European Biophysics Journal最新文献

In the vast majority of biologically relevant cases of receptor-ligand complex formation, the binding site of the receptor is a small part of its surface, and moreover, formation of a biologically active complex often requires a specific orientation of the ligand relative to the binding site. Before the formation of the initial form of the complex, only long-range, electrostatic and hydrodynamic interactions can act between the ligand approaching the binding site and the receptor. In this context, the question arises whether as a result of these interactions, there is a pre-orientation of the ligand towards the binding site, which to some extent would accelerate the formation of the complex. The role of electrostatic interactions in the orientation of the ligand relative to the binding site of the receptor is well documented. The analogous role of hydrodynamic interactions, although assessed as very significant by Brune and Kim (PNAS 91, 2930–2934, (1994)), is still debatable. In this article, I present the current state of knowledge on this subject and consider the possibilities of demonstrating the orienting effect of hydrodynamic interactions in the processes of receptor–ligand association, in an experimental way supported by computer simulations.

One of the main concerns of Anfinsen was to reveal the connection between the amino-acid sequence and their biologically active conformation. This search gave rise to two crucial questions in structural biology, namely, why the proteins fold and how a sequence encodes its folding. As to the why, he proposes a plausible answer, namely, the thermodynamic hypothesis. As to the how, this remains an unsolved challenge. Consequently, the protein folding problem is examined here from a new perspective, namely, as an ‘analytic whole’. Conceiving the protein folding in this way enabled us to (i) examine in detail why the force-field-based approaches have failed, among other purposes, in their ability to predict the three-dimensional structure of a protein accurately; (ii) propose how to redefine them to prevent these shortcomings, and (iii) conjecture on the origin of the state-of-the-art numerical-methods success to predict the tridimensional structure of proteins accurately.

The biotech industry has great interest in investigating therapeutic proteins in high concentration environments like human serum. The fluorescence detection system (Aviv-FDS) allows the performance of analytical ultracentrifuge (AUC) sedimentation velocity (SV) experiments in tracer or BOLTS protocols. Here, we compare six pooled human serum samples by AUC SV techniques and demonstrate the potential of this technology for characterizing therapeutic antibodies in serum. Control FDS SV experiments on serum alone reveal a bilirubin–HSA complex whose sedimentation is slowed by solution nonideality and exhibits a Johnston–Ogston (JO) effect due to the presence of high concentrations of IgG. Absorbance SV experiments on diluted serum samples verify the HSA–IgG composition as well as a significant IgM pentamer boundary at 19 s. Alexa-488 labeled Simponi (Golimumab) is used as a tracer to investigate the behavior of a therapeutic monoclonal antibody (mAb) in serum, and the sedimentation behavior of total IgG in serum. Serum dilution experiments allow extrapolation to zero concentration to extract s^o, while global direct boundary fitting with SEDANAL verifies the utility of a matrix of self- and cross-term phenomenological nonideality coefficients (k_s and BM₁) and the source of the JO effect. The best fits include weak reversible association (~ 4 × 10³ M⁻¹) between Simponi and total human IgG. Secondary mAbs to human IgG and IgM verify the formation of a 10.2 s 1:1 complex with human IgG and a 19 s complex with human IgM pentamers. These results demonstrate that FDS AUC allows a range of approaches for investigating therapeutic antibodies in human serum.

A novel approach is presented that increases sensitivity and specificity for detecting minimal traces of DNA in liquid and on solid samples. Förster Resonance Energy Transfer (FRET) from YOYO to Ethidium Bromide (EtBr) substantially increases the signal from DNA-bound EtBr highly enhancing sensitivity and specificity for DNA detection. The long fluorescence lifetime of the EtBr acceptor, when bound to DNA, allows for multi-pulse pumping with time gated (MPPTG) detection, which highly increases the detectable signal of DNA-bound EtBr. A straightforward spectra/image subtraction eliminates sample background and allows for a huge increase in the overall detection sensitivity. Using a combination of FRET and MPPTG detection an amount as small as 10 pg of DNA in a microliter sample can be detected without any additional sample purification/manipulation or use of amplification technologies. This amount of DNA is comparable to the DNA content of a one to two human cells. Such a detection method based on simple optics opens the potential for robust, highly sensitive DNA detection/imaging in the field, quick evaluation/sorting (i.e., triaging) of collected DNA samples, and can support various diagnostic assays.

Graphical abstract

Determination of the size, density, and mass of viral particles can provide valuable information to support process and formulation studies in clinical development. Analytical ultracentrifugation (AUC), as a first principal method, has been shown to be a beneficial tool for the characterization of the non-enveloped adeno associated virus (AAV). Here, we demonstrate the suitability of AUC for the challenging characterization of a representative for enveloped viruses, which usually are expected to exhibit higher dispersity than non-enveloped viruses. Specifically, the vesicular stomatitis virus (VSV)-based oncolytic virus VSV-GP was used to evaluate potential occurrence of non-ideal sedimentation by testing different rotor speeds and loading concentrations. The partial specific volume was determined via density gradients and density contrast experiments. Additionally, nanoparticle tracking analysis (NTA) was used to determine the hydrodynamic diameter of VSV-GP particles to calculate their molecular weight via the Svedberg equation. Overall, this study demonstrates the applicability of AUC and NTA for the characterization of size, density, and molar mass of an enveloped virus, namely VSV-GP.

Viral vector-based gene therapies and vaccines require accurate characterization of capsid species. The current gold standard for assessing capsid loading of adeno-associated virus (AAV) is sedimentation velocity analytical ultracentrifugation (SV-AUC). However, routine SV-AUC analysis is often size-limited, especially without the use of advanced techniques (e.g., gravitational-sweep) or when acquiring the multiwavelength data needed for assessing the loading fraction of viral vectors, and requires analysis by specialized software packages. Density gradient equilibrium AUC (DGE-AUC) is a highly simplified analytical method that provides high-resolution separation of biologics of different densities (e.g., empty and full viral capsids). The analysis required is significantly simpler than SV-AUC, and larger viral particles such as adenovirus (AdV) are amenable to characterization by DGE-AUC using cesium chloride gradients. This method provides high-resolution data with significantly less sample (estimated 56-fold improvement in sensitivity compared to SV-AUC). Multiwavelength analysis can also be used without compromising data quality. Finally, DGE-AUC is serotype-agnostic and amenable to intuitive interpretation and analysis (not requiring specialized AUC software). Here, we present suggestions for optimizing DGE-AUC methods and demonstrate a high-throughput AdV packaging analysis with the AUC, running as many as 21 samples in 80 min.

Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260 nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles.

Nowadays, reports of antimicrobial resistance (AMR) against many antibiotics are increasing because of their misapplication. With this rise, there is a serious decrease in the discovery and development of new types of antibiotics amid an increase in multi-drug resistance. Unfermented Acinetobacter baumannii from gram-negative bacteria, which is one of the main causes of nosocomial infections and multi-drug resistance, has 4 main kinds of antibiotic resistance mechanism: inactivating antibiotics by enzymes, reduced numbers of porins and changing of their target or cellular functions due to mutations, and efflux pumps. In this study, characterization of the possible mutations in OprD (OccAB1) porins from hospital strains of A. baumannii were investigated using single channel electrophysiology and compared with the standard OprD isolated from wild type ATCC 19,606. For this aim, 5 A. baumannii bacteria samples were obtained from patients infected with A. baumannii, after which OprD porins were isolated from these A. baumannii strains. OprD porins were then inserted in an artificial lipid bilayer and the current–voltage curves were obtained using electrical recordings through a pair of Ag/AgCl electrodes. We observed that each porin has a characteristic conductance and single channel recording, which then leads to differences in channel diameter. Finally, the single channel data have been compared with the gene sequences of each porin. It was interesting to find out that each porin isolated has a unique porin diameter and decreased anion selectivity compared to the wild type.

A recent investigation was aimed at obtaining structural information on a highly extended protein via SEC-MALS-SAXS. Significantly broadened elution peaks were observed, reminiscent of a phenomenon known as viscous fingering. This phenomenon is usually observed above 50 mg/mL for proteins like bovine serum albumin (BSA). Interestingly, the highly extended protein (Brpt5.5) showed viscous fingering at concentrations lower than 5 mg/mL. The current study explores this and other non-ideal behavior, emphasizing the presence of these effects at relatively low concentrations for extended proteins. BSA, Brpt5.5, and a truncated form of Brpt5.5 referred to as Brpt1.5 are studied systematically using size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (AUC), and viscosity. The viscous fingering effect is quantified using two approaches and is found to correlate well with the intrinsic viscosity of the proteins—Brpt5.5 exhibits the most severe effect and is the most extended protein tested in the study. By AUC, the hydrodynamic non-ideality was measured for each protein via global analysis of a concentration series. Compared to BSA, both Brpt1.5 and Brpt5.5 showed significant non-ideality that could be easily visualized at concentrations at or below 5 mg/mL and 1 mg/mL, respectively. A variety of relationships were examined for their ability to differentiate the proteins by shape using information from AUC and/or viscosity. Furthermore, these relationships were also tested in the context of hydrodynamic modeling. The importance of considering non-ideality when investigating the structure of extended macromolecules is discussed.

The recent surge of therapeutic interest in recombinant adeno-associated viral (AAV) vectors for targeted DNA delivery has brought analytical ultracentrifugation (AUC) into the spotlight. A major concern during formulation of AAV therapeutics is purity of the active species (DNA-containing capsid, or “filled capsids”). Insertion of DNA into AAV is not a highly efficient process; thus, a significant amount of empty and partial/intermediate AAV molecules may exist. Recent guidance from the FDA includes limiting the presence of empty AAV capsids and other impurities to reduce immunotoxicity. While chromatographic techniques (SEC, SEC–MALS, AEX) are often used for empty and full capsid quantitation due to the ease of accessibility and familiarity among most biochemists, the resolution and sensitivity attained by sedimentation velocity (SV-AUC) in the formulation buffer and purification buffers is unmatched. Approaches for using SV-AUC to determine the empty-to-full capsid ratio have already been discussed by others; however, in this report, we focus on the importance of characterizing other impurities, such as free DNA, partially filled capsids, and aggregates that are recognized as species of concern for immunotoxicity. We also demonstrate the usefulness of applying multiple analyses (e.g., c(s), g(s*), WDA) in confirming the presence of and determining the hydrodynamic parameters of these various species.