Resolving power of dynamic light scattering for protein and polystyrene nanoparticles.

Dynamic light scattering (DLS) is a non-invasive, label-free technique for the characterization of particles ranging from nanometer to micrometer size. It is widely used for the analysis of proteins to assess association states and the nature of protein aggregates. Despite its frequent use, little quantitative information on its size resolution capabilities, in particular for protein material, is available. This study explores the resolving power of a standard DLS setup for binary mixtures of latex standard particles and mixtures of protein monomer and protein particles made from cross-linked protein material. At constant instrument settings, the resolving power depends on the size ratio and the mass ratio of the species in a mixture as well as on the total concentration and the scattering characteristics of the material. In this study, we provide a summary at which parameter combinations resolution of two species with varying size is possible. These data guide the quantitative evaluation of DLS results for mixtures. We found that a mixture of an antibody monomer and protein particles of an average hydrodynamic diameter of 50 nm can be resolved at a 1-20-fold excess of monomer (by mass). A mixture of monomer and 70 nm particles can be resolved at a 2-30-fold excess, a mixture of monomer and 190 nm particles at a 200-1700-fold excess of monomer. The findings allow to better judge DLS results for protein samples of unknown composition.

A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN.

Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrate.

Cooperative binding of ATP and RNA induces a closed conformation in a DEAD box RNA helicase.

RNA helicases couple the energy from ATP hydrolysis with structural changes of their RNA substrates. DEAD box helicases form the largest class of RNA helicases and share a helicase core comprising two RecA-like domains. An opening and closing of the interdomain cleft during RNA unwinding has been postulated but not shown experimentally. Single-molecule FRET experiments with the Bacillus subtilis DEAD box helicase YxiN carrying donor and acceptor fluorophores on different sides of the interdomain cleft reveal an open helicase conformation in the absence of nucleotides, or in the presence of ATP, or ADP, or RNA. In the presence of ADP and RNA, the open conformation is retained. By contrast, cooperative binding of ATP and RNA leads to a compact helicase structure, proving that the ATP- and ADP-bound states of RNA helicases display substantially different structures only when the RNA substrate is bound. These results establish a closure of the interdomain cleft in the helicase core at the beginning of the unwinding reaction, and suggest a conserved mechanism of energy conversion among DEAD box helicases across kingdoms.

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins.

DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

Different species of alpha-synuclein oligomers induce calcium influx and seeding.

Aggregation of alpha-synuclein (alpha-syn) has been linked to the pathogenesis of Parkinson's disease (PD) and other neurodegenerative diseases. Increasing evidence suggests that prefibrillar oligomers and protofibrils, rather than mature fibrils of alpha-syn, are the pathogenic species in PD. Despite extensive effort on studying oligomerization of alpha-syn, no studies have compared different oligomer species directly on a single-particle level and investigated their biological effects on cells. In this study, we applied a novel highly sensitive single molecule detection system that allowed a direct comparison of different oligomer types. Furthermore, we studied biological effects of different oligomer types on cells. For this purpose, we developed new oligomerization protocols, that enabled the use of these different oligomers in cell culture. We found that all of our three aggregation protocols resulted in heterogeneous populations of oligomers. Some types of oligomers induced cell death via disruption of cellular ion homeostasis by a presumably pore-forming mechanism. Other oligomer types could directly enter the cell resulting in increased alpha-syn aggregation. Based on our results, we propose that under various physiological conditions, heterogeneous populations of oligomeric forms will coexist in an equilibrium. These different oligomer types lead directly or indirectly to cell damage. Our data indicate that inhibition of early alpha-syn aggregation events would consequently prevent all alpha-syn oligomer related toxicities. This has important implications for the development of disease-modifying drugs for the treatment of PD and other synucleinopathies.

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

Nanoparticle tracking analysis of particle size and concentration detection in suspensions of polymer and protein samples: Influence of experimental and data evaluation parameters.

Nanoparticle Tracking Analysis (NTA) is an emerging technique for detecting simultaneously sub-micron particle size distributions and particle concentrations of a sample. This study deals with the performance evaluation for the detection and characterisation of various particles by NTA. Our investigation focusses on the NTA measurement parameter set-ups, as will be shown in this study, are very crucial parameters to correctly analyse and interpret the data. In order to achieve this, we used (i) polystyrene standard particles as well as (ii) protein particles. We show the highly precise and reproducible detection of particle size and concentration in monodisperse polystyrene particle systems, under specified and constant parameter settings. On the other hand, our results exemplify potential risks and errors while setting inadequate parameters with regards to the results and thus interpretation thereof. In particular changes of the parameters, camera level (CL) and detection threshold (DT), led to significant changes in the determined particle concentration. We propose defined and specified "optimal" camera levels for monodisperse particle suspension characterisations in the size range of 20-1000nm. We illustrate that the results of polydisperse polystyrene standard particle solution measurements, highly depend on the used parameter settings, which are rarely published with the data. Changes in these settings led to the "appearance" or "disappearance" of particle populations ("peaks") for polydisperse systems. Thus, a correct evaluation of the particle size populations in the sample becomes very challenging. For the use of NTA in biopharmaceutical analysis, proteinaceous samples were investigated. We analysed protein particle suspensions and compared unstressed and stressed (formation of aggregates) protein samples similar to polystyrene particle analysis. We also studied these samples in two different measuring modes (general capture mode and live monitoring mode) that the commercially available analysis software is offering. Our results stated the live monitoring mode as more suitable for protein samples, as the results were more reproducible and less operator-depending. In conclusion, NTA is a potential technique and unique in quantitative evaluation of particle suspensions in the subvisible size range, especially for monodisperse suspensions. We strongly urge on not underestimating the influence of the measuring parameters on the obtained results, which should be presented with the data in order to better judge and interpret the NTA results.

Buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: from protein buffer capacity prediction to bioprocess applications.

Protein therapeutics, including monoclonal antibodies (mAbs), have significant buffering capacity, particularly at concentrations>50 mg/mL. This report addresses pH-related issues critical to adoption of self-buffered monoclonal antibody formulations. We evaluated solution conditions with protein concentrations ranging from 50 to 250 mg/mL. Samples were both buffer-free and conventionally buffered with citrate. Samples were non-isotonic or adjusted for isotonicity with NaCl or trehalose. Studies included accelerated temperature stability tests, shaking stability studies, and pH changes in infusion media as protein concentrate is added. We present averaged buffering slopes of capacity that can be applied to any mAb and present a general method for calculating buffering capacity of buffer-free, highly concentrated antibody liquid formulations. In temperature stability tests, neither buffer-free nor conventionally buffered solution conditions showed significant pH changes. Conventionally buffered solutions showed significantly higher opalescence than buffer-free ones. In general, buffer-free solution conditions showed less aggregation than conventionally buffered solutions. Shaking stability tests showed no differences between buffer-free and conventionally buffered solutions. "In-use" preparation experiments showed that pH in infusion bag medium can rapidly approximate that of self-buffered protein concentrate as concentrate is added. In summary, the buffer capacity of proteins can be predicted and buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions.

Boosting antibody developability through rational sequence optimization.

The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.

Buffer capacity of biologics--from buffer salts to buffering by antibodies.

Controlling pH is essential for a variety of biopharmaceutical process steps. The chemical stability of biologics such as monoclonal antibodies is pH-dependent and slightly acidic conditions are favorable for stability in a number of cases. Since control of pH is widely provided by added buffer salts, the current study summarizes the buffer characteristics of acetate, citrate, histidine, succinate, and phosphate buffers. Experimentally derived values largely coincide with values calculated from a model that had been proposed in 1922 by van Slyke. As high concentrated protein formulations become more and more prevalent for biologics, the self-buffering potential of proteins becomes of relevance. The current study provides information on buffer characteristics for pH ranges down to 4.0 and up to 8.0 and shows that a monoclonal antibody at 50 mg/mL exhibits similar buffer capacity as 6 mM citrate or 14 mM histidine (pH 5.0-6.0). Buffer capacity of antibody solutions scales linearly with protein concentration up to more than 200 mg/mL. At a protein concentration of 220 mg/mL, the buffer capacity resembles the buffer capacity of 30 mM citrate or 50 mM histidine (pH 5.0-6.0). The buffer capacity of monoclonal antibodies is practically identical at the process relevant temperatures 5, 25, and 40°C. Changes in ionic strength of ΔI=0.15, in contrast, can alter the buffer capacity up to 35%. In conclusion, due to efficient self-buffering by antibodies in the pH range of favored chemical stability, conventional buffer excipients could be dispensable for pH stabilization of high concentrated protein solutions.

A structural model for the DEAD box helicase YxiN in solution: localization of the RNA binding domain.

DEAD box proteins consist of a common helicase core formed by two globular RecA domains that are separated by a cleft. The helicase core acts as a nucleotide-dependent switch that alternates between open and closed conformations during the catalytic cycle of duplex separation, thereby providing basic helicase activity. Flanking domains can direct the helicase core to a specific RNA substrate by mediating high-affinity or high-specificity RNA binding. In addition, they may position RNA for the helicase core or may directly contribute to unwinding. While structures of different helicase cores have been determined previously, little is known about the orientation of flanking domains relative to the helicase core. YxiN is a DEAD box protein that consists of a helicase core and a C-terminal RNA binding domain (RBD) that mediates specific binding to hairpin 92 in 23S rRNA. To provide a framework for understanding the functional cooperation of the YxiN helicase core and the RBD, we mapped the orientation of the RBD in single-molecule fluorescence resonance energy transfer experiments. We present a model for the global conformation of YxiN in which the RBD lies above a slightly concave patch that is formed by flexible loops on the surface of the C-terminal RecA domain. The orientation of the RBD is different from the orientations of flanking domains in the Thermus thermophilus DEAD box protein Hera and in Saccharomyces cerevisiae Mss116p, in line with the different functions of these DEAD box proteins and of their RBDs. Interestingly, the corresponding patch on the C-terminal RecA domain that is covered by the YxiN RBD is also part of the interface between the translation factors eIF4A and eIF4G. Possibly, this region constitutes an adaptable interface that generally allows for the interaction of the helicase core with additional domains or interacting factors.