Non-thermal effects of electromagnetic fields at mobile phone frequency on the refolding of an intracellular protein: myoglobin

Non-thermal effects induced by exposure to microwave electromagnetic field (MW-EMF) at 1.95 MHz, a frequency used in mobile communication, have been observed on the refolding kinetics of the heme binding site in an intracellular protein: tuna myoglobin, starting from acidic conditions. We have selected myoglobin because it can be considered a good model to study protein interactions with MW-EMF for its well-known high-resolution crystallographic structure. Myoglobin solutions at pH 3.0 were subjected to 3 h exposure to microwave field (with a specific absorption rate of 51 +/- 1 mW/g); the heme site refolding has been followed by measuring the molecular absorption in the Soret spectral region and the data were fitted to a bi-exponential model. The kinetics of exposed samples appear to be slowered by MW-EMF action. Moreover, the tryptophanyl lifetime distribution of the exposed protein, as deduced by the analysis of the fluorescence emission decay from its single tryptophan, appears sharper if compared to non-exposed protein samples. This observation suggests that the presence of MW-EMF could affect the propensity of protein molecules to populate specific conformational substates among which myoglobin molecules fluctuate at acidic pH. Changes in the structural fluctuation caused by MW perturbation can affect differently the aggregation process that occurs competitively during the protein folding, so representing a potential risk for protein "misfolding." These data suggest that MW-EMF could have also biochemical and, consequently, biological effects on eukaryotic cells that are still under investigation.     

Folding myoglobin within a sol-gel glass: protein folding constrained to a small volume

The unfolding and refolding reaction of myoglobin was examined in solution and within a porous silica sol-gel glass. The sol-gel pores constrain the protein to a volume that is the same size and shape as the folded native state accompanied by a few layers of water solvation. Denaturants such as low pH buffers can be diffused through the gel pores to the protein to initiate unfolding and refolding. Acid-induced unfolding was hindered by the steric constraints imposed by the gel pores such that more denaturing conditions were required within the gel than in solution to create the unfolded state. No new folding intermediates were observed. Refolding of myoglobin was not complete in millimolar pH 7 buffer alone. Addition of 25% glycerol to the pH 7 buffer resulted in nearly complete refolding, and the use of 1 M phosphate buffer resulted in complete refolding. The role of this cosolvent and salt in disrupting the ordered water surrounding the protein within the gel is discussed in light of the Hofmeister series and entropic trapping via a diminished hydrophobic effect within the gel. These results are consistent with the premises of folding models in which secondary and tertiary structures are considered to form within a compact conformation of the protein backbone.     

High‐throughput protein refolding screening method using zeolite

We established a 96‐well‐plate‐based refolding screening system using zeolite. In this system, protein denatured and solubilized with 6 M guanidine hydrochloride is adsorbed onto zeolite placed in a 96‐well plate. The refolding conditions can be tested by incubating the samples with refolding buffers under various conditions of pH, salts, and additives. In this study, we chose green fluorescent protein as the model protein. Green fluorescent protein was expressed as inclusion bodies, and we tested the effects of four pH conditions and six additives on its refolding. The results demonstrate that green fluorescent protein was more efficiently refolded with zeolite than with the conventional dilution method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Monoclonal antibodies assisting refolding of firefly luciferase

The reactivation efficiency in the refolding of denatured luciferase in the presence and the absence of monoclonal antibodies (mAbs) has been studied. Luciferase could be partially reactivated when the protein was denatured in high concentrations of guanidium chloride (GdmCl; >4.5 M) and the refolding was carried out in very low protein concentrations. The refolding yield was, however, significantly lower when it was performed on luciferase that had been denatured with lower concentrations of GdmCl. The efficiency of refolding decreases when the formation of aggregates increases. Three of the five luciferase mAbs tested (4G3, N2E3, S2G10) dramatically increased the yield of reactivation and simultaneously eliminated the formation of aggregates. It is proposed that these mAbs assisted the refolding of luciferase by binding to the exposed hydrophobic surface of the refolding intermediate, thus preventing it from aggregating. The epitopes interacting with these refolding-assisting mAbs are all located in the A-subdomain of the N-terminal region of luciferase. These results have also shed light on the structural features of the intermediate and its interface involved in protein aggregate formation, contributing to the understanding of the protein folding mechanism.     

Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone‐ion exchange chromatography

Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone‐ion exchange chromatography (AMC‐IEC). This new method is applied to the refolding of a widely used model protein, urea‐denatured/dithiothreitol‐reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, β‐cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC‐IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC‐IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL^?1, the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC‐IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC‐IEC is a high efficient and low cost protein refolding method. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Application of a chaperone-based refolding method to two- and three-dimensional off-lattice protein models

A model of protein-chaperone interaction as a two-phase (unfolding/refolding) iterative annealing mechanism able to promote structural segregation of hydrophobic and hydrophilic monomers and thereby facilitate access to nativelike states has recently been applied successfully to two 22-mers of the Honeycutt and Thirumalai BLN (hydrophobic, hydrophilic, neutral) heteropolymer model. This technique is here applied to a much wider data set: 94 8-mers of the off-lattice protein model originally presented in two dimensions by Stillinger and Head-Gordon, and later extended into three dimensions by Irb?ck and Potthast; the model chaperone is shown to be equally successful, and by progressive elaboration of the chaperone model as in the earlier BLN model work, to be utilizing very similar underlying mechanisms. It is demonstrated that on average, contacts with the model chaperone give rise to a consistent movement in structure space in the direction of more nativelike structures; this method of global minimization does not therefore rely fundamentally on random search. Insofar as the responses to the chaperone of the two- and three-dimensional forms of the substrate model do differ, this can be interpreted as reflecting the different handling of hydrophilic monomers in the models-in particular, whether there is active repulsion between these and monomers of hydrophobic character. The chaperone-induced refolding method is also tested on a set of 220 9-mer chains of each version of the substrate model, where it is seen that the two-dimensional model, with its more clearly distinguished roles for the hydrophobic and hydrophilic monomers, shows a more favorable scaling behavior.     

Time-resolved fluorescence study during denaturation and renaturation of curcumin-myoglobin complex

Curcumin influences the transition point, the concentration of denaturant required to effect 50% of the total change, of myoglobin denaturation. Curcumin enhances absorbance of myoglobin at 280 nm with a binding constant K=3.0×10(4) M(-1) whereas fluorescence of curcumin is quenched by myoglobin with a Stern-Volmer association constant of 2.5×10(5) M(-1). Unfolding process of myoglobin-curcumin induces a recovery in fluorescence lifetime loss. The gain in time-resolved fluorescence lifetime during unfolding has been again lost during refolding of curcumin-myoglobin complex by dilution process suggesting partial reversibility of unfolding process for both myoglobin and curcumin-myoglobin complex.     

Experimental characterization of the folding kinetics of the notch ankyrin domain

Proteins constructed from linear arrays of tandem repeats provide a simplified architecture for understanding protein folding. Here, we examine the folding kinetics of the ankyrin repeat domain from the Drosophila Notch receptor, which consists of six folded ankyrin modules and a seventh partly disordered N-terminal ankyrin repeat sequence. Both the refolding and unfolding kinetics are best described as a sum of two exponential phases. The slow, minor refolding phase is limited by prolyl isomerization in the denatured state (D). The minor unfolding phase, which appears as a lag during fluorescence-detected unfolding, is consistent with an on-pathway intermediate (I). This intermediate, although not directly detected during refolding, is shown to be populated by interrupted refolding experiments. When plotted against urea, the rate constants for the major unfolding and refolding phases define a single non-linear v-shaped chevron, as does the minor unfolding phase. These two chevrons, along with unfolding amplitudes, are well-fitted by a sequential three-state model, which yields rate constants for the individual steps in folding and unfolding. Based on these fitted parameters, the D to I step is rate-limiting, and closely matches the major observed refolding phase at low denaturant concentrations. I appears to be midway between N and D in folding free energy and denaturant sensitivity, but has Trp fluorescence properties close to N. Although the Notch ankyrin domain has a simple architecture, folding is slow, with the limiting refolding rate constant as much as seven orders of magnitude smaller than expected from topological predictions.     

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.     

Conformational propagation with prion-like characteristics in a simple model of protein folding

Protein refolding/misfolding to an alternative form plays an aetiologic role in many diseases in humans, including Alzheimer's disease, the systemic amyloidoses, and the prion diseases. Here we have discovered that such refolding can occur readily for a simple lattice model of proteins in a propagatable manner without designing for any particular alternative native state. The model uses a simple contact energy function for interactions between residues and does not consider the peculiarities of polypeptide geometry. In this model, under conditions where the normal (N) native state is marginally stable or unstable, two chains refold from the N native state to an alternative multimeric energetic minimum comprising a single refolded conformation that can then propagate itself to other protein chains. The only requirement for efficient propagation is that a two-faced mode of packing must be in the ground state as a dimer (a higher-energy state for this packing leads to less efficient propagation). For random sequences, these ground-state dimeric configurations tend to have more beta-sheet-like extended structure than almost any other sort of dimeric ground-state assembly. This implies that propagating states (such as for prions) are beta-sheet rich because the only likely propagating forms are beta-sheet rich. We examine the details of our simulations to see to what extent the observed properties of prion propagation can be predicted by a simple protein folding model. The formation of the alternative state in the present model shows several distinct features of amyloidogenesis and of prion propagation. For example, an analog of the phenomenon of conformationally distinct strains in prions is observed. We find a parallel between 'glassy' behavior in liquids and the formation of a propagatable state in proteins. This is the first report of simulation of conformational propagation using any heteropolymer model. The results imply that some (but not most) small protein sequences must maintain a sequence signal that resists refolding to propagatable alternative native states and that the ability to form such states is not limited to polypeptides (or reliant on regular hydrogen bonding per se) but can occur for other protein-like heteropolymers.     

Assisted folding of D-glyceraldehyde-3-phosphate dehydrogenase by trigger factor

The Escherichia coli trigger factor is a peptidyl-prolyl cis-trans isomerase that catalyzes proline-limited protein folding extremely well. Here, refolding of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of trigger factor was investigated. The regain of activity of GAPDH was markedly increased by trigger factor after either long- or short-term denaturation, and detectable aggregation of GAPDH intermediates was prevented. In both cases, time courses of refolding of GAPDH were decelerated by trigger factor. The reactivation yield of GAPDH showed a slow down-turn when molar ratios of trigger factor to GAPDH were above 5, due to tight binding between trigger factor and GAPDH intermediates. Such inactive bound GAPDH could be partially rescued from trigger factor by addition of reduced alphaLA as competitor, by further diluting the refolding mixture, or by disrupting hydrophobic interactions in the complexes. A model for trigger factor assisted refolding of GAPDH is proposed. We also suggest that assisted refolding of GAPDH is due mainly to the chaperone function of trigger factor.     

Effect of rate of chemical or thermal renaturation on refolding and aggregation of a simple lattice protein

We used dynamic Monte Carlo simulation to investigate how changing the rate of chemical or thermal renaturation affects the folding and aggregation behavior of a system of simple, two-dimensional lattice protein molecules. Four renaturation methods were simulated: infinitely slow cooling; slow but finite cooling; quenching; and pulse renaturation. The infinitely slow cooling method, which is equivalent to dialysis or diafiltration, provides refolding yields that are relatively high and aggregates that are relatively small (mostly dimers or trimers). The slow but finite cooling method, which is equivalent to multiple-step dilution, provides refolding yields that are almost as high as those observed in the infinitely slow cooling case, but in a relatively short period of time. Quenching, which is equivalent to one-step dilution or quick quenching, is extremely slow and has low re- folding yields. A maximum appears in the refolding yield as a function of denaturant concentration in the simulation but disappears after a very long duration. Finally, the pulse renaturation method provides refolding yields that are substantially higher than those observed in the other three methods, even at high packing fractions. As in the early stages of quenching, there is a maximum in the refolding yield as a function of denaturant concentration when relatively large numbers of denatured chains are added to the refolding solution at each step.     

Titin (Visco-) Elasticity in Skeletal Muscle Myofibrils

Titin is the third most abundant protein in sarcomeres and fulfills a number of mechanical and signaling functions. Specifically, titin is responsible for most of the passive forces in sarcomeres and the passive visco-elastic behaviour of myofibrils and muscles. It has been suggested, based on mechanical testing of isolated titin molecules, that titin is an essentially elastic spring if Ig domain un/refolding is prevented either by working at short titin lengths, prior to any unfolding of Ig domains, or at long sarcomere (and titin) lengths when Ig domain un/refolding is effectively prevented. However, these properties of titin, and by extension of muscles, have not been tested with titin in its natural structural environment within a sarcomere. The purpose of this study was to gain insight into the Ig domain un/refolding kinetics and test the idea that titin could behave essentially elastically at any sarcomere length by preventing Ig domain un/refolding during passive stretch-shortening cycles. Although not completely successful, we demonstrate here that titin’s visco-elastic properties appear to depend on the Ig domain un/refolding kinetics and that indeed, titin (and thus myofibrils) can become virtually elastic when Ig domain un/refolding is prevented.     

Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins

Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (相似文献   

A novel system for continuous protein refolding and on-line capture by expanded bed adsorption

A novel two-step protein refolding strategy has been developed, where continuous renaturation-bydilution is followed by direct capture on an expanded bed adsorption (EBA) column. The performance of the overall process was tested on a N-terminally tagged version of human beta2-microglobulin (HAT-hbeta2m) both at analytical, small, and preparative scale. In a single scalable operation, extracted and denatured inclusion body proteins from Escherichia coli were continuously diluted into refolding buffer, using a short pipe reactor, allowing for a defined retention and refolding time, and then fed directly to an EBA column, where the protein was captured, washed, and finally eluted as soluble folded protein. Not only was the eluted protein in a correctly folded state, the purity of the HAThbeta2m was increased from 34% to 94%, and the product was concentrated sevenfold. The yield of the overall process was 45%, and the product loss was primarily a consequence of the refolding reaction rather than the EBA step. Full biological activity of HAT-hbeta2m was demonstrated after removal of the HAT-tag. In contrast to batch refolding, a continuous refolding strategy allows the conditions to be controlled and maintained throughout the process, irrespective of the batch size; i.e., it is readily scalable. Furthermore, the procedure is fast and tolerant toward aggregate formation, a common complication of in vitro protein refolding. In conclusion, this system represents a novel approach to small and preparative scale protein refolding, which should be applicable to many other proteins.     

Antiperoxidase antibodies enhance refolding of horseradish peroxidase

The effect of monoclonal antibodies on protein folding was studied using horseradish peroxidase refolding from guanidine hydrochloride as a model process. Among the five antiperoxidase clones tested, one was found to increase the yield of catalytically active peroxidase after guanidine treatment. The same clone also increased the activity of the native peroxidase by a factor of 2-2.5. While peroxidase refolding under standard conditions resulted in the recovery of only 7-8% of the initial catalytic activity, antibody-assisted refolding increased the yield to 50-100% (or 20-40% from the activity of native enzyme with antibodies). Kinetics of autorefolding and antibody-assisted refolding differed significantly. In the course of autorefolding the catalytic activity was recovered within the first 2.5 min and did not change further within a 2.5- to 60-min interval, whereas in the course of antibody-assisted refolding maximal catalytic activity was attained only in 60 min. The yield of active peroxidase for the antibody-assisted refolding depended linearly on the antibody concentration. The observed effect was strongly specific. Other antiperoxidase clones tested as well as nonspecific antithyroglobulin antibody affected neither kinetics, no the yield of peroxidase refolding.     

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Changes in circular dichroic and absorption spectra of myoglobin induced by carboxymethylation

Changes in the circular dichroic and absorption spectra were studied on solutions of myoglobin whose histidine residues had been modified by carboxymethylation under denaturing conditions. Carboxymethylation resulted in a dramatic decrease in the molar extinction coefficient in the Soret region indicative of a major change in the heme environment. This was accompanied by a remarkable change in the secondary structure of the protein involving helix-to-random coil transition, indicating that extensive histidine modification prevented unfolded myoglobin from refolding to its native conformation.     

Interaction between Protein Subunits from Model Studies

A molecular model of hemoglobin was constructed which made it possible to visualize the relation between various amino acid residues in the molecule. The model indicates that electrostatic forces might play a significant role in holding the subunits of hemoglobin together. This would explain why myoglobin does not form a tetramer while four β-chains, which are structurally similar to myoglobin, do assemble into a hemoglobin H molecule. Also, as far as the primary structures of hemoglobin chains of various species are known, the proposed ionic links between subunits are consistent with the fact that mammalian hemoglobins form stable tetramers while the peptide chains of lamprey hemoglobin are only weakly associated. The different behavior of hemoglobin H and of normal hemoglobin upon oxygen uptake is briefly discussed in terms of allosteric effects.