Transformation of the Nonprocessive Fast Skeletal Myosin II into a Processive Motor

Myosin family motors play diverse cellular roles. Precise insights into how the light chains contribute to the functional variabilities among myosin motors, however, remain unresolved. Here, it is demonstrated that the fast skeletal muscle myosin II isoform myosin heavy chain (MHC‐IID) can be transformed into a processive motor, by simply replacing the native regulatory light chain MLC2f with the regulatory light chain variant MLC2v from the slow muscle myosin II. Single molecule kinetic analyses and optical trapping measurements of the hybrid motor reveal marked changes such as increased association rate of myosin toward adenosine triphosphate (ATP) and actin by more than twofold. The direct consequence of high adenosine diphosphate (ADP) affinity and increased actin rebinding is the altered overall actomyosin association time during the cross‐bridge cycle. The data indicate that the MLC2v influences the duty ratio in the hybrid motor, suggestive of promoting interhead communication and enabling processive movement. This finding establishes that the regulatory light chain fine‐tunes the motor's mechanical output that may have important implications under physiological conditions. Furthermore, the success of this approach paves the way to engineer motors from a known motor protein element to assemble highly specialized biohybrid machines for potential applications in nano‐biomedicine and engineering.     

Changes in supramolecular structure and improvement in reactivity of dissolving pulp via enzymatic pretreatment with processive endoglucanase EG1 from Volvaria volvacea

Abstract

Processive endoglucanase EG1 and its core domain, EG1(CD), were used to pretreat the commercial dissolving pulp to improve cellulose reactivity. The Fock reactivity of the pulp which was treated with EG1 and EG1 (CD) at 50?U/g enzyme loading increased from 74.3% of the control to 90.6% and 88.4%, respectively. Refining also improved the Fock reactivity of the pulp, but not as effective as EG1 or EG1(CD) treatment. Refining prior to EG1 or EG1(CD) treatment could slightly further improve the Fock reactivity, to 91.6% and 90.0%, respectively. After enzymatic treatment and (or) refining, the water retention value, differential scanning calorimetry and alkaline solubility analysis indicated that enzyme treatment, especially by EG1, significantly increased the accessibility of fibers to reaction reagents. Combined with the characteristics of soluble reducing sugar produced by EG1 treatment and the changes of degree of polymerization, it is inferred that a small fraction of cellulose crystallization regions are destroyed in the enzymatic hydrolysis process due to the processive acting ability of EG1, and some microchannels in the fiber cell wall were created, which is similar to the effect of “drilling holes”, so that the reaction reagent can reach the inside of the cell wall evenly, thus obviously improving the reactivity of the dissolved pulp.     

Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 β‐elimination contributes to the rate‐limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59–1.83 fold change in the rate of catalysis, thus suggesting that β‐elimination is partly rate‐limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing β‐elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods.     

Vitamin K-Dependent Protein Activation: Normal Gamma-Glutamyl Carboxylation and Disruption in Disease

Vitamin K-dependent (VKD) proteins undergo an unusual post-translational modification, which is the conversion of specific Glu residues to carboxylated Glu (Gla). Gla generation is required for the activation of VKD proteins, and occurs in the endoplasmic reticulum during their secretion to either the cell surface or from the cell. The gamma-glutamyl carboxylase produces Gla using reduced vitamin K, which becomes oxygenated to vitamin K epoxide. Reduced vitamin K is then regenerated by a vitamin K oxidoreductase (VKORC1), and this interconversion of oxygenated and reduced vitamin K is referred to as the vitamin K cycle. Many of the VKD proteins support hemostasis, which is suppressed during therapy with warfarin that inhibits VKORC1 activity. VKD proteins also impact a broad range of physiologies beyond hemostasis, which includes regulation of calcification, apoptosis, complement, growth control, signal transduction and angiogenesis. The review covers the roles of VKD proteins, how they become activated, and how disruption of carboxylation can lead to disease. VKD proteins contain clusters of Gla residues that form a calcium-binding module important for activity, and carboxylase processivity allows the generation of multiple Glas. The review discusses how impaired carboxylase processivity results in the pseudoxanthoma elasticum-like disease.     

A Novel Low Temperature PCR Assured High-Fidelity DNA Amplification

As previously reported, a novel low temperature (LoTemp) polymerase chain reaction (PCR) catalyzed by a moderately heat-resistant (MHR) DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94–96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV) type 52 (HPV-52) as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3′–5′ exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.