy data have been used as restraints in molecular dynamics trajectories to develop structural ensembles for p47-p97 complexes in adenosine diphosphate (ADP)- and adenosine triphosphate (ATP)-bound conformations, highlighting differences in interactions in the two states. Our study establishes the importance of intrinsically disordered regions in p47 for the formation of functional p47-p97 complexes.

The intrinsic kinase activity of BRD4 spans its BD2-B-BID domains.

Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that is a therapeutic target in many cancers and inflammatory diseases. BRD4 plays important roles in transcription as an active kinase, which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II), the proto-oncogene c-MYC, and transcription factors TAF7 and CDK9. BRD4 is also a passive scaffold that recruits transcription factors. Despite these well-established functions, there has been little characterization of BRD4's biophysical properties or its kinase activity. We report here that the 156 kD mouse BRD4 exists in an extended dimeric conformation with a sedimentation coefficient of ∼6.7 S and a high frictional ratio. Deletion of the conserved B motif (aa 503-548) disrupts BRD4's dimerization. BRD4 kinase activity maps to amino acids 351 to 598, which span bromodomain-2, the B motif, and the BID domain (BD2-B-BID) and contributes to the in vivo phosphorylation of its substrates. As further assessed by analytical ultracentrifugation, BRD4 directly binds purified Pol II CTD. Importantly, the conserved A motif of BRD4 is essential for phosphorylation of Pol II CTD, but not for phosphorylation of TAF7, mapping its binding site to the A motif. Peptides of the viral MLV integrase (MLVIN) protein and cellular histone lysine methyltransferase, NSD3, which have been shown by NMR to bind to the extra-terminal (ET) domain, also are phosphorylated by BRD4. Thus, BRD4 has multiple distinct substrate-binding sites and a common kinase domain. These results provide new insights into the structure and kinase function of BRD4.

Distinct disease features in chimpanzees infected with a precore HBV mutant associated with acute liver failure in humans.

Transmission to chimpanzees of a precore hepatitis B virus (HBV) mutant implicated in acute liver failure (ALF) in humans did not cause ALF nor the classic form of acute hepatitis B (AHB) seen upon infection with the wild-type HBV strain, but rather a severe AHB with distinct disease features. Here, we investigated the viral and host immunity factors responsible for the unusual severity of AHB associated with the precore HBV mutant in chimpanzees. Archived serial serum and liver specimens from two chimpanzees inoculated with a precore HBV mutant implicated in ALF and two chimpanzees inoculated with wild-type HBV were studied. We used phage-display library and next-generation sequencing (NGS) technologies to characterize the liver antibody response. The results obtained in severe AHB were compared with those in classic AHB and HBV-associated ALF in humans. Severe AHB was characterized by: (i) the highest alanine aminotransferase (ALT) peaks ever seen in HBV transmission studies with a significantly shorter incubation period, compared to classic AHB; (ii) earlier HBsAg clearance and anti-HBs seroconversion with transient or undetectable hepatitis B e antigen (HBeAg); (iii) limited inflammatory reaction relative to hepatocellular damage at the ALT peak with B-cell infiltration, albeit less extensive than in ALF; (iv) detection of intrahepatic germline antibodies against hepatitis B core antigen (HBcAg) by phage-display libraries in the earliest disease phase, as seen in ALF; (v) lack of intrahepatic IgM anti-HBcAg Fab, as seen in classic AHB, but at variance with ALF; and (vi) higher proportion of antibodies in germline configuration detected by NGS in the intrahepatic antibody repertoire compared to classic AHB, but lower than in ALF. This study identifies distinct outcome-specific features associated with severe AHB caused by a precore HBV mutant in chimpanzees, which bear closer resemblance to HBV ALF than to classic AHB. Our data suggest that precore HBV mutants carry an inherently higher pathogenicity that, in addition to specific host factors, may play a critical role in determining the severity of acute HBV disease.

The biofilm adhesion protein Aap from Staphylococcus epidermidis forms zinc-dependent amyloid fibers.

The skin-colonizing commensal bacterium Staphylococcus epidermidis is a leading cause of hospital-acquired and device-related infections. Its pathogenicity in humans is largely due to its propensity to form biofilms, surface-adherent bacterial accumulations that are remarkably resistant to chemical and physical stresses. Accumulation-associated protein (Aap) from S. epidermidis has been shown to be necessary and sufficient for mature biofilm formation and catheter infection. Aap contains up to 17 tandem B-repeat domains, capable of zinc-dependent assembly into twisted, rope-like intercellular filaments in the biofilm. Using microscopic and biophysical techniques, we show here that Aap B-repeat constructs assemble further into zinc-dependent functional amyloid fibers. We observed such amyloid fibers by confocal microscopy during both early and late stages of S. epidermidis biofilm formation, and we confirmed that extracellular fibrils from these biofilms contain Aap. Unlike what has been observed for amyloidogenic biofilm proteins from other bacteria, which typically use chaperones or initiator proteins to initiate amyloid assembly, our findings indicate that Aap from S. epidermidis requires Zn2+ as a catalyst that drives amyloid fiber formation, similar to many mammalian amyloid-forming proteins that require metals for assembly. This work provides detailed insights into S. epidermidis biofilm formation and architecture that improve our understanding of persistent staphylococcal infections.

Determining the Stoichiometry of a Protein-Polymer Conjugate Using Multisignal Sedimentation Velocity Analytical Ultracentrifugation.

Advances in polymer science have broadened the applications of protein-polymer conjugates as biopharmaceuticals and biotechnology reagents. The complex nature of these conjugates makes characterization challenging. Here, we describe the use of multisignal sedimentation velocity analytical ultracentrifugation to measure the polymer-to-protein ratio. To demonstrate the principle, we applied this approach to a series of antibody-drug conjugates with different polymer-to-protein ratios and various degrees of heterogeneity, and validated results with orthogonal analytical techniques. We found that multisignal sedimentation velocity can provide accurate information on key attributes including polymer-to-protein ratio, which is important for maximizing the therapeutic potential of future protein-polymer conjugates.

Calibrating analytical ultracentrifuges.

Analytical ultracentrifugation (AUC) is based on the concept of recording and analyzing macroscopic macromolecular redistribution that results from a centrifugal force acting on the mass of suspended macromolecules in solution. Since AUC rests on first principles, it can provide an absolute measurement of macromolecular mass, sedimentation and diffusion coefficients, and many other quantities, provided that the solvent density and viscosity are known, and provided that the instrument is properly calibrated. Unfortunately, a large benchmark study revealed that many instruments exhibit very significant systematic errors. This includes the magnification of the optical detection system used to determine migration distance, the measurement of sedimentation time, and the measurement of the solution temperature governing viscosity. We have previously developed reference materials, tools, and protocols to detect and correct for systematic measurement errors in the AUC by comparison with independently calibrated standards. This 'external calibration' resulted in greatly improved precision and consistency of parameters across laboratories. Here we detail the steps required for calibration of the different data dimensions in the AUC. We demonstrate the calibration of three different instruments with absorbance and interference optical detection, and use measurements of the sedimentation coefficient of NISTmAb monomer as a test of consistency. Whereas the measured uncorrected sedimentation coefficients span a wide range from 6.22 to 6.61 S, proper calibration resulted in a tenfold reduced standard deviation of sedimentation coefficients. The calibrated relative standard deviation and mean error of 0.2% and 0.07%, respectively, is comparable with statistical errors and side-by-side repeatability in a single instrument.

A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA.

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.

Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation.

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.

Role of humoral immunity against hepatitis B virus core antigen in the pathogenesis of acute liver failure.

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome leading to death or liver transplantation in 80% of cases. Due to the extremely rapid clinical course, the difficulties in obtaining liver specimens, and the lack of an animal model, the pathogenesis of ALF remains largely unknown. Here, we performed a comprehensive genetic and functional characterization of the virus and the host in liver tissue from HBV-associated ALF and compared the results with those of classic acute hepatitis B in chimpanzees. In contrast with acute hepatitis B, HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and miRNA expression profiling revealed a dominant B cell disease signature, with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, and complement deposition. Thus, HBV ALF appears to be an anomalous T cell-independent, HBV core-driven B cell disease, which results from the rare and unfortunate encounter between a host with an unusual B cell response and an infecting virus with a highly mutated core antigen.

All tubulins are not alike: Heterodimer dissociation differs among different biological sources.

Tubulin, the subunit of microtubules, is a noncovalent heterodimer composed of one α- and one ß-tubulin monomer. Both tubulins are encoded by multiple genes or composed of different isotypes, which are differentially expressed in different tissues and in development. Tubulin αß dimers are found throughout the eukaryotes and, although very similar, are known to differ among organisms. We seek to investigate tubulins from different tissues and different organisms for a basic physical characteristic: heterodimer stability and monomer exchange between heterodimers. We previously showed that mammalian brain tubulin heterodimers reversibly dissociate, following the mass action law. Dissociation yields native monomers that can exchange with added tubulin to form new heterodimers. Here, we compared the dissociation of tubulins from multiple sources, including mammalian (rat) brain, cultured human cells (HeLa cells), chicken brain, chicken erythrocytes, and the protozoan Leishmania We used fluorescence-detected analytical ultracentrifugation to measure tubulin dissociation over a >1000-fold range in concentration and found that tubulin heterodimers from different biological sources differ in Kd by as much as 150-fold under the same conditions. Furthermore, when fluorescent tracer tubulins from various sources were titrated with unlabeled tubulin from a single source (rat brain tubulin), heterologous dimerization occurred, exhibiting similar affinities, in some cases binding even more strongly than with autologous tubulin. These results provide additional insight into the regulation of heterodimer formation of tubulin from different biological sources, revealing that monomer exchange appears to contribute to the sorting of α- and ß-tubulin monomers that associate following tubulin folding.

Nucleic acid-induced dimerization of HIV-1 Gag protein.

HIV-1 Gag is a highly flexible multidomain protein that forms the protein lattice of the immature HIV-1 virion. In vitro, it reversibly dimerizes, but in the presence of nucleic acids (NAs), it spontaneously assembles into virus-like particles (VLPs). High-resolution structures have revealed intricate details of the interactions of the capsid (CA) domain of Gag and the flanking spacer peptide SP1 that stabilize VLPs, but much less is known about the assembly pathway and the interactions of the highly flexible NA-binding nucleocapsid (NC) domain. Here, using a novel hybrid fluorescence proximity/sedimentation velocity method in combination with calorimetric analyses, we studied initial binding events by monitoring the sizes and conformations of complexes of Gag with very short oligonucleotides. We observed that high-affinity binding of oligonucleotides induces conformational changes in Gag accompanied by the formation of complexes with a 2:1 Gag/NA stoichiometry. This NA-liganded dimerization mode is distinct from the widely studied dimer interface in the CA domain and from protein interactions arising in the SP1 region and may be mediated by protein-protein interactions localized in the NC domain. The formation of the liganded dimer is strongly enthalpically driven, resulting in higher dimerization affinity than the CA-domain dimer. Both detailed energetic and conformational analyses of different Gag constructs revealed modulatory contributions to NA-induced dimerization from both matrix and CA domains. We hypothesize that allosterically controlled self-association represents the first step of VLP assembly and, in concert with scaffolding along the NA, can seed the formation of two-dimensional arrays near the NA.

Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.

Membrane-less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher-order assemblies influences the recruitment of the speckle-type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin-3-RING ubiquitin ligase (CRL3) substrate adaptor, self-associates into higher-order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild-type SPOP localizes to liquid nuclear speckles, self-association-deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB-mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration-dependent populations of the resulting oligomeric species. Higher-order oligomerization of SPOP stimulates CRL3(SPOP) ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher-order protein self-association may be a general mechanism to concentrate functional components in membrane-less cellular bodies.

Measuring Ultra-Weak Protein Self-Association by Non-ideal Sedimentation Velocity.

Ultra-weak self-association can govern the macroscopic solution behavior of concentrated macromolecular solutions ranging from food products to pharmaceutical formulations and the cytosol. For example, it can promote dynamic assembly of multi-protein signaling complexes, lead to intracellular liquid-liquid phase transitions, and seed crystallization or pathological aggregates. Unfortunately, weak self-association is technically extremely difficult to study, as it requires very high protein concentrations where short intermolecular distances cause strongly correlated particle motion. Additionally, protein samples near their solubility limit in vitro frequently show some degree of polydispersity. Here we exploit the strong mass-dependent separation of assemblies in the centrifugal field to study ultra-weak binding, using a sedimentation velocity technique that allows us to determine particle size distributions while accounting for colloidal hydrodynamic interactions and thermodynamic non-ideality (Chaturvedi, S. K.; et al. Nat. Commun. 2018, 9, 4415; DOI: 10.1038/s41467-018-06902-x ). We show that this approach, applied to self-associating proteins, can reveal a time-average association state for rapidly reversible self-associations from which the free energy of binding can be derived. The method is label-free and allows studying mid-sized proteins at millimolar protein concentrations in a wide range of solution conditions. We examine the performance of this method with hen egg lysozyme as a model system, reproducing its well-known ionic-strength-dependent weak self-association. The application to chicken γS-crystallin reveals weak monomer-dimer self-association with KD = 24 mM, corresponding to a standard free energy change of approximately -9 kJ/mol, which is a large contribution to the delicate balance of forces ensuring eye lens transparency.

Enhanced Sample Handling for Analytical Ultracentrifugation with 3D-Printed Centerpieces.

The centerpiece of the sample cell assembly in analytical ultracentrifugation holds the sample solution between windows, sealed against high vacuum, and is shaped such that macromolecular migration in centrifugal fields exceeding 200 000g can proceed undisturbed by walls or convection while concentration profiles are imaged with optical detection systems aligned perpendicular to the plane of rotation. We have recently shown that 3D printing using various materials allows inexpensive and rapid manufacturing of centerpieces. In the present work, we expand this endeavor to examine the accuracy of the measured sedimentation process, as well as short-term durability of the centerpieces. We find that 3D-printed centerpieces can be used many times and can provide data equivalent in quality to commonly used commercial epoxy resin centerpieces. Furthermore, 3D printing enables novel designs adapted to particular experimental objectives because they offer unique opportunities, for example, to create well-defined curved surfaces, narrow channels, and embossed features. We present examples of centerpiece designs exploiting these capabilities for improved AUC experiments. This includes narrow sector centerpieces that substantially reduce the required sample volume while maintaining the standard optical path length; thin centerpieces with integrated window holders to provide very short optical pathlengths that reduce optical aberrations at high macromolecular concentrations; long-column centerpieces that increase the observable distance of macromolecular migration for higher-precision sedimentation coefficients; and three-sector centerpieces that allow doubling the number of samples in a single run while reducing the sample volumes. We find each of these designs allows unimpeded macromolecular sedimentation and can provide high-quality sedimentation data.