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.

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.

Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

In vivo human gracilis whole muscle passive stress-sarcomere strain relationship

We measured the passive mechanical properties of intact, living human gracilis muscles (n=11 individuals, 1 female, age: 33±12years, mass: 89±23kg, height: 177±8cm). Measurements were performed in patients undergoing surgery for free functioning myocutaneous tissue transfer of the gracilis muscle to restore elbow flexion after brachial plexus injury. Whole muscle force of the gracilis tendon was measured in four joint configurations (JC1-JC4) with a buckle force transducer placed at the distal tendon. Sarcomere length was also measured by biopsy from the proximal gracilis muscle. After the muscle was removed a three-dimensional volumetric reconstruction of the muscle was created via photogrammetry. Muscle length from JC1 to JC4 increased by 3.3±1.0 cm, 7.7±1.2 cm, 10.5±1.3 cm and 13.4±1.2 cm respectively, corresponding to 15%, 34%, 46% and 59% muscle fiber strain respectively. Muscle volume and an average optimal fiber length of 23.1±0.7 cm yielded an average muscle physiological cross-sectional area of 6.8±0.7 cm2 which is approximately three times that measured previously from cadaveric specimens. Absolute passive tension increased from 0.90±0.21 N in JC1 to 16.50±2.64 N in JC4. As expected, sarcomere length also increased from 3.24±0.08 µm at JC1 to 3.63±0.07 µm at JC4, which are on the descending limb of the human sarcomere length-tension curve. Peak passive muscle stress was 27.8±5.5 kPa in JC4 and muscle modulus ranged from 44.8 MPa in JC1 to 125.7 MPa in JC4. Compared to other mammalian species, human muscle passive mechanical properties are more similar to rodent muscle than rabbit muscle. These data provide direct measurements of whole human muscle passive mechanical properties that can be used in modeling studies and for understanding comparative passive mechanical properties among mammalian muscles.

Discovery and evaluation of novel benzazepinone derivatives as glycogen phosphorylase inhibitors with potent activity

Glycogen phosphorylase (GP) is a key enzyme of glycogen catabolism, so it is significant to discover a new GP inhibitor. A series of benzazepinone derivatives were discovered as GP inhibitors with potent activity. Among these derivatives, compound 5d showed significant potential against rabbit muscle GPa (IC50 = 0.25 ± 0.05 μM) and cellular efficacy. The in vivo study revealed that 5d significantly inhibited increases in fasting blood glucose level in two kinds of hyperglycemic mice models. The possible binding mode of compound 5d was explored based on molecular docking simulations. These results indicated that derivatives with benzazepinone were potential chemical entities against hyperglycemia.

Stability and in vivo safety of gold, titanium nitride and parylene C coatings on NdFeB magnets implanted in muscles towards a new generation of myokinetic prosthetic limbs

NdFeB magnets implantation in muscles could enable limb prostheses control by means of a myokinetic interface. Parylene C proved as optimal coating for corrosion resistance, in vitro biocompatibility and safe implantability in rabbit muscle.

Mechanochemical Synthesis and reactivity of 1,2,3-Triazole Carbohydrate Derivatives as Glycogen Phosphorylase Inhibitors

Aims: In this aim, we have developed this work to recommend an original route for the preparation of triazole derivatives. Background: Carbohydrates containing 1,2,3-triazole derivatives have various biological activities. Due to their advantageous and biological property, they are eye-catching synthetic targets in the arsenal of organic chemistry. Thus, finding green and efficient methods, as well as using ball millig procedure for the synthesis of these heterocycles is of interest to organic chemistry researchers. Objective: The objective of this study was to synthesize carbohydrate-derived triazoles under high-speed vibration milling conditions and investigate their properties. Materials and Method: A mixture of glycoside azide derivatives (1 mmol) and prop-2-yn-1-ol (1.5 mmol) in the presence of copper (I) was vigorously shaken under vibration milling conditions at 650 rpm with three balls for 15 min. The deprotection of the resulting triazole derivatives was effected by treatment with 4M hydrochloric acid in methanol under reflux. Results and Discussion: A short and convenient route to synthesize carbohydrate-derived triazoles, based in a ball-mill via 1,3-dipolar cycloaddition reactions to prop-2-yn-1-ol was developed. Cleavage of the isopropylidene protecting group provided water-soluble triazoles, evaluated as glycogen phosphorylase inhibitors. 1-[6-(4-Hydroxymethyl-[1,2,3]triazol-1-yl)- 2,2-dimethyl-tetrahydro-furo[3,4-d][1,3]dioxol-4-yl]-ethane-1,2-diol was the best inhibitor of rabbit muscle glycogen phosphorylase b (IC50 = 60 μM). Conclusion: In summary, we developed new, short and convenient routes to glucose-derived 1,2,3-triazole based on 1,3- dipolar cycloaddition reactions flowed by ball milling. Use of isopropylidene protective groups gave access to the analogous deprotected water-soluble motifs, analogous to known inhibitors of glycogen phosphorylase.

Synthesis, Kinetic and Conformational Studies of 2-Substituted-5-(β-d-glucopyranosyl)-pyrimidin-4-ones as Potential Inhibitors of Glycogen Phosphorylase

Dysregulation of glycogen phosphorylase, an enzyme involved in glucose homeostasis, may lead to a number of pathological states such as type 2 diabetes and cancer, making it an important molecular target for the development of new forms of pharmaceutical intervention. Based on our previous work on the design and synthesis of 4-arylamino-1-(β-d-glucopyranosyl)pyrimidin-2-ones, which inhibit the activity of glycogen phosphorylase by binding at its catalytic site, we report herein a general synthesis of 2-substituted-5-(β-d-glucopyranosyl)pyrimidin-4-ones, a related class of metabolically stable, C-glucosyl-based, analogues. The synthetic development consists of a metallated heterocycle, produced from 5-bromo-2-methylthiouracil, in addition to protected d-gluconolactone, followed by organosilane reduction. The methylthio handle allowed derivatization through hydrolysis, ammonolysis and arylamine substitution, and the new compounds were found to be potent (μM) inhibitors of rabbit muscle glycogen phosphorylase. The results were interpreted with the help of density functional theory calculations and conformational analysis and were compared with previous findings.