Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fast protein size-exclusion liquid chromatography (SEC-FPLC) was used to study solvent-induced unfolding of six proteins. Two of them (sperm whale myoglobin and hen white lysozyme) denature on the simple N (native)<-->U (completely unfolded) scheme. The other four proteins [bovine and human alpha-lactalbumin, bovine carbonic anhydrase B (BCAB), and beta-lactamase from Staphylococcus aureus] denature through the molten globule (MG) state (i.e., on the N<-->MG<-->U denaturation scheme). We have shown that the permeation properties of the Superose 12 columns are practically independent of temperature, pH, and denaturants in wide concentration intervals. In the case of myoglobin and lysozyme denaturation at 4 degrees C (when the exchange between the native and unfolded states is slower than the characteristic time of chromatography), a bimodal distribution on molecular dimensions in the transition region was observed. This indicates that, under denaturant action, protein molecules can only be in one of the two states with different compactness. In other words, this shows that FPLC is one of the most direct approaches to establish the "all-or-none" mechanism of the equilibrium solvent-induced denaturation of globular proteins. The curves of guanidinium hydrochloride- (GdmHCl) or urea-induced unfolding (N<-->U or MG<-->U transitions) of a protein on a column (monitored either by the relative areas of two peaks or--for fast exchange--by the position of the average peak) coincide with those monitored by far-UV CD in solution. The Stokes radius values obtained with the use of FPLC for the molten globule states of BCAB (1.6 M GdmHCl in 0.1 M sodium phosphate, pH 6.8, and acid form at pH 3.6) and for the human alpha-lactalbumin molten globule (2.0 M GdmHCl in 0.1 M sodium phosphate, pH 6.8) coincide with those known from literature. Thus, it has been shown that fast protein size-exclusion liquid chromatography (FPLC) is an "inert" technique, i.e., it does not shift the equilibrium between N, MG, and U states and, therefore, can be used for qualitative and quantitative studies of protein denaturation.
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PMID:Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule. 824 Nov 85

A new two-step procedure of protein refolding in vitro, proposed by Rozema and Gellman and named artificial chaperone-assisted refolding, is discussed. The new approach has been inspired by the two-step mechanism of the GroE system. In the first step, the protein is captured by a detergent under conditions that would normally lead to irreversible protein aggregation (heating or denaturant removal). In the second step, removal of detergent from the protein--detergent complex is triggered by addition of a cyclodextrin which is capable of forming "inclusion complexes" with detergent, allowing the protein to refold. The protein refolded with artificial chaperones (detergent and cyclodextrin) may be purified via a two-step protocol. After refolding was complete, the solution was passed through a 0. 22-micro(m) filter, to remove aggregated protein, and then through a M = 10 kD cutoff filter. The second filtration was intended to allow the low-molecular-weight artificial chaperones to pass, but to retain the refolded enzyme. The application of the above procedure for refolding of carbonic anhydrase B from human erythrocytes, hen egg white lysozyme, pig heart citrate synthase, and creatine kinase from rabbit skeletal muscles (MM isoenzyme) is discussed.
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PMID:Artificial chaperone-assisted refolding of proteins. 955 24

Alcohols have been shown to cause a conformational transition of proteins into a new stable conformational state resembling that of the "molten globule intermediate" characterized by high alpha-helical content and disrupted tertiary structure. We have studied the effect of monohydric alcohols on the stability and structural characteristics of small globular protein hen egg white lysozyme by the combined use of differential scanning calorimetry, circular dichroism, and nuclear magnetic resonance spectroscopy. The protein stability was found to be significantly decreased with increasing alcohol concentration, and, in presence of moderate to higher alcohol concentrations, depending on the pH and alcohol studied, the protein was found to be unfolded even at 4 degrees C. Correlation between thermal stability and alpha-helicity of several small globular proteins like hen egg white lysozyme, horse heart cytochrome C, and bovine carbonic anhydrase B, observed in presence of increasing alcohol concentrations, suggests that probably alcohols induce helical structures in unfolded protein. The temperature-dependent near- and far-UV circular dichroism and proton nuclear magnetic resonance spectroscopic studies on lysozyme in the presence of 2,2,2-trifluoroethanol and methanol, respectively, showed that alcohols do induce significantly higher helical structures in unfolded protein compared to folded protein. The results presented in this paper suggest that the molten globule intermediate of proteins in the presence of high alcohols as reported earlier is due to alcohol-induced local folding rather than global folding of unfolded protein and hence is an off-pathway product and not a real folding intermediate.
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PMID:Alcohol-induced molten globule intermediates of proteins: are they real folding intermediates or off pathway products? 973 68

The power of genetic engineering methods, along with increasing genomic information, makes heterologous expression of proteins an extremely important biochemical tool. Unfortunately, proteins obtained in this way often are not in their native form, and folding becomes a crucial step in protein production. We have recently developed a strategy that promotes the folding of chemically denatured proteins via the sequential addition of low molecular weight "artificial chaperones." Here we describe in detail the application of this method to porcine heart citrate synthase. Refolding yields of as high as 65% have been achieved. Mechanistic studies indicate that there are significant differences between artificial chaperone-assisted refolding of citrate synthase and artificial chaperone-assisted refolding of two other proteins that have been examined, carbonic anhydrase B (Rozema, D., and Gellman, S. H. (1996) J. Biol. Chem. 271, 3478-3487) and lysozyme (Rozema, D., and Gellman, S. H. (1996) Biochemistry 35, 15760-15771). The differences among these three test proteins reveal the range of procedural variation that must be considered in the application of the artificial chaperone method to new proteins.
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PMID:Artificial chaperone-assisted refolding of citrate synthase. 985 49

Production of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation. Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen. The screen includes 12 factors shown by previous experiments to enhance protein folding and it incorporates the 12 factors into 16 different folding conditions. By examining a 1/256th fraction of the full factorial, multiple folding conditions were determined for the ligand binding domains from glutamate and kainate receptors, and for lysozyme and carbonic anhydrase B. The impact of each factor on the formation of biologically active material was estimated by calculating factor main effects. Factors and corresponding levels such as pH (8.5) and L-arginine (0.5 M) consistently had a positive effect on protein folding, whereas detergent (0.3 mM lauryl maltoside) and nonpolar additive (0.4 M sucrose) were detrimental to the folding of these four proteins. One of the 16 conditions yielded the most folded material for three out of the four proteins. Our results suggest that this protein folding screen will be generally useful in determining whether other proteins will fold in vitro and, if so, what factors are important. Furthermore, fractional factorial folding screens are well suited to the evaluation of previously untested factors on protein folding.
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PMID:A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase. 1042 36

High molecular weight cyclic alpha-1,4-glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form.
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PMID:Cycloamylose as an efficient artificial chaperone for protein refolding. 1111 53

Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. Beta-cyclodextrin-acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.
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PMID:Solid-phase artificial chaperone-assisted refolding using insoluble beta-cyclodextrin-acrylamide copolymer beads. 1567 15