Although overexpression and 15N enrichment facilitate the observation of resonances from disordered proteins in Escherichia coli, alone is insufficient for detecting most globular proteins. Here, we explain this dichotomy overcome problem while extending capability in-cell NMR by using 19F-labeled Resonances small (∼10 kDa) containing amino acid analogue 3-fluoro-tyrosine can be observed cells, but larger 19F are broadened beyond detection. Incorporating trifluoromethyl-l-phenylalanine...

Fibrils of the intrinsically disordered protein alpha-synuclein are hallmarks Parkinson's disease. The fluorescent dye thioflavin T is often used to characterize fibrillation, but this assay may not provide quantitative information about structure and mechanism. To gain such information, we incorporated 19F-labeled amino acid, 3-fluorotyrosine, into recombinant human at its endogenous tyrosine residues. Tyrosine 39 in positively charged N-terminal region 140-residue protein. other three...

Almost everything we know about protein biophysics comes from studies on purified proteins in dilute solution. Most proteins, however, operate inside cells where the concentration of macromolecules can be >300 mg/mL. Although reductionism-based approaches have served science well for more than a century, biochemists now tools to study under these physiologically relevant conditions. We review part this burgeoning postreductionist landscape by focusing high-resolution nuclear magnetic...

To understand how macromolecular crowding affects enzyme activity, we quantified the Michaelis-Menten kinetics of mitochondrial malate dehydrogenase (MDH) in presence hen egg white (HEW), lysozyme, bovine serum albumin (BSA), gum arabic, poly(vinylpyrrolidone) (PVP), and dextrans various molecular weights. Although tended to decrease Km Vmax values, magnitude depended on agent, reaction direction, isozyme (mitochondrial porcine heart or thermophlic TaqMDH from Thermus flavus). Crowding...

Enzymes operate in a densely packed cellular environment that rarely matches the dilute conditions under which they are studied. To better understand ramifications of this crowding, Michaelis–Menten kinetics yeast alcohol dehydrogenase (YADH) were monitored spectrophotometrically presence high concentrations dextran. Crowding decreased maximal rate reaction by 40% for assays with ethanol, primary substrate YADH. This observation was attributed to slowed release reduced β-nicotinamide adenine...

Living cells are crowded. The presence of so many macromolecules packed into a confined environment affects the structure and function biological components, kinetics thermodynamics biochemical reactions. In fact, crowding studies have already led to important insights, such as existence metabolons other temporary protein complexes that play essential roles in metabolism, cell signaling, organization intracellular environment. Even so, this topic is rarely addressed chemistry biochemistry...

Although it is well-known that the environment of mitochondria a densely packed network macromolecules, kinetics essential metabolic enzyme, citrate synthase, has been studied only under dilute conditions. To understand how this crowded impacts behavior Michaelis–Menten were measured spectrophotometrically in presence synthetic crowders as function size, concentration, and identity. The largest factor contributing to crowding effects was overlap concentration (c*), above which polymers begin...

Fluorescence recovery after photobleaching was used to measure the diffusion coefficient of green fluorescent protein (GFP, 27 kDa) in Escherichia coli presence or absence four coexpressed proteins: cytoplasmic maltose binding (42 kDa), tau-40 (45 alpha-synuclein (14 calmodulin (17 kDa). The GFP remains constant regardless type coexpresseed and whether not induced. We conclude that expression these soluble proteins has little no effect on GFP. These results have implications for utility...

To understand the consequences of macromolecular crowding, studies have largely employed in vitro experiments with synthetic polymers assumed to be both pure and "inert". These alter enzyme kinetics by excluding volume that would otherwise available enzymes, substrates, products. Presented here is evidence other factors, addition excluded volume, must considered interpretation crowding polymers. Dextran has a weaker effect on Michaelis-Menten kinetic parameters yeast alcohol dehydrogenase...

Fluorescence recovery after photobleaching and fluorescence correlation spectroscopy are the primary means for studying translational diffusion in biological systems. Both techniques, however, present numerous obstacles measuring mobility structures only slightly larger than optical resolution. We report a new method using through-prism total internal reflection microscopy with continuous to overcome these obstacles. Small structures, such as prokaryotic cells or isolated eukaryotic...

In order to better understand how the complex, densely packed, heterogeneous milieu of a cell influences enzyme kinetics, we exposed opposing reactions catalyzed by yeast alcohol dehydrogenase (YADH) both synthetic and protein crowders ranging from 10 550 kDa. The results reveal that effects macromolecular crowding depend on direction reaction. presence polymers, Ficoll dextran, decrease Vmax Km for ethanol oxidation. contrast, these have little effect or even increase kinetic parameters...

Abstract Background The NAD + -dependent histone deacetylases, known as "sirtuins", participate in a variety of processes critical for single- and multi-cellular life. Recent studies have elucidated the importance sirtuin activity development, aging, disease; yet, underlying mechanistic pathways are not well understood. Specific sirtuins influence chromatin structure gene expression, but differences their they relate to distinct functions just beginning emerge. To further define range global...

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to Protein Nuclear Magnetic Resonance under Physiological ConditionsGary J. Pielak*, Conggang Li, Andrew C. Miklos, Alexander P. Schlesinger, Kristin M. Slade, Gui-Fang Wang, and Imola G. ZigoneanuCite this: Biochemistry 2009, 48, 38, 9170Publication Date (Web):September 8, 2009Publication History Received21 August 2009Published online8 September inissue 29...

Abstract The measurement of pH is one the most basic and necessary skills in a life science laboratory. function physical characteristics biological molecules are highly sensitive to environment. Common buffers must be prepared with appropriate pH, usually close neutral for applications. This unit includes discussion different instrumentation, notably electrodes. It also relates theory critical parameters technique, covers correct use, handling, storage instruments. Curr. Protoc. Essential...

Most of our understanding enzyme kinetics is based on experiments conducted in dilute solution. However, these conditions do not accurately represent the crowded cellular environment, which contains high concentrations (300–400g/L) various macromolecules such as proteins, carbohydrates and ribosomes. This crowding reduces volume solvent available for other molecules solution has previously been shown to impact behavior enzymes. To study potential consequences, Michaelis‐Menten yeast alcohol...

Most of our understanding enzyme kinetics is based on experiments conducted in dilute solution, which does not accurately represent crowded cellular environments. Cells contain high concentrations (300-400g/L) various macromolecules such as proteins, carbohydrates and ribosomes. This crowding reduces the volume solvent available for other molecules solution has previously been shown to impact behavior enzymes. To study these potential consequences, Michaelis-Menten glutamate dehydrogenase...