Resolution of Cys and Lys labeling of alpha-crystallin with site-sensitive fluorescent 3-hydroxyflavone dye

Ratiometric fluorescent probes based on 3-hydroxyflavone (3HF) are highly sensitive tools for studying polarity, hydration, electronic polarizability, and electrostatics in different microheterogeneous systems, including protein molecules. In the present work, a reactive derivative of 3HF, 6-bromomethyl-4'-diethylamino-3-hydroxyflavone, recently synthesized in our group, was applied to label covalently bovine lens alpha-crystallin. The labeling of SH and NH(2) groups are clearly distinguished by spectroscopic criteria. We observe that the NH(2) labeling creates the positive charge in the proximity to fluorophore, which results in strong internal Stark effect producing the shift in excitation spectrum by ca. 15 nm. Analysis of excitation-dependent fluorescence spectra allows separation of the emission profiles of these SH- and NH(2)-labeled species. Applying recently developed multiparametric analysis of the obtained emission spectra, we described the physicochemical properties of the sites of SH and NH(2) labeling in alpha-crystallin. The site of SH labeling has medium-low polarity (dielectric constant, epsilon = 4.9 +/- 0.9) is protic, and does not contain proximal aromatic residues (according to the obtained refractive index, n = 1.41 +/- 0.14). The site of NH(2) labeling is also of medium-low polarity. The novel label due to its two-wavelength ratiometric response and high sensitivity to the type of labeling may offer new possibilities in the studies of structure, dynamics, and interactions of proteins by probing their SH- and NH(2)-labeling sites.     

Hsc70 Rapidly Engages Tau after Microtubule Destabilization

The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7–9 interact with residues 312–342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.     

Entropy capacity determines protein folding

Search and study of the general principles that govern kinetics and thermodynamics of protein folding generate a new insight into the factors controlling this process. Here, based on the known experimental data and using theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue-that is, an entropy capacity-for fast protein folding. Statistical analysis of conformational entropy and number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, alpha/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts (class alpha/beta has the largest number of contacts) and average conformational entropy per residue (class all-alpha has the largest number of rotatable angles phi, psi, and chi per residue). These class-specific features determine the folding rates: alpha proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones. Our result is in agreement with the experimental folding rates for 60 proteins. This suggests that structural and sequence properties are important determinants of protein folding rates.     

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses.     

One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains

Amino acid selective isotope labeling is a useful approach to simplification of nuclear magnetic resonance (NMR) spectra of large proteins. Cell-free protein synthesis offers essentially unlimited flexibility of labeling patterns but is labor-intensive and expensive. In vivo labeling is simple in principle but generally requires auxotrophic strains, inhibitors of amino acid synthesis, or complex media formulations. We describe a simple procedure for amino acid selective labeling of proteins expressed in prototrophic Escherichia coli strains. Excellent labeling selectivity was achieved for histidine, lysine, methionine, and alanine. Simplicity and robustness of this protocol make it a useful tool for protein NMR.     

Predicting Calendar Aging in Lithium Metal Secondary Batteries: The Impacts of Solid Electrolyte Interphase Composition and Stability

Calendar aging of lithium metal batteries, in which cells' components degrade internally due to chemical reactions while no current is being applied, is a relatively unstudied field. In this work, a model to predict calendar aging of lithium metal cells is developed using two sets of readily obtainable data: solid electrolyte interphase (SEI) layer composition (measured via X‐ray photoelectron spectroscopy) and SEI stability (measured as a degradation rate using a simple constant current–constant voltage charging protocol). Electrolyte properties such as volume and salt concentration are varied in order to determine their effect on SEI stability and composition, with subsequent impacts to calendar aging. Lower salt concentrations produce a solvent‐based, more soluble SEI, while the highest concentration produces a salt‐based, less soluble SEI. Higher electrolyte volumes promote dissolution of the SEI and thus decrease its stability. The model predicts that lithium metal would be the limiting factor in calendar aging, depleting long before the electrolyte does. Additionally, the relative composition of the electrolyte during aging is modeled and found to eventually converge to the same value independent of initial salt concentration.     

Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency: A ROLE FOR GLOBAL CONFORMATIONAL CHANGES*

The serine hydrolase monoacylglycerol lipase (MGL) functions as the main metabolizing enzyme of 2-arachidonoyl glycerol, an endocannabinoid signaling lipid whose elevation through genetic or pharmacological MGL ablation exerts therapeutic effects in various preclinical disease models. To inform structure-based MGL inhibitor design, we report the direct NMR detection of a reversible equilibrium between active and inactive states of human MGL (hMGL) that is slow on the NMR time scale and can be modulated in a controlled manner by pH, temperature, and select point mutations. Kinetic measurements revealed that hMGL substrate turnover is rate-limited across this equilibrium. We identify a network of aromatic interactions and hydrogen bonds that regulates hMGL active-inactive state interconversion. The data highlight specific inter-residue interactions within hMGL modulating the enzymes function and implicate transitions between active (open) and inactive (closed) states of the hMGL lid domain in controlling substrate access to the enzymes active site.     

Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells

Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.