Surfactant-induced refolding of lysozyme

The surfactant-lysozyme interaction was investigated by circular dichroism, fluorescence, UV, dynamic light scattering, surface tension, turbidity measurements and lysozyme activity assay. A new way of refolding of lysozyme was found. It was shown that the lysozyme unfolded by anionic surfactants could be renatured by adding cationic surfactants. That is, lysozyme formed precipitate with anionic surfactants, the precipitates could be dissolved by adding a cationic surfactant solution, and then the lysozyme was refolded to its native state spontaneously. Different couples of anionic surfactants and cationic surfactants including C10SO3/C10NE, C12SO3/C10NE, C10SO3/C12NE, C10SO3/C12NB, C10SO4/C10NE and C12SO4/C10NE (C(n)SO3, C(n)SO4, C(n)NE and C(n)NB represent sodium alkyl sulfonate/sulfate, alkyl triethyl/butyl ammonium bromide respectively) were investigated, all of them gave similar results. The results were explained in terms of the differences between the interaction of anionic-cationic surfactants and that of surfactant-lysozyme. It was thought that the formation of mixed micelles of anionic-cationic surfactants is a more favorable process than that of lysozyme-surfactant complexes, which induces the dissociation of lysozyme-surfactant complexes when cationic surfactants were added.     

Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme.

Solvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D2O compared with H2O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources. The rates were found to be essentially independent of whether backbone amide nitrogens were protiated or deuterated, indicating that making and breaking of their hydrogen bonding interactions is not associated with a substantial isotope effect. Neither were the rates significantly affected by adding moderate concentrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H2O and D2O are also unlikely to explain the isotope effects. The data suggest that different factors, acting in opposing directions, may be dominant under different conditions. Thus, the isotope effect on the rate-determining step was found to be qualitatively reversed on going to low pH, suggesting that one component is probably associated with changes in the environments of carboxylate groups in forming the folding transition state. This term disappears at low pH as these groups are protonated and an opposing effect then dominates. It was not possible to identify this other effect on the basis of the present data, but a dependence of the hydrophobic interaction on solvent isotopic composition is a likely candidate.     

Molecularly imprinted polymer-assisted refolding of lysozyme

For production of active proteins using heterologous expression systems, refolding of proteins from inclusion bodies often creates a bottleneck due to its poor yield. In this study, we show that molecularly imprinted polymer (MIP) toward native lysozyme promotes the folding of chemically denatured lysozyme. The MIP, which was prepared with 1 M acrylamide, 1 M methacrylic acid, 1 M 2-(dimethylamino)ethyl methacrylate, and 5 mg/mL lysozyme, successfully promoted the refolding of lysozyme, whereas the non-imprinted polymer did not. The refolding yield of 90% was achieved when 15 mg of the MIP was added to 0.3 mg of the unfolded lysozyme. The parallel relationship between the refolding yield and the binding capacity of the MIP suggests that MIP promotes refolding through shifting the folding equilibrium toward the native form by binding the refolded protein.     

Oxidative refolding of amyloidogenic variants of human lysozyme

The oxidative refolding of human lysozyme and its two best characterised amyloidogenic variants, Ile56Thr and Asp67His, has been investigated in vitro by means of the concerted application of a range of biophysical techniques. The results show that in each case the ensemble of reduced denatured conformers initially collapses into a large number of unstructured intermediates with one or two disulphide bonds, the majority of which then fold to form the native-like three-disulphide intermediate, des-[77-95]. The slow step in the overall folding reaction involves the rearrangement of the latter to the fully oxidised native protein containing four disulphide bonds. The Ile56Thr and Asp67His variants were found to fold faster than the wild-type protein by a factor of 2 and 3 respectively, an observation that can be attributed primarily to the reduction in the barriers to conformational rearrangements that results from both the mutations. The efficient folding of these variants despite their enhanced propensities to aggregate when compared to the wild-type protein is consistent with their ability to be secreted in sufficient quantities to give rise to the systemic amyloidoses with which they are associated.     

NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 degree C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116-Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis-trans equilibrium. The rate of trans-->cis isomerization in the native-like nuclease intermediate is approximately 100-fold faster than that observed in a Lys-Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116-Pro 117 peptide bond is comparable to that observed for other X-Pro isomerizations.     

A new kinetic scheme for lysozyme refolding and aggregation

The competing first- and third-order reaction scheme for lysozyme is shown to not predict fed-batch lysozyme refolding when the model is parameterized using independent batch experiments, even when variations in chemical composition during the fed-batch experiment are accounted for. A new kinetic scheme is proposed that involves rapid partitioning between the alternative fates of refolding and aggregation, and which allows for aggregation via a sequential mechanism. The model assumes that monomeric lysozyme in different states, including native, is able to aggregate with intermediates, accounting for recent experimental evidence that native protein can be incorporated into aggregates and explaining why native protein in the refolding buffer reduces yield. Stopped-flow light-scattering measurements were used to measure the association rate for the sequential aggregation mechanism, and refolding rate constants were determined in a series of batch experiments designed to be "snapshots" of the composition during a fed-batch experiment. The new kinetic scheme gave a good a priori prediction of fed-batch refolding performance.     

High-pressure refolding of disulfide-cross-linked lysozyme aggregates: thermodynamics and optimization

Previous exploratory work revealed that high pressure (200 MPa), in combination with oxido-shuffling agents such as glutathione, effectively refolds covalently cross-linked aggregates of lysozyme into catalytically active native molecules, at concentrations up to 2 mg/mL (1). To understand further and optimize this process, in the current study we varied the redox conditions and levels of guanidine hydrochloride (GdnHCl) in the refolding buffer. Maximum refolding yields of 80% were seen at 1 M GdnHCl; higher concentrations did not increase refolding yields further. A maximum in refolding yield was observed at redox conditions with a 1:1 ratio of oxidized to reduced glutathione (GSSG:GSH). Yields decreased dramatically at more oxidizing conditions ([GSSG] > [GSH]). Kinetics of dissolution and refolding of covalently cross-linked aggregates of lysozyme depended strongly on redox conditions. At GSSG:GSH ratios of 4:1, 1:1, and 1:16, lysozyme dissolved and refolded with time constants of 62, 20, and 8 h, respectively. Estimates of the free energy of unfolding of lysozyme in GdnHCl solutions at 200 MPa suggested that the native state of lysozyme is strongly favored (ca.18.6 kJ/mol) under the conditions used for dissolution and refolding.     

Continuous refolding of lysozyme with fed-batch addition of denatured protein solution

A continuous refolding method with addition of denatured protein solution in a fed-batch manner through a ceramic membrane tube was developed. Denatured and fully reduced lysozyme was continuously refolded with high refolding efficiencies. In this method, a denatured lysozyme solution was gradually added from the outer surface of the membrane tube into a refolding buffer flowing continuously inside the tube under controlled mixing conditions. The refolding efficiencies of lysozyme in this continuous refolding were higher than those in a batch dilution method. This method has applicability to large-scale downstream processes and can attain a high efficiency and protein concentration in refolding. Refolded proteins can be supplied continuously following purification steps.     

The influence of operational parameters on lysozyme refolding using size-exclusion chromatography

The influence of several parameters on the gel filtration refolding of hen egg white lysozyme from a starting concentration of 40 mg/ml was investigated. Refolding was found to be unaffected by temperature between 30 and 50°C, giving 100% recovered specific activity. At 10°C a 20% reduction in refolding yield was observed. Refolding was carried out successfully with both acrylamide (Sephacryl S100)- and dextran (Superdex 75)-based gel media. At the isoelectric pH of lysozyme, aggregation was suppressed in the column method, whereas protein aggregates were formed during dilution-based refolding. A number of compounds (carboxymethyl cellulose, dextran, sucrose) were added to the mobile phase to reduce the relative viscosity between the sample and mobile phase. Only sucrose, up to 20% (wt), was found not to interfere with lysozyme refolding.     

A near-native state on the slow refolding pathway of hen lysozyme

The refolding of four disulfide lysozyme (at pH 5.2, 20 degrees C) involves parallel pathways, which have been proposed to merge at a near-native state. This species contains stable structure in the alpha- and beta-domains but lacks a functional active site. Although previous experiments have demonstrated that the near-native state is populated on the fast refolding pathway, its relevance to slow refolding molecules could not be directly determined from previous experiments. In this paper, we describe experiments that investigate the effect of added salts on the refolding pathway of lysozyme at pH 5.2, 20 degrees C. We show, using stopped flow tryptophan fluorescence, inhibitor binding, and circular dichroism (CD), that the rate of formation of native lysozyme on the slow refolding track is significantly reduced in solutions of high ionic strength in a manner dependent on the position of the anion in the Hofmeister series. By contrast, the rate of evolution of hydrogen exchange (HX) protection monitored by electrospray ionization mass spectrometry (ESI MS) is unchanged under the refolding conditions studied. The data show, therefore, that at high ionic strengths beta-domain stabilization and native state formation on the slow refolding pathway become kinetically decoupled such that the near-native state becomes significantly populated. Thus, by changing the energy landscape with the addition of salts new insights into the relevance of intermediate states in lysozyme refolding are revealed.     

Critical analysis of methods for analysing human calcium kinetics

A comparison has been made between several different compartmental and non-compartmental methods for analyzing human calcium kinetics. Using data from studies in six normal subjects, plus a computer-generated set of “error-free” data, the bone accretion rate and the exchangeable calcium pool size have been calculated by each method, along with their corresponding uncertainties. The effects of selective deletions of data have also been determined for the various methods.The results are highly dependent upon the model employed and the parameter investigated. In general, the non-compartmental models provide accretion rates which are less sensitive to measurement errors, and have less stringent requirements as to the necessary duration of an experimental study. Among compartmental models, a three-compartment model based on data collected from two hours to twenty days after isotopic calcium injection gives estimates of skeletal accretion rate and exchangeable pool size very similar to those resulting from a four-compartment model that includes additional earlier data.The relative advantages of various compartmental and non-compartmental methods of analysis are discussed in relation to these results, and practical recommendations offered to the clinical investigator.     

Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation

It is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding.     

Comparison of the kinetics of S-S bond, secondary structure, and active site formation during refolding of reduced denatured hen egg white lysozyme

To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism (CD), fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray-mass spectrometry. Thus, it was possible to monitor the appearance and/or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far-UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme.     

Kinetics of unfolding and refolding of proteins. 3. Results for lysozyme

The kinetics of the reversible denaturation of lysozyme by guanidine hydrochloride have been studied over a wide range of pH and denaturant concentration. The results contrast sharply with those observed for cytochrome c. The interconversion of native (N) and denatured (D) states is strictly first-order in both directions under most conditions, showing that no intermediate forms of any kind accumulate to a significant extent during the reaction. At one pH (pH 2.6) experiments were extended to extreme denaturant concentrations, and under these conditions kinetic intermediates were observed. Analysis by the procedures of the first paper of this series Ikai &; Tanford 1973 showed that the principal intermediate observed at low denaturant concentration must be different from that observed at high denaturant concentration, and that both must be on the direct pathway from N to D. This suggests that the over-all reaction mechanism is of the form

and this mechanism is able to account for all of the observed results. The intermediate X₁ which accumulates transiently in the renaturation reaction at low concentrations of guanidine hydrochloride is spectrally very similar to the native state, i.e. it is probably a highly ordered state, but most of the interactions between surface acidic and basic groups, which are responsible for the anomalous titration behavior of native lysozyme, do not yet exist in this state. It is probable that the difference between the kinetics of refolding of lysozyme and cytochrome c may be ascribed to the existence of disulfide cross-links in lysozyme, which were intact in these experiments. These cross-links greatly limit the possible pathways for folding and thus make it much less likely that incorrectly folded states on dead-end pathways are readily accessible.     

GroEL accelerates the refolding of hen lysozyme without changing its folding mechanism.

The chaperonin GroEL binds folding intermediates of four-disulfidehen lysozyme transiently within its central cavity. Using stopped flow fluorescence we show that GroEL binds early intermediates in folding and accelerates the slow kinetic phase that reflects the reversal of non-native interactions involving tryptophan residues and the formation of the native state. Pulsed hydrogen exchange monitored by electrospray ionization mass spectrometry demonstrates that GroEL does not alter the folding mechanism, nor are protected species unfolded by the chaperonin. The data suggest a mechanism for GroEL-assisted folding in which the reorganization of non-native tertiary interactions is facilitated but domain folding is unperturbed.     

Probabilistic approach to lysozyme crystal nucleation kinetics

Nucleation of lysozyme crystals in quiescent solutions at a regime of progressive nucleation is investigated under an optical microscope at conditions of constant supersaturation. A method based on the stochastic nature of crystal nucleation and using discrete time sampling of small solution volumes for the presence or absence of detectable crystals is developed. It allows probabilities for crystal detection to be experimentally estimated. One hundred single samplings were used for each probability determination for 18 time intervals and six lysozyme concentrations. Fitting of a particular probability function to experimentally obtained data made possible the direct evaluation of stationary rates for lysozyme crystal nucleation, the time for growth of supernuclei to a detectable size and probability distribution of nucleation times. Obtained stationary nucleation rates were then used for the calculation of other nucleation parameters, such as the kinetic nucleation factor, nucleus size, work for nucleus formation and effective specific surface energy of the nucleus. The experimental method itself is simple and adaptable and can be used for crystal nucleation studies of arbitrary soluble substances with known solubility at particular solution conditions.     

Efficient lysozyme refolding at a high final concentration and a low dilution factor

Of the various protein refolding methods, direct dilution is one of the simplest and easiest for scaling up the refolding process. However, it requires a large amount of refolding buffer, often utilizes a number of chemicals, and results in a low final protein concentration. In this report, we demonstrate that reduced dithiothreitol (DTT^red), a carryover from denaturation, is a crucial and adverse factor in lysozyme refolding. Accordingly, we proposed a method of using high concentration of oxidized glutathione (GSSG) in the refolding buffer to eliminate excess DTT^red and aid in the refolding of lysozyme. The efficiency of this method is 84%, which resulted in a high final refolded protein concentration of 1.5 g/l and required only a low dilution factor (4×). Furthermore, compared with the traditional 50× direct dilution (resulting in a similar yield of 74%), the low dilution factor required much less GSSG and other constituents.     

The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase.

The oxidative refolding of hen lysozyme has been studied by a variety of time-resolved biophysical methods in conjunction with analysis of folding intermediates using reverse-phase HPLC. In order to achieve this, refolding conditions were designed to reduce aggregation during the early stages of the folding reaction. A complex ensemble of relatively unstructured intermediates with on average two disulfide bonds is formed rapidly from the fully reduced protein after initiation of folding. Following structural collapse, the majority of molecules slowly form the four-disulfide-containing fully native protein via rearrangement of a highly native-like, kinetically trapped intermediate, des-[76-94], although a significant population (approximately 30%) appears to fold more quickly via other three-disulfide intermediates. The folding catalyst PDI increases dramatically both yields and rates of lysozyme refolding, largely by facilitating the conversion of des-[76-94] to the native state. This suggests that acceleration of the folding rate may be an important factor in avoiding aggregation in the intracellular environment.