Spectroscopic Studies of Structure‐Function Relationships in Free and Immobilized Alcohol Dehydrogenase^a

Protein Conformational Dynamics upon Association with the Surfaces of Lipid Membranes and Engineered Nanoparticles: Insights from Electron Paramagnetic Resonance Spectroscopy

“…Based on this, it was discovered that although LADH is a homodimer, with each protomer having one tightly coordinated zinc atom, the protomers in some LADH dimers adopt distinct conformations. Furthermore, the immobilization did not affect the populations of these states for both surfaces [126]. However, after the CW EPR spectra were decomposed, it become apparent that the spectral components for soluble and immobilized enzymes were not identical, with greater spectral broadening observed in the case of immobilized proteins.…”

“…Clark and colleagues applied extensively CW EPR spectroscopy to study the effects of immobilization on the conformational dynamics of enzymes and enzyme active sites [90][91][92]126]. The technique was applied to horse liver alcohol dehydrogenase (LADH) to find the relationship between protein activity and its structure [126]. LADH, an oxoreductase homodimer, catalyzes the interconversions between aldehydes and alcohols and is active toward a broad spectrum of alcohols, aldehydes, and ketones.…”

“…Therefore, the efforts have been made to understand how to optimize its application to industrial biotechnology. The effect of immobilization was studied [126] for two surfaces, namely CNBr-Sepharose (via covalent attachment) and Octyl-Sepharose (by adsorption). It was found that the enzyme-specific activity has decreased significantly upon immobilization, as compared with the enzyme in a solution; and the decrease was greater in the case of Octyl-Sepharose.…”

Protein Adsorption and Conformational Changes

“…Indeed, the mode of association with lipid surfaces modulates αS aggregation in different ways [91,115,124,125]. Furthermore, it has been suggested that fibrillization in the presence of lipid molecules may result in the formation of protein-lipid co-aggregates [126][127][128] and that the morphology of fibrils is modulated by the relative proportion of protein and lipids [129].…”

Kinetics and Active Fraction Determination of a Protease Enzyme Immobilized on Functionalized Membranes: Mathematical Modeling and Experimental Results

“…The percentages of the two subpopulations in different samples of the immobilized enzyme were delineated, assuming that the enzyme existed in the same two states in all samples, only differing in relative amounts of the two populations. Since this EPR investigation, the technique has been used to study changes in conformation of different kinds of immobilized enzymes, including alcohol dehydrogenase, β‐galactosidase, subtilisin, papain, and glutamate dehydrogenase (Marg et al, 1986; Skerker and Clark, 1987; Clark et al, 1987, 1988; Telo et al, 1990; Zhuang and Butterfield, 1992; Butterfield et al, 1994; Ganapathi et al, 1995; Bhardwaj et al, 1996; O'Connor and Bailey, 1989; Viswanath et al, 1998). In most EPR reports of enzymes on supports, two subpopulations have been discerned, but just one population and as many as three subpopulations have also been observed (Zhuang and Butterfield, 1992; Song et al, 1992).…”