AIMS Biophysics最新文献

Although Mg²⁺ is the metal ion that functions as the cofactor for DNA polymerases (DNA pols) in vivo, Mn²⁺ can also serve in this capacity but it reduces base discrimination. Metal ions aside from Mg²⁺ or Mn²⁺ can act as cofactors for some DNA pols but not for others. Here we report on the ability of several divalent metal ions to substitute for Mg²⁺ or Mn²⁺ with BST DNA polymerase (BST pol), an A family DNA pol. We selected the metal ions based on whether they had previously been shown to be effective with other DNA pols. We found that Co²⁺ and Cd²⁺ were the only cations tested that could replace Mg²⁺ or Mn²⁺. When Co²⁺ was substituted for Mg²⁺, the incorporation efficiency for correct dNTPs increased 6-fold but for incorrect dNTPs there was a decrease which depended on the incoming dNTP. With Mn²⁺, base selectivity was impaired compared to Co²⁺ and Cd²⁺. In addition, Co²⁺ and Mn²⁺ helped BST pol to catalyze primer-extension past a mismatch. Finally both Co²⁺ and Mn²⁺ enhanced ground-state binding of both correct and incorrect dNTPs to BST pol: Dideoxy terminated primer-template complexes.

There is increasing interest in the use of DNA polymerases (DNA pols) in next-generation sequencing strategies. These methodologies typically rely on members of the A and B family of DNA polymerases that are classified as high-fidelity DNA polymerases. These enzymes possess the ability to selectively incorporate the correct nucleotide opposite a templating base with an error frequency of only 1 in 10⁶ insertion events. How they achieve this remarkable fidelity has been the subject of numerous investigations, yet the mechanism by which these enzymes achieve this level of accuracy remains elusive. Several smFRET assays were designed to monitor the conformational changes associated with the nucleotide selection mechanism(s) employed by DNA pols. smFRET has also been used to monitor the movement of DNA pols along a DNA substrate as well as to observe the formation of proof-reading complexes. One member among this class of enzymes, the large fragment of Bacillus stearothermophilus DNA polymerase I (Bst pol I LF), contains both 5'→3' polymerase and 3'→5' exonuclease domains, but reportedly lacks exonuclease activity. We have designed a smFRET assay showing that Bst pol I LF forms proofreading complexes. The formation of proofreading complexes at the single molecule level is strongly influenced by the presence of the 3' hydroxyl at the primer-terminus of the DNA substrate. Our assays also identify an additional state, observed in the presence of a mismatched primer-template terminus, that may be involved in the transfer of the primer-terminus from the polymerase to the exonuclease active site.

G protein-coupled receptors (GPCR) comprise a diverse superfamily of over 800 proteins that have gained relevance as biological targets for pharmaceutical drug design. Although these receptors have been investigated for decades, three-dimensional structures of GPCR have only recently become available. In this review, we focus on the technological advancements that have facilitated efforts to gain insights into GPCR structure. Progress in these efforts began with the initial crystal structure determination of rhodopsin (PDB: 1F88) in 2000 and has continued to the most recently published structure of the A_1AR (PDB: 5UEN) in 2017. Numerous experimental developments over the past two decades have opened the door for widespread GPCR structural characterization. These efforts have resulted in the determination of three-dimensional structures for over 40 individual GPCR family members. Herein we present a comprehensive list and comparative analysis of over 180 individual GPCR structures. This includes a summary of different GPCR functional states crystallized with agonists, dual agonists, partial agonists, inverse agonists, antagonists, and allosteric modulators.

A breakthrough has been discovered in pathology chemistry related to increasing molecular structure that can interfere with oxygen diffusion through cell membranes. Free radicals can crosslink unsaturated low-viscosity fatty acid oils by chain-growth polymerization into more viscous liquids and even solids. Free radicals are released by mitochondria in response to intermittent hypoxia that can increase membrane molecular organization to reduce fluidity and oxygen diffusion in a possible continuing vicious cycle toward pathological disease. Alternate computational chemistry demonstrates molecular bond dynamics in free energy for cell membrane physiologic movements. Paired electrons in oxygen and nitrogen atoms require that oxygen bonds rotate and nitrogen bonds invert to seek polar nano-environments and hide from nonpolar nano-environments thus creating fluctuating instability at a nonpolar membrane and polar biologic fluid interface. Subsequent mechanomolecular movements provide free energy to increase diffusion by membrane transport of molecules and oxygen into the cell, cell-membrane signaling/recognition/defense in addition to protein movements for enzyme mixing. In other chemistry calcium bonds to membrane phosphates primarily on the outer plasma cell membrane surface to influence the membrane firing threshold for excitability and better seal out water permeation. Because calcium is an excellent metal conductor and membrane phosphate headgroups form a semiconductor at the biologic fluid interface, excess electrons released by mitochondria may have more broad dissipation potential by safe conduction through calcium atomic-sized circuits on the outer membrane surface. Regarding medical conditions, free radicals are known to produce pathology especially in age-related disease in addition to aging. Because cancer cell membranes develop extreme polymorphism that has been extensively followed in research, accentuated easily-visualized free-radical models are developed. In terms of treatment, use of vitamin nutrient supplements purported to be antioxidants that remove free radicals has not proved worthwhile in clinical trials presumably due to errors with early antioxidant measurements based on inaccurate colorimetry tests. However, newer covalent-bond shrinkage tests now provide accurate measurements for free-radical inhibitor hydroquinone and other molecules toward drug therapy.

Photobiomodulation (PBM) also known as low-level level laser therapy is the use of red and near-infrared light to stimulate healing, relieve pain, and reduce inflammation. The primary chromophores have been identified as cytochrome c oxidase in mitochondria, and calcium ion channels (possibly mediated by light absorption by opsins). Secondary effects of photon absorption include increases in ATP, a brief burst of reactive oxygen species, an increase in nitric oxide, and modulation of calcium levels. Tertiary effects include activation of a wide range of transcription factors leading to improved cell survival, increased proliferation and migration, and new protein synthesis. There is a pronounced biphasic dose response whereby low levels of light have stimulating effects, while high levels of light have inhibitory effects. It has been found that PBM can produce ROS in normal cells, but when used in oxidatively stressed cells or in animal models of disease, ROS levels are lowered. PBM is able to up-regulate anti-oxidant defenses and reduce oxidative stress. It was shown that PBM can activate NF-kB in normal quiescent cells, however in activated inflammatory cells, inflammatory markers were decreased. One of the most reproducible effects of PBM is an overall reduction in inflammation, which is particularly important for disorders of the joints, traumatic injuries, lung disorders, and in the brain. PBM has been shown to reduce markers of M1 phenotype in activated macrophages. Many reports have shown reductions in reactive nitrogen species and prostaglandins in various animal models. PBM can reduce inflammation in the brain, abdominal fat, wounds, lungs, spinal cord.

To date, X-ray crystallography remains the gold standard for the determination of macromolecular structure and protein substrate interactions. However, the unpredictability of obtaining a protein crystal remains the limiting factor and continues to be the bottleneck in determining protein structures. A vast amount of research has been conducted in order to circumvent this issue with limited success. No single method has proven to guarantee the crystallization of all proteins. However, techniques using antibody fragments, lipids, carrier proteins, and even mutagenesis of crystal contacts have been implemented to increase the odds of obtaining a crystal with adequate diffraction. In addition, we review a new technique using the scaffolding ability of PDZ domains to facilitate nucleation and crystal lattice formation. Although in its infancy, such technology may be a valuable asset and another method in the crystallography toolbox to further the chances of crystallizing problematic proteins.