Characterisation of the dominant oxidative folding intermediate of hen lysozyme 1 1Edited by P. E. Wright

Berg, Bert van den; Chung, Evonne W.; Robinson, Carol V.; Dobson, Christopher M.

doi:10.1006/jmbi.1999.2915

Cited by 80 publications

(99 citation statements)

References 37 publications

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“…However, this phase diagram method can detect an intermediate for the oxidative refolding of reduced, denatured lysozyme because this oxidative refolding is much slower (ϳ4 -24 h) than the refolding of lysozyme with four disulfides intact. An intermediate with highly native-like structure in the ␣-and ␤-domains of the enzyme while lacking the Cys 76 -Cys 94 disulfide bond was isolated during the oxidative refolding of reduced, denatured lysozyme (30). As shown in a recent study (42), an intrinsically disordered protein FlgM gains structure in living Escherichia coli cells and under physiologically relevant conditions in vitro, which suggests a reason for the observation that some proteins are only folded under physiologically relevant conditions and proves the biological relevance of studying proteins in vivo and at physiologically relevant solute concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…Preparing Reduced, Denatured Lysozyme-Reduced, denatured lysozyme was generated as described (30,31). Lysozyme (20 mg/ml) was dissolved in 100 mM Tris-HCl buffer (pH 8.5) containing 8 M urea, 100 mM NaCl, and 1 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

“…Oxidative refolding of the reduced, denatured protein was initiated by a rapid 90 -110-fold dilution of the concentrated solution into the refolding buffer, effected by vigorous agitation with a Vortex mixer for 3-5 s, giving reduced, denatured lysozyme a final concentration of 5 M. The refolding buffer (pH 8.5), optimized to give the highest refolding yield (30), consisted of 100 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 2 M urea (30), reduced/oxidized glutathione, and different concentrations of mixed or single crowding agents. Stock solutions of reduced/oxidized glutathione in 100 mM Tris-HCl at pH 8.5 were made immediately prior to use, and the final concentrations of reduced and oxidized glutathione in the refolding experiment were 1.33 and 0.27 mM, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Lysozyme is a small single chain protein which has been studied in detail in vitro both under conditions where the native disulfide bonds are maintained throughout the refolding reaction and under oxidative conditions (25)(26)(27)(28)(29)(30). In contrast to the oxidized lysozyme, the reduced, denatured lysozyme is susceptible to aggregation (31).…”

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Mixed Macromolecular Crowding Accelerates the Oxidative Refolding of Reduced, Denatured Lysozyme

Zhou

Liang

et al. 2004

Journal of Biological Chemistry

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The oxidative refolding of reduced, denatured hen egg white lysozyme in the presence of a mixed macromolecular crowding agent containing both bovine serum albumin (BSA) and polysaccharide has been studied from a physiological point of view. When the total concentration of the mixed crowding agent is 100 g/liter, in which the weight ratio of BSA to dextran 70 is 1:9, the refolding yield of lysozyme after refolding for 4 h under this condition increases 24% compared with that in the presence of BSA and 16% compared with dextran 70. A remarkable increase in the refolding yield of lysozyme by a mixed crowding agent containing BSA and Ficoll 70 is also observed. Further folding kinetics analyses show that these two mixed crowding agents accelerate the oxidative refolding of lysozyme remarkably, compared with single crowding agents. These results suggest that the stabilization effects of mixed macromolecular crowding agents are stronger than those of single polysaccharide crowding agents such as dextran 70 and Ficoll 70, whereas the excluded volume effects of mixed macromolecular crowding agents are weaker than those of single protein crowding agents such as BSA. Both the refolding yield and the rate of the oxidative refolding of lysozyme in these two mixed crowded solutions with suitable weight ratios are higher than those in single crowded solutions, indicating that mixed macromolecular crowding agents are more favorable to lysozyme folding and can be used to simulate the intracellular environments more accurately than single crowding agents do.The main question that protein folding encountered in vivo is that the intracellular environment is highly crowded because of the presence of high concentrations of soluble and insoluble macromolecules, which include proteins, nucleic acids, ribosomes, and carbohydrates (polysaccharides) (1-3). It has been estimated that the concentration of macromolecules in cytoplasm is in the range of 80 -400 g/liter (3-5), and all the macromolecules in physiological fluid media collectively occupy a lower limit of ϳ10% and a upper limit of ϳ40% of total fluid volume (1, 2, 6). These media are termed "crowded" or "volumeoccupied" rather than "concentrated" (7,8). The biophysical theory of macromolecular crowding has been well developed by a thermodynamic approach (2, 8 -10). The effect of excluded volume upon protein stability and conformation has been discussed using a simplified statistical thermodynamic model (11). One pertinent prediction of this model is that intramolecular excluded volume will increase the chemical potential of the unfolded state relative to that of the native or compact nonnative state, resulting in a relative stabilization of the native or compact non-native state relative to a fully unfolded state (12).Most of our knowledge about protein folding comes from biophysical studies of the refolding of pure denatured proteins at low concentrations in simple defined buffers carried out in the past three decades since the pioneering studies of Anfinsen (13). However, intra...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mixed Macromolecular Crowding Accelerates the Oxidative Refolding of Reduced, Denatured Lysozyme

Zhou

Liang

et al. 2004

Journal of Biological Chemistry

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“…Epidermal growth factor (EGF) (13) forms both non-native three-disulfide isomers as well as a predominant species with two native disulfides (EGF-II). There are other proteins with predominant oxidative folding interme-diates that are only partially oxidized and contain only native disulfide bonds, for example hen egg white lysozyme (19), trypsin-specific squash inhibitor from Ecballium elaterium (EETI-II) (20), the cyclotide kalata B1 (21), and insulin-like growth factor-I (22). In contrast to the formation and reshuffling of disulfides commonly studied using acid trapping and RP-HPLC, the change in conformation at the level of amino acid residues, along the folding pathway, is poorly understood.…”

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confidence: 99%

Oxidative Folding of Amaranthus α-Amylase Inhibitor

Cemazar

Zahariev

Pongor

et al. 2004

Journal of Biological Chemistry

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Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus ␣-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process.Cracking the code for folding a string of amino acid residues into a biologically active three-dimensional structure is still a major challenge of both chemical and biological interest. The information that determines the folding of a protein is contained solely in its amino acid residues (1). In proteins that contain cysteine residues the formation of disulfide bonds is an additional degree of freedom in the folding process. In these proteins a fusion between the recovery of the native tertiary structure (conformational folding) and the regeneration of native disulfide bonds is coupled in the oxidative folding process (2).Disulfide formation is important for in vivo folding of a considerable number of eukaryotic proteins and is a key posttranslational modification for the stabilization of folded structures. The study of oxidative folding in vitro can provide a detailed structural description in terms of the disulfide intermediate species present along the pathway of the complex folding process. A thorough account of an oxidative folding reaction should combine a study of the formation of covalently stable intermediate species and their disulfide bond connectivity and an analysis of the conformational assembly of these species. An established method for elucidation of the exact role that disulfides play in the process of achieving...

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Mass Spectrometry‐Based Approaches to Study Biomolecular Dynamics: Equilibrium Intermediates

2005

Mass Spectrometry in Biophysics

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In the preceding chapter, we surveyed various MS-based approaches to study higher-order structure of proteins under native conditions. For many decades, such well-defined and highly organized structures were thought of as the most important (if not the only) determinants of protein function. Protein folding was often considered a linear process leading from fully unstructured (and, therefore, dysfunctional) states to the highly organized native (function-competent) state. The advent of NMR has changed our perception of what "functional" protein states are, with the realization that native proteins are very dynamic species. Perhaps the most illustrious examples of the intimate link between protein dynamics and function were found in enzyme catalysis, where the chemical conversion of substrate to product is often driven by relatively small-scale dynamic events within (and often beyond) the active site. It became clear in recent years that large-scale macromolecular dynamics may also be an important determinant of protein function. A growing number of proteins are found to be either partially or fully unstructured under native conditions, and such flexibility (intrinsic disorder) appears to be vital for their function. Proteins that do have native folds under physiological conditions can also exhibit dynamic behavior via local structural fluctuations or by sampling alternative (higher-energy or "activated") conformations transiently. In many cases, such activated (non-native) states are functionally important despite their low Boltzmann weight. Realization of the importance of transient non-native protein structures for their function not only greatly advanced our understanding of processes as diverse as recognition, signaling, and transport Mass

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Characterisation of the dominant oxidative folding intermediate of hen lysozyme 1 1Edited by P. E. Wright

Cited by 80 publications

References 37 publications

Mixed Macromolecular Crowding Accelerates the Oxidative Refolding of Reduced, Denatured Lysozyme

Mixed Macromolecular Crowding Accelerates the Oxidative Refolding of Reduced, Denatured Lysozyme

Oxidative Folding of Amaranthus α-Amylase Inhibitor

Mass Spectrometry‐Based Approaches to Study Biomolecular Dynamics: Equilibrium Intermediates

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