A case for glycerol as an acceptable additive for single-particle cryoEM samples

Buffer-composition and sample-preparation guidelines for cryo-electron microscopy are geared towards maximizing imaging contrast and reducing electron-beam-induced motion. These pursuits often involve the minimization or the complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, it is shown that the inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by an ∼2.3 Å resolution reconstruction of mouse apoferritin (∼500 kDa) and an ∼3.3 Å resolution reconstruction of rabbit muscle aldolase (∼160 kDa) in the presence of 20%(v/v) glycerol. While it was found that generating thin ice that is amenable to high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion or an inability to pick particles. Overall, these findings indicate that glycerol should not be discounted as a cryoEM sample-buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.

Experimental platform for the functional investigation of membrane proteins in giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) are micrometer-sized model membrane systems that can be viewed directly under the microscope. They serve as scaffolds for the bottom-up creation of synthetic cells, targeted drug delivery and have been used in many in vitro studies of membrane related phenomena. GUVs are also of interest for the functional investigation of membrane proteins that carry out many key cellular functions. A major hurdle to a wider application of GUVs in this field is the diversity of existing protocols that are optimized for individual proteins. Here, we compare PVA assisted and electroformation techniques for GUV formation under physiologically relevant conditions, and analyze the effect of immobilization on vesicle structure and membrane tightness towards small substrates and protons. There, differences in terms of yield, size, and leakage of GUVs produced by PVA assisted swelling and electroformation were found, dependent on salt and buffer composition. Using fusion of oppositely charged membranes to reconstitute a model membrane protein, we find that empty vesicles and proteoliposomes show similar fusion behavior, which allows for a rapid estimation of protein incorporation using fluorescent lipids.

Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse ge-netic disorders. However, for gene therapy applications an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. Once there, the ribonucleoprotein complex (RNP) can be transported into the nucleus. Lipid nanoparticles (LNP) serve as promising candidates for delivery of CRISPR-Cas9 RNP. These delivery vehicles have been optimized for the delivery of nucleic acids, such as mRNA. Co-delivery of Cas9 encoding mRNA with the accompanying sgRNA leads to translation of the Cas9 protein and formation of the Cas9 RNP inside the cell. Only recently, direct delivery of the CRISPR-Cas9 RNP complexes has been explored, which requires adjustments to the LNP formulation. In this study, the importance of buffer composition and cationic charge during RNP and ssDNA en-trapment in LNP are demonstrated. After optimizing several formulation parameters, LNP were prepared that were colloidally stable in human plasma and efficiently deliver the SpCas9 RNP and ssDNA for HDR-correction in reporter cells. Under optimal formulation conditions, gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively were achieved at nanomolar CRISPR-Cas9 RNP concentrations.

Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays—Protein and Buffer Composition Contribution

A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.

Buffer composition affects rose bengal dialysis rate through cellulose membrane

Due to its fluorescent and phototoxic properties, rose bengal (RB) is used in photodynamic therapy. To improve the delivery of RB to its site of action, the application of nanocarrier systems has been proposed. The most promising approach includes the use of pH-responsive nanoparticles. To evaluate the pattern of drug release in different buffers, equilibrium dialysis is commonly used. Here, we used water and two buffers to determine the impact of solvent composition on the aggregation and dialysis rate of RB through a cellulose membrane. The results show that buffer composition does not influence the fluorescent properties of RB. However, the presence of additional ions causes a change in diffusion rate that is most probably linked to the size of RB aggregates.

Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin

Due to fast relaxation processes of transition metal ions, electron paramagnetic resonance (EPR) spectroscopy of metalloproteins needs to be performed at cryogenic temperatures. To avoid damaging the biological system upon freezing, a cryoprotectant is generally added to the sample as a glassing agent. Even though cryoprotectants are expected to be inert substances, evidences in literature show their non-innocent role in altering the shape of EPR spectra of proteins and biological objects in general. In this work we conduct a systematic study on the impact of several experimental factors—such as buffer composition, choice of cryoprotectant, pH and temperature—on the EPR spectrum of myoglobin, taken as a reference system for being a well-characterized heme-containing protein. We focus on high-pH buffers to induce and investigate the alkaline transition of ferric myoglobin (pKa ~ 8.9). A combined approach of continuous-wave EPR and UV–visible absorption spectroscopy shows that using particular pairs of buffers and cryoprotectants determines a considerable pH variation in the sample and that this effect is enhanced at cryogenic temperature. In addition, phase memory times were measured to evaluate the efficiency of different cryoprotectants and compared with spectral linewidths in continuous-wave EPR. Our findings suggest that among the selected cryoprotectants ethylene glycol is rather effective, even more than the widely used glycerol, without having unwanted effects.

A case for glycerol as an acceptable additive to cryoEM samples

Buffer composition and sample preparation guidelines for cryo-electron microscopy are geared toward maximizing imaging contrast and reducing electron beam-induced motion. These pursuits often involve the minimization or complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, we show that inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by a ~2.3 Å reconstruction of mouse apoferritin (~500 kDa) and a ~3.3 Å reconstruction of rabbit muscle aldolase (~160 kDa) in presence of 20% v/v glycerol. While we found that generating thin ice that is amenable for high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion nor an inability to pick particles. Overall, our findings indicate glycerol should not be discounted as a cryoEM sample buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.

Detection of the Benthic Dinoflagellates, Ostreopsis cf. ovata and Amphidinium massartii (Dinophyceae), Using Loop-Mediated Isothermal Amplification

For the in situ and sensitive detection of benthic dinoflagellates, we have established an integrated loop-mediated isothermal amplification (LAMP) assay based on Ostreopsis cf. ovata and Amphidinium massartii. To detect the two species, a set of species-specific primers was constructed between the ITS gene and D1–D6 LSU gene, and the reaction temperature, time, and buffer composition were optimized to establish this method. In addition, the specificity of the LAMP primers was verified both in strains established in the laboratory and in field samples collected from the Jeju coastal waters, Korea. With the LAMP assay, the analysing time was within 45 to 60 min, which may be shorter than that with the conventional PCR. The detection sensitivity of the LAMP assay for O. cf. ovata or A. massartii was comparable to other molecular assays (PCR and quantitative PCR (qPCR)) and microscopy examination. The detection limit of LAMP was 0.1 cell of O. cf. ovata and 1 cell of A. massartii. The optimized LAMP assay was successfully applied to detect O. cf. ovata and A. massartii in field samples. Thus, this study provides an effective method for detecting target benthic dinoflagellate species, and could be further implemented to monitor phytoplankton in field surveys as an altenative.

Purification of polyclonal Immunoglobulin G from human serum using LigaGuardTM and LigaTrapTM adsorbents

This study presents the chromatographic purification of immunoglobulin G (IgG) from human plasma using a two-column process integrating the peptide-based adsorbents LigaGuardTM, which captures non-Ig plasma proteins in flow-through mode, and LigaTrapTM, which isolates IgG in bind-and-elute. Buffer composition and column loading were optimized for both adsorbents. Two process configurations were evaluated. In the first design, plasma was fed to a LigaGuardTM column to capture plasma proteins, the effluent was loaded on the LigaTrapTM column, and the bound IgG was eluted with 63.8% global recovery and 99.7% purity; in comparison, Protein G agarose afforded ~67% recovery and 97.2% purity. In the alternative design, the LigaGuardTM column was utilized to polish the LigaTrapTM elution stream, affording 82.3% global recovery and 98.8% purity. Collectively, these results demonstrate the potential of a fully chromatographic process for purifying polyclonal IgG from plasma feedstocks.

Exploring the in Vitro Operating Window of Glycosyltransferase PtUGT1 from Polygonum Tinctorium for a Biocatalytic Route to Indigo Dye

The eobiotic compound indican lends itself to a compelling biocatalytic dyeing strategy for denim, in which the formation of corrosive by-products is avoided. However, the efficient and scalable production of indican remains a key bottleneck. This work focuses on the in vitro characterization of PtUGT1, a glycosyltransferase from Polygonum tinctorium that catalyzes the formation of indican via the glycosylation of indoxyl. Here, the buffer composition and enzyme concentration were identified as key parameters for enzyme activity and stability. The short lifetime of the enzyme under reaction conditions initiated an immobilization study. As a consequence, an amino-functionalized methacrylate resin was identified as a highly functional option for efficient immobilization of PtUGT1, allowing immobilization yields of > 98% for enzyme loadings up to 7.6 w-%. We further report a stabilization factor of 47 and significantly improved overall biocatalytic productivity. The straightforward handling and reuse of the described heterogeneous biocatalyst is demonstrated.