Exploring the uncharted territory of the potential protein-protein interactions of cytosolic malate dehydrogenase.

In this review, we examine the protein-protein interactions of cytosolic malate dehydrogenase (MDH), an under-studied area in cellular metabolism. We provide a comprehensive overview of MDH involvement in metabolism, especially its interactions with metabolic partners and dynamics of changing metabolism. We present an analysis of the biophysical nature of these interactions and the current methods used to study them.

Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase.

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) to both synthetic and protein crowders ranging from 10 to 550 kDa. The results reveal that the effects from macromolecular crowding depend on the direction of the reaction. The presence of the synthetic polymers, Ficoll and dextran, decrease V and K for ethanol oxidation.

Overlap Concentration and the Effect of Macromolecular Crowding on Citrate Synthase Activity.

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

Effects of Macromolecular Crowding on Alcohol Dehydrogenase Activity Are Substrate-Dependent.

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

Slowed Diffusion and Excluded Volume Both Contribute to the Effects of Macromolecular Crowding on Alcohol Dehydrogenase Steady-State Kinetics.

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 polymers alter enzyme kinetics by excluding volume that would otherwise be available to the enzymes, substrates, and products. Presented here is evidence that other factors, in addition to excluded volume, must be considered in the interpretation of crowding studies with synthetic polymers.

Macromolecular crowding and the steady-state kinetics of malate dehydrogenase.

To understand how macromolecular crowding affects enzyme activity, we quantified the Michaelis-Menten kinetics of mitochondrial malate dehydrogenase (MDH) in the presence of hen egg white (HEW), lysozyme, bovine serum albumin (BSA), gum arabic, poly(vinylpyrrolidone) (PVP), and dextrans of various molecular weights. Although crowding tended to decrease Km and Vmax values, the magnitude depended on the crowding agent, reaction direction, and isozyme (mitochondrial porcine heart or thermophlic TaqMDH from Thermus flavus). Crowding slowed oxaloacetate reduction more significantly than malate oxidation, which may suggest that mitochondrial enzymes have evolved to function optimally under the crowded constraints in which they are immersed.

Sirtuin-mediated nuclear differentiation and programmed degradation in Tetrahymena.

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 of sirtuin activity in development, aging, and disease; yet, underlying mechanistic pathways are not well understood. Specific sirtuins influence chromatin structure and gene expression, but differences in their pathways as they relate to distinct chromatin functions are just beginning to emerge.

Protein (19)F NMR in Escherichia coli.

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

19F NMR studies of alpha-synuclein conformation and fibrillation.

Fibrils of the intrinsically disordered protein alpha-synuclein are hallmarks of 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 the 19F-labeled amino acid, 3-fluorotyrosine, into recombinant human alpha-synuclein at its endogenous tyrosine residues.

Effects of recombinant protein expression on green fluorescent protein diffusion in Escherichia coli.

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

Quantifying green fluorescent protein diffusion in Escherichia coli by using continuous photobleaching with evanescent illumination.

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 for measuring translational mobility in structures only slightly larger than optical resolution. We report a new method using through-prism total internal reflection fluorescence microscopy with continuous photobleaching to overcome these obstacles.

Protein nuclear magnetic resonance under physiological conditions.

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 protein science well for more than a century, biochemists now have the tools to study proteins under these more physiologically relevant conditions.