A convenient method for the relative and absolute quantification of lipid components in liposomes by 1H- and 31P NMR-spectroscopy.

OBJECTIVE: Liposomes are promising delivery systems for pharmaceutical applications and have been used in medicine in the recent past. Preparation of liposomes requires reliable characterization and quantification of the phospholipid components for which the traditional cumbersome molybdate method is used frequently. The objective was to improve relative and absolute quantification of lipid components from liposomes. METHODS: A reliable method for quantification of lipid composition in liposome formulations in the 1-10 µmol range with 1H- and 31P NMR spectroscopy at 600 MHz has been developed. The method is based on three crystalline small-molecule standards (Ph3PO4, (Tol)3PO4, and Ph3PO) in CDCl3. RESULTS: Excellent calibration linearity and chemical stability of the standards was observed. The method was tested in blind fashion on liposomes containing POPC, PEG-ceramide and a pH-sensitive trans-aminocyclohexanol-based amphiphile (TACH).1 Relative quantification (percentage of components) as well as determination of absolute lipid amount was possible with excellent reproducibility with an average error of 5%. Quantification (triplicate) was accomplished in 15 min based on 1H NMR and in 1 h based on 31P NMR. Very little change in mixture composition was observed over multiple preparative steps. CONCLUSION: Liposome preparations containing POPC, POPE, DOPC, DPPC, TACH, and PEG-ceramide can be reliably characterized and quantified by 1H NMR and 31P NMR spectroscopy at 600 MHz in the µmol range.

The development of tissue handling skills is sufficient and comparable after training in virtual reality or on a surgical robotic system: a prospective randomized trial.

BACKGROUND: Virtual reality is a frequently chosen method for learning the basics of robotic surgery. However, it is unclear whether tissue handling is adequately trained in VR training compared to training on a real robotic system. METHODS: In this randomized controlled trial, participants were split into two groups for "Fundamentals of Robotic Surgery (FRS)" training on either a DaVinci VR simulator (VR group) or a DaVinci robotic system (Robot group). All participants completed four tasks on the DaVinci robotic system before training (Baseline test), after proficiency in three FRS tasks (Midterm test), and after proficiency in all FRS tasks (Final test). Primary endpoints were forces applied across tests. RESULTS: This trial included 87 robotic novices, of which 43 and 44 participants received FRS training in VR group and Robot group, respectively. The Baseline test showed no significant differences in force application between the groups indicating a sufficient randomization. In the Midterm and Final test, the force application was not different between groups. Both groups displayed sufficient learning curves with significant improvement of force application. However, the Robot group needed significantly less repetitions in the three FRS tasks Ring tower (Robot: 2.48 vs. VR: 5.45; p < 0.001), Knot Tying (Robot: 5.34 vs. VR: 8.13; p = 0.006), and Vessel Energy Dissection (Robot: 2 vs. VR: 2.38; p = 0.001) until reaching proficiency. CONCLUSION: Robotic tissue handling skills improve significantly and comparably after both VR training and training on a real robotic system, but training on a VR simulator might be less efficient.

Controlling the interaction between CaMKII and Calmodulin with a photocrosslinking unnatural amino acid.

Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild-type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium-dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Furthermore, we were able to achieve activation of CaMKII L308BzF by UV-induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto-inhibitory role of the regulatory segment, inter-subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKIIL308BzF in vitro, and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light.

The effect of the bgs13 mutation on the structure of the reporter protein beta-lactoglobulin: Influence on folding and aggregation in Pichia pastoris.

Pichia pastoris, a methylotrophic yeast used for recombinant protein expression, has the capability of performing many eukaryotic post-translational modifications, growing to high cell densities, and producing proteins in a cost-effective manner. However, P. pastoris's secretion properties are not always efficient, and its secretory pathway mechanisms have not been thoroughly elucidated. A previously identified mutant strain, bgs13, was found to efficiently secrete most recombinant proteins tested, raising the possibility that this bgs13 mutant is a universal super secreter. In this study, we used a reporter protein, ß-lactoglobulin (b-LG), to perform structural analysis of the protein secreted from wild type and mutant bgs13 strains to investigate the secretory mechanism. Primary, secondary, and tertiary structures of b-LG were examined using Edman sequencing, circular dichroism, tryptophan fluorescence, and temperature induced aggregation analysis. Our results demonstrate that the bgs13 produced more b-LG than the wt strain and that this protein was functionally folded similar to the wt. Surprisingly, we also found that the bgs13 b-LG was more resistant to aggregation, providing another example of the superior qualities of this strain for enhanced secreted protein production.

CaMKII autophosphorylation can occur between holoenzymes without subunit exchange.

The dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naive holoenzymes, is broadly termed subunit exchange. In contrast to the expectations of previous work, we found little evidence for subunit exchange upon activation, and no effect of restraining subunits to their parent holoenzymes. Rather, mass photometry, crosslinking mass spectrometry, single molecule TIRF microscopy and biochemical assays identify inter-holoenzyme phosphorylation (IHP) as the mechanism for spreading phosphorylation. The transient, activity-dependent formation of groups of holoenzymes is well suited to the speed of neuronal activity. Our results place fundamental limits on the activation mechanism of this kinase.

Underrepresented Impurities in 4-Hydroxyphenylpyruvate Affect the Catalytic Activity of Multiple Enzymes.

Macrophage migration inhibitory factor (MIF) is a key immunostimulatory protein with regulatory properties in several disorders, including inflammation and cancer. All the reported inhibitors that target the biological activities of MIF have been discovered by testing against its keto/enol tautomerase activity. While the natural substrate is still unknown, model MIF substrates are used for kinetic experiments. The most extensively used model substrate is 4-hydroxyphenyl pyruvate (4-HPP), a naturally occurring intermediate of tyrosine metabolism. Here, we examine the impact of 4-HPP impurities in the precise and reproducible determination of MIF kinetic data. To provide unbiased evaluation, we utilized 4-HPP powders from five different manufacturers. Biochemical and biophysical analyses showed that the enzymatic activity of MIF is highly influenced by underrepresented impurities found in 4-HPP. Besides providing inconsistent turnover results, the 4-HPP impurities also influence the accurate calculation of ISO-1's inhibition constant, an MIF inhibitor that is broadly used for in vitro and in vivo studies. The macromolecular NMR data show that 4-HPP samples from different manufacturers result in differential chemical shift perturbations of amino acids in MIF's active site. Our MIF-based conclusions were independently evaluated and confirmed by 4-hydroxyphenylpyruvate dioxygenase (HPPD) and D-dopachrome tautomerase (D-DT); two additional enzymes that utilize 4-HPP as a substrate. Collectively, these results explain inconsistencies in previously reported inhibition values, highlight the effect of impurities on the accurate determination of kinetic parameters, and serve as a tool for designing error-free in vitro and in vivo experiments.

Branch point strength controls species-specific CAMK2B alternative splicing and regulates LTP.

Regulation and functionality of species-specific alternative splicing has remained enigmatic to the present date. Calcium/calmodulin-dependent protein kinase IIß (CaMKIIß) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific CAMK2B alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strengths during evolution globally renders constitutive exons alternative, thus providing novel mechanistic insight into cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9, we introduce a weaker, human branch point sequence into the mouse genome, resulting in strongly altered Camk2b splicing in the brains of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point-controlled CAMK2B alternative splicing with a fundamental function in learning and memory.

NMR analysis and molecular dynamics conformation of α-1,6-linear and α-1,3-branched isomaltose oligomers as mimetics of α-1,6-linked dextran.

The conformational preferences of several α-1,6-linear and α-1,3-branched isomalto-oligosaccharides were investigated by NMR and MD-simulations. Right-handed helical structure contributed to the solution geometry in isomaltotriose and isomaltotetraose with one nearly complete helix turn and stabilizing intramolecular hydrogen bonds in the latter by MD-simulation. Decreased helix contribution was observed in α-1,3-glucopyranosyl- and α-1,3-isomaltosyl-branched saccharide chains. Especially the latter modification was predicted to cause a more compact structure consistent with literature rheology measurements as well as with published dextranase-resistant α-1,3-branched oligosaccharides. The findings presented here are significant because they shed further light on the conformational preference of isomalto-oligosaccharides and provide possible help for the design of dextran-based drug delivery systems or for the targeted degradation of capsular polysaccharides by dextranases in multi-drug resistant bacteria.

A Conserved Kinase-Based Body-Temperature Sensor Globally Controls Alternative Splicing and Gene Expression.

Homeothermic organisms maintain their core body temperature in a narrow, tightly controlled range. Whether and how subtle circadian oscillations or disease-associated changes in core body temperature are sensed and integrated in gene expression programs remain elusive. Furthermore, a thermo-sensor capable of sensing the small temperature differentials leading to temperature-dependent sex determination (TSD) in poikilothermic reptiles has not been identified. Here, we show that the activity of CDC-like kinases (CLKs) is highly responsive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation segment. Lower body temperature activates CLKs resulting in strongly increased phosphorylation of SR proteins in vitro and in vivo. This globally controls temperature-dependent alternative splicing and gene expression, with wide implications in circadian, tissue-specific, and disease-associated settings. This temperature sensor is conserved across evolution and adapted to growth temperatures of diverse poikilotherms. The dynamic temperature range of reptilian CLK homologs suggests a role in TSD.

Role of BGS13 in the Secretory Mechanism of Pichia pastoris.

The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker's yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain's cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale.IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.

NMR solution geometry of saccharides containing the 6-O-(α-D-glucopyranosyl)-α/ß-D-glucopyranose (isomaltose) or 6-O-(α-D-galactopyranosyl)-α/ß-D-glucopyranose (melibiose) core.

The solution geometries of D-Glcp, Me-D-Glcp, 6-O-Me-D-Glcp, Me-6-O-Me-D-Glcp, D-Glcp-(α-1,6)-D-Glcp (isomaltose), D-Glcp-(α-1,6)-D-Glcp-(α-1,6)-D-Glcp (isomaltotriose), D-Galp-(α-1,6)-D-Glcp (melibiose), D-Galp-(α-1,6)-D-Glcp-(α-1,2)-D-Fruf (raffinose), and D-Galp-(α-1,6)-D-Galp-(α-1,6)-D-Glcp-(α-1,2)-D-Fruf (stachyose) in water are described by NMR spectroscopy, molecular dynamic simulations and quantum mechanical calculations. Overall, a change in anomeric configuration at the reducing end and/or anomeric substitution (methylation) changed the conformational space of the terminal CH2OH group significantly. Conformational analysis of the free monosaccharides matched literature results very well. Dihedral angle histograms weighted against published Karplus equations yielded excellent matches of experimental J-values in some cases but significant deviations in other. The anomeric hemiacetal configuration appeared to have a significant remote influence on the conformational space of the α-1,6-glycosidic linkage. Rigid glycosidic φ-conformations (g+) combined with mostly st-conformations for glycosidic ψ-angles from computations matched experimental nuclear Overhauser enhancements in all cases. While the investigated Glcp-α-1,6-Glcp linkages were nearly identical in φ/ψ-conformation, differences were apparent in the Galp-α-1,6-Galp linkage of stachyose. Of twenty-one crystal structures, a total of fourteen had ligand conformations corresponding to the most abundant or second-most abundant solution geometry determined in this study.

Synthesis and conformational analysis of d-gluco-pyranosyl-(6,6')-d-gluco-pyranuronate, a model compound for the inter-glycan 6,6'-ester linkage.

The synthesis of a 6,6'-ester linked disaccharide analog model compound was achieved in five steps from d-glucose and featured a key oxidative esterification transformation. The synthesized d-gluco-pyranosyl-(6,6')-d-gluco-pyranuronate was characterized in D2O using NMR spectroscopy. Using the experimental data together with molecular dynamics simulations (TIP3P, water), a model of the compound's conformational behavior was established. The effect of the 6,6'-ester linkage on the solution phase structure was compared to that of the previously reported 6,6'-ether linkage in a disaccharide analog. Based on the established models, the ester linkage was found to have a profound effect on the overall shape of the molecule.

Fliposomes: trans-2-aminocyclohexanol-based amphiphiles as pH-sensitive conformational switches of liposome membrane - a structure-activity relationship study.

Recently developed lipids with the trans-2-aminocyclohexanol (TACH) moiety represent unique pH-sensitive conformational switches ("flipids") that can trigger the membrane of liposome-based drug delivery systems at lowered pH as seen in many pathological scenarios. A library of flipids with various TACH-based headgroups and hydrocarbon tails were designed, prepared, and characterized to systematically elucidate the relationship between their chemical structures and their ability to form and to trigger liposomes. Liposomes (fliposomes) consisting of a flipid, POPC and PEG-ceramide were stable at 4°C, pH 7.4 for up to several months and yet released the encapsulated fluorophore in seconds upon acidification. The colloidal properties and encapsulation efficiencies of the fliposomes depended on the structure features of the flipids such as the polarity of the headgroups and the shape and fluidity of the lipid tails. The pH-triggered release also depended on the flipid structure, where shorter linear tails yielded more efficient release. The release of fliposomes was enhanced at different narrow pH ranges, depending on the basicity of the flipid headgroup, which can be estimated either by calculated pKa or by acid/base titration of the flipids while its conformation is monitored by 1H NMR. The structure-activity relationship of the flipids supports "lipid tail conformational shortening" as the mechanism to disrupt lipid membranes and would provide great flexibility in the design of pH-sensitive drug delivery systems.

Functional role of the three conserved cysteines in the N domain of visual arrestin-1.

Arrestins specifically bind active and phosphorylated forms of their cognate G protein-coupled receptors, blocking G protein coupling and often redirecting the signaling to alternative pathways. High-affinity receptor binding is accompanied by two major structural changes in arrestin: release of the C-tail and rotation of the two domains relative to each other. The first requires detachment of the arrestin C-tail from the body of the molecule, whereas the second requires disruption of the network of charge-charge interactions at the interdomain interface, termed the polar core. These events can be facilitated by mutations destabilizing the polar core or the anchoring of the C-tail that yield "preactivated" arrestins that bind phosphorylated and unphosphorylated receptors with high affinity. Here we explored the functional role in arrestin activation of the three native cysteines in the N domain, which are conserved in all arrestin subtypes. Using visual arrestin-1 and rhodopsin as a model, we found that substitution of these cysteines with serine, alanine, or valine virtually eliminates the effects of the activating polar core mutations on the binding to unphosphorylated rhodopsin while only slightly reducing the effects of the C-tail mutations. Thus, these three conserved cysteines play a role in the domain rotation but not in the C-tail release.

Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.

The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris.

trans-2-Aminocyclohexanol-based amphiphiles as highly efficient helper lipids for gene delivery by lipoplexes.

Lipidic amphiphiles equipped with the trans-2-aminocyclohexanol (TACH) moiety are promising pH-sensitive conformational switches ("flipids") that can trigger a lipid bilayer perturbation in response to increased acidity. Because pH-sensitivity was shown to improve the efficiency of several gene delivery systems, we expected that such flipids could significantly enhance the gene transfection by lipoplexes. Thus a series of novel lipids with various TACH-based head groups and hydrocarbon tails were designed, prepared and incorporated into lipoplexes that contain the cationic lipid 1,2-dioleoyl-3-trimethylammonio-propane (DOTAP) and plasmid DNA encoding a luciferase gene. B16F1 and HeLa cells were transfected with such lipoplexes in both serum-free and serum-containing media. The lipoplexes consisting of TACH-lipids exhibited up to two orders of magnitude better transfection efficiency and yet similar toxicity compared to the ones with the conventional helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol. Thus, the TACH-lipids can be used as novel helper lipids for efficient gene transfection with low cytotoxicity.

A biomimetic approach for enhancing the in vivo half-life of peptides.

The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of peptides without compromising their potency. Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo. We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.

Efficient Photochemical Synthesis of Peptide-α-Phenylthioesters.

Low yields and substantial epimerization of peptide-α-thioesters often compromise the overall efficiency of native chemical ligation (NCL). Peptide arylthioesters are more reactive than peptide alkylthioesters in NCL, but are also more difficult to handle due to their propensity to hydrolyze, and are therefore often generated in situ. However, pre-prepared peptide arylthioesters are required for some NCL applications. Here we present a 7-nitroindoline-based photochemical method that generates protected peptide phenylthioesters under neutral reaction conditions via their activated esters from photoreactive peptide precursors in high isolated yields, and with low levels of epimerization. This method is fully compatible with Fmoc-strategy solid-phase peptide synthesis. Global deprotection with trifluoroacetic acid furnishes peptide phenylthioesters for NCL. Photoreactive peptide precursors can also be converted into their hydrazides in two steps by this method.

Fliposomes: pH-triggered conformational flip of new trans-2-aminocyclohexanol-based amphiphiles causes instant cargo release in liposomes.

A new type of pH-sensitive liposomes (fliposomes) was designed based on the amphiphiles that are able to perform a pH-triggered conformational flip (flipids). This flip disrupts the liposome membrane and causes rapid release of the liposome cargo, specifically in response to lowered pH. The flipids (1) and (2) are equipped with a trans-2-aminocyclohexanol conformational switch. pH-sensitive fliposomes containing one or both of these flipids, as well as POPC and PEG ceramide, were constructed and characterized. These compositions were stable at 4°C and pH 7.4 for several months. Fliposomes loaded with ANTS/DPX performed an unusually quick content release within a few seconds at pH below 8.5 (in case of 2) and 6.0 (in case of 1). This difference in pH sensitivity demonstrates a potential for the custom design of flipids by variation of the amino group to target areas with specific pH values. The pH titration curves for the fliposome leakage parallel the curves for the acid-induced conformational flip of 1 and 2 studied by ¹H NMR. A plausible mechanism of pH sensitivity starts with an acid-triggered conformational flip of 1 or 2, which changes the molecular size and shape, shortens the lipid tails, and perturbs the liposome membrane, resulting in the content leakage.

Fliposomes: pH-controlled release from liposomes containing new trans-2-morpholinocyclohexanol-based amphiphiles that perform a conformational flip and trigger an instant cargo release upon acidification.

A new type of pH-sensitive liposome (fliposomes) was designed based on the amphiphiles that are able to perform a pH-triggered conformational flip (flipids). This flip disrupts the liposome membrane and causes rapid release of the liposome cargo, specifically in the areas of increased acidity. The flipids (1-3) are equipped with a trans-2-morpholinocyclohexanol conformational switch, pH-Sensitive fliposomes containing one of these flipids, POPC and PEG-ceramide (molar ratio 50/45/5) were constructed and characterized. These compositions were stable at 4 degrees C and pH 7.4 for several months. Fliposomes loaded with ANTS/DPX demonstrated an unusually quick content release (in a few seconds) at pH below 5.5, which was more efficient in the case of flipid 1 with the shorter linear C12-tails. The pH-titration curve for the fliposome leakage paralleled the curve for the acid-induced conformational flip of 1-3 studied by 1H NMR. A plausible mechanism of the pH-sensitivity starts with an acid-triggered conformational flip of 1, 2 or 3, which changes the molecular size and shape, shortens the lipid tails, and perturbs the liposome membrane resulting in the content leakage.