Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

Monitoring of actin unfolding by room temperature tryptophan phosphorescence

Slow intramolecular mobility of native and inactivated actin from rabbit skeletal muscle during the process of protein unfolding induced by GdnHCl was studied using tryptophan room temperature phosphorescence (RTP). By this method, the conclusion was confirmed that an essentially unfolded intermediate preceded the formation of inactivated actin [Turoverov et al. Biochemistry (2002) 41, 1014-1019]. It was found that the kinetic intermediate generated at the early stage of protein denaturation has no tryptophan RTP, suggesting the high lability of its structure. Symbate changes of integral intensity and the mean lifetime of RTP during the U* --> I transition suggests a gradual increase of the number of monomers incorporated in the associate (U* --> I(1)... --> I(n)... --> I(15)), which is accompanied by an increase of structural rigidity. The rate of inactivated actin formation (I identical with I(15)) is shown to increase with the increase of protein concentration. It is shown that, no matter what the means of inactivation, actin transition to the inactivated state is accompanied by a significant increase of both integral intensity and the mean lifetime of RTP, suggesting that inactivated actin has a rigid structure.     

Physical-chemical properties of actin in different structural states. New ideas about its folding-unfolding pathways

Results of actin folding-unfolding pathways examination and characterization of intermediate and misfolded states are summarized. Properties of microenvironments and peculiarities of location of tryptophan residues in protein are analysed in detail. This allowed to conclude that the main contribution to the bulk fluorescence of native protein is made by internal tryptophan residues Trp 340 and Trp 356, localized in hydrophobic regions, while tryptophan residues Trp 79 and Trp 86 are quenched. It has been shown that inactivated actin, previously regarded as an intermediate state between native and completely unfolded state of protein is in reality a misfolded aggregated state. The properties of actin in this state were characterized in detail. In particular, it is shown that inactivated actin is a monodisperse associate consisting of 15 monomer unit. Two earlier unknown intermediate states, which precede completely unfolding of protein macromolecule and formation of inactivated actin, were visualized. A new scheme of folding-unfolding processes was proposed. It is shown that the reason of anomalous effects, which are recorded for actin in solutions with small concentrations of GdnHCl, is a specific interaction of actin with a denaturant.     

The structure and dynamics of partially folded actin.

Steady-state and time-resolved intrinsic fluorescence, fluorescence quenching by acrylamide, and surface testing by hydrophobic label ANS were used to study the structure of inactivated alpha-actin. The results are discussed together with that of earlier experiments on sedimentation, anisotropy of fluorescence, and CD spectrum in the near- and far-UV regions. A dramatic increase in ANS binding to inactivated actin in comparison with native and unfolded protein indicates that the inactivated actin has solvent-exposed hydrophobic clusters on the surface. It results in specific association of actin macromolecules (sedimentation constants for native and inactivated actin are 3 and 20 S, respectively) and, consequently, in irreversibility of native-inactivated actin transition. It was found that, though the fluorescence spectrum of inactivated actin is red-shifted, the efficiency of the acrylamide collision quenching is even lower than that of the intact protein. It suggests that tryptophan residues of inactivated actin are located in the inner region of protein formed by polar groups, which are highly packed. It correlates with the pronounced near-UV CD spectrum of inactivated actin. The experimentally found tryptophan fluorescence lifetimes allowed evaluation rotational correlation times on the basis of Perrin plots. It is found that oscillations of tryptophan residues in inactivated actin are restricted in comparison with native one. The inactivated actin properties were invariant with experimental conditions (ionic strength, the presence of reducing agents), the way of inactivation (Ca2+ and/or ATP removal, heating, 3-5 M urea or 1.5 M GdmCl treatment), and protein concentration (within the limits 0.005-1.0 mg/mL). The same state of actin appears on the refolding from the completely unfolded state. Thermodynamic stability, pronounced secondary structure, and the existing hydrophobic clusters, tested by ANS fluorescence and reversibility of transition inactivated-unfolded forms, allowed us to suggest that inactivated actin can be intermediate in the folding-unfolding pathway.     

Kinetics of inactivation of lectin: analysis of a method of tryptophan phosphorescence at room temperature

The internal dynamics of muscle actin during inactivation induced by guanidine hydrochloride (0.5-1.8 M) was studied by the method of room-temperature tryptophan phosphorescence (RTTP). It was shown that the essentially unfolded actin intermediate, which appears within the first minutes of incubation with guanidine hydrochloride, exhibits no RTTP, suggesting a high lability of its structure. Subsequent accumulation of associates of inactivated actin is accompanied by a significant increase in the intensity and decay time of RTTP, which is caused by the rigidity of the structure of inactivated actin. The kinetic dependencies of the intensity and lifetime of RTTP of actin during its inactivation depended on the concentration of the protein and guanidine hydrochloride.     

Room temperature phosphorescence of amorphous aggregates and amyloid fibrils resulting from protein misfolding

Using actin, alpha-lactalbumin and insulin as examples, it was shown that the formation of amorphous aggregates of proteins and amyloid fibrils leads to an increase in the rigidity of tryprophan and tyrosine residues micro-environment and, consequently, to the appearance of tryptophan (tyrosine) room temperature phosphorescence (RTP). RTP was used for examining a slow intramolecular mobility of native (G-, F-form) and inactivated (I) rabbit skeletal muscle actin during the process of GdnHCl induced protein unfolding. This method made it possible to confirm that an essentially unfolded intermediate precedes the formation of inactivated actin. It has been found that the kinetic intermediate generated at the early stage of protein denaturation has no tryptophan RTP, suggesting a high lability of its structure. Symbate changes of integral intensity (relative quantum yield) and the mean lifetime of RTP during the U*-->I transition suggest a gradual increase of the number of monomers incorporated in the associate (U*-->11...-->In...-->I15), which is accompanied by an increase of protein structural rigidity. The rate of inactivated actin formation (I-->I15) is shown to increase with the increase of protein concentration. It is shown that, no matter what method of inactivation was employed (1--2 M GdnHCl or 3.0-3.5 M urea, Ca2+ removal, incubation at 70 degrees C, refolding from completely unfolded state by dialysis from 8 M urea or 6 M GdnHCl), actin transition to the inactivated state is accompanied by a significant increase in both integral intensity and the mean lifetime of RTP, suggesting the rigid structure of inactivated actin. It is shown that the lifetime of inactivated actin RTP does not depend on GdnHCl concentration within the limits from 0 to 4 M. On using insulin and alpha-lactalbumin as examples, it is shown that RTP can be used in studies of fibrillogenesis and properties of amyloid fibrils.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

Tryptophan phosphorescence of nascent and inactivated actin at the room temperature]

Millisecond internal dynamics of native and inactivated actin from rabbit skeletal muscle was examined using room temperature phosphorescence. Inactivated actin was prepared by incubation of G-actin at 70 degrees C, by treatment with 4 M urea or 1.5 M guanidinium hydrochloride, renaturation from fully unfolded state or by Ca2+ ion removal. It was shown that inactivation of actin, irrespective of the denaturation procedure applied, leads to a sharp decrease of millisecond fluctuations of the protein structure. Restriction of the slow intramolecular mobility in inactivated actin can result from changes of the protein conformation and/or specific association of macromolecules.     

Features of the structural organization of inactivated actin--an intermediate form of the protein during the folding-unfolding process

The structure of inactivated actin was studied by the methods of intrinsic fluorescence upon stationary and pulse excitation, selective fluorescence quenching with acrylamide, and testing the protein surface with a hydrophobic probe, 8-anilino-1-naphthalenesulfonic acid (ANS). The results are discussed along with earlier data on actin sedimentation, near- and far-UV CD spectra, and fluorescence anisotropy. The thermodynamic stability of inactivated actin, the presence of a secondary structure characteristic of the native protein, and the reversibility of the inactivated actin-completely unfolded actin transition allow inactivated actin to be considered an intermediate form in the process of protein folding into the native globular structure. In vitro actin inactivation is accompanied by specific association of actin macromolecules resulting in the formation of homogeneous stable complexes. The tendency toward aggregation (or specific association, in the case of actin), which is determined by the presence of extended hydrophobic clusters on the molecule surface, appears to be one of the intrinsic properties of any protein in the intermediate state. The mobility of the amino acid side chains in the inactivated actin differs considerably from that in the completely unfolded actin. The relaxation properties of the microenvironment of tryptophan residues determine relatively long-wave fluorescence spectra of the inactivated actin. However, the mobility observed is insufficient to compensate the asymmetry of the microenvironment of aromatic residues, which is confirmed by a characteristic and intense CD spectrum in the near-UV region. The mobility of the indole rings of tryptophans located in the internal regions of the inactivated actin that are solvent-inaccessible although polar is even considerably lower than that in the native actin.     

Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

Characterization of an actin flow artifact that occurs during the presteady state dissociation of actosubfragment-1

Models for the activation of the myosin subfragment-1 (S-1) ATPase activity by actin describe transitions that occur between kinetic intermediate states during steady state hydrolysis of ATP. These states consist of myosin-nucleotide complexes in rapid equilibrium binding with actin, but steady state measurements of actin binding during hydrolysis lead only to a weighted average of the individual binding constants involved. In the current work, in order to determine the individual binding constants involved in the activation process, we have investigated the presteady state kinetics of the dissociation of actomyosin by ATP. We find that an actin flow artifact appears to dominate the time course of dissociation, and characterization of this artifact reveals that its magnitude rises linearly (approximately) with the concentration of bound S-1. Attempts to subtract the actin flow artifact from the actoS-1 dissociation signal were not entirely successful due at least partially to the transient nature of the bound S-1 concentration in the first few milliseconds. However, further studies reveal that if the order of addition of actin, ATP, and S-1 are varied, the observed light scattering transients are essentially superimposable. One possible explanation of these data is that the binding constants for myosin-ATP and myosin-ADP-Pi to actin are equal. However, it is also possible that the flow artifact is so large that further analysis is precluded. In addition, we show that the actin flow artifact has little effect on the fluorescence measurements of the phosphate burst reported previously. Therefore, the prior interpretation of the fluorescence data remains unchanged.     

Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.

High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.     

Contribution of the 6-120 disulfide bond of alpha-lactalbumin to the stabilities of its native and molten globule states.

The unfolding and refolding of a derivative of alpha-lactalbumin, in which the disulfide bond between Cys6 and Cys120 is selectively reduced and S-carboxymethylated, are investigated by equilibrium and kinetic circular dichroism measurements. The native conformation of this derivative is known to be essentially identical to that of intact alpha-lactalbumin. The equilibrium unfolding of the derivative involves a stable intermediate, which is also similar to the molten globule state of the disulfide intact protein. The results of stopped-flow circular dichroism experiments show that the same intermediate is formed rapidly as a transient intermediate in kinetic refolding. The conformational stabilities for the native and intermediate states have been estimated and compared with the stabilities for the corresponding states of intact alpha-lactalbumin. The stabilization of the native state by the disulfide has been interpreted in terms of a decrease in chain entropy in the unfolded state and elimination of the strain imposed on the disulfide bond in the native state. The molten globule state is also stabilized by the disulfide bond, although the degree of stabilization of the molten globule state is smaller than of the native state. The results suggest that, in the molten globule state, some ordered structures are present within the loop moiety formed by the 6-120 disulfide.     

Apoflavodoxin folding mechanism: an alpha/beta protein with an essentially off-pathway intermediate.

The folding reaction of Anabaena apoflavodoxin has been studied by stopped-flow kinetics and site-directed mutagenesis. Although the urea unfolding equilibrium is two-state, a transient intermediate accumulates during the folding reaction. The intermediate is monomeric, and it is not related to proline isomerization. Unlike many cases where the presence of an intermediate has been detected either by a burst phase or by the curvature, at low urea concentration, of the otherwise only observable kinetic phase, two kinetic phases are observed in apoflavodoxin folding whose total amplitude equals the amplitude of unfolding. To determine the role of the intermediate in the folding reaction, the apoflavodoxin kinetic data have been fitted to all conceivable three-species kinetic models (either linear or triangular). Using a stepwise fitting procedure, we find that the off-pathway mechanism explains most of the kinetic data (not a slow unfolding phase), the on-pathway mechanism being rejected. By using global analysis, good overall agreement between data and fit is found when a triangular mechanism is considered. The fitted values of the microscopic constants indicate that most of the unfolded molecules refold from the denatured state. Apoflavodoxin thus folds via a triangular, but essentially off-pathway, mechanism. We calculate that the retardation of the folding caused by the off-pathway intermediate is not large. Some unusual properties of the intermediate are discussed.     

Detection of conformational changes in actin by proteolytic digestion: Evidence for a new monomeric species

When KCl is added to a solution of G-actin to induce full polymerization, a decrease in the rate at which actin undergoes enzymatic proteolysis occurs. This decrease cannot be accounted for by factors affecting the enzymes employed, but rather appears to be due to a change in the conformation of G-actin. Partially polymerized actin solutions also show a reduction in digestibility which is dependent on the F-actin content, suggesting that F-actin is essentially indigestible. Moreover, low rates of digestion were also observed at sub-critical actin concentrations, where actin in the presence of 0.1 m-KCl does not polymerize. This indicates that a confomational change occurs in G-actin before the polymerization step.At sub-critical concentrations in 0.1 m-KCl, actin is in a truly monomeric state as judged by its viscosity characteristics, its inability to enhance the rate of polymerization of G-actin and its possession of ATP as the actin-bound nucleotide. These data support the existence of a new species of actin, called F-ATP-actin monomer, which has the same physical properties and the same bound nucleotide as G-actin, but digestion characteristics like F-actin. Since F-ATP-actin monomers have the same low susceptibility to proteolysis as F-ADP-actin polymers, and because both G-ATP-actin and G-ADP-actin have similar high rates of digestion, the observed change in the conformation of actin cannot be due to the phosphorylated state of the actin-bound nucleotide. Instead, the conformational change appears to be caused by the addition of KCl to G-actin.The newly-detected monomeric species is considered to be an intermediate in the polymerization process where F-ATP-actin monomers form a population of polymerizable molecules which must reach a critical concentration before nucleation and F-actin polymer formation begin.     

Cation-induced toroidal condensation of DNA studies with Co3+(NH3)6

The unfolding and refolding of Staphylococcus aureus penicillinase have been followed by urea-gradient electrophoresis. Unfolding of the native state proceeds by an all-or-none transition to fully unfolded protein, with no detectable accumulation of partially unfolded states. In contrast, refolding is complex and proceeds by very rapid, reversible formation of a partially folded state, H, which had been detected and characterized previously, as it is the most stable conformation at intermediate denaturant concentrations. At very low urea concentrations, a more compact conformational state was observed as a transient intermediate in refolding. There was little kinetic heterogeneity of the unfolded protein, as is normally observed with proteins containing proline residues.     

Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.     

Destabilization of the Escherichia coli RNase H kinetic intermediate: switching between a two-state and three-state folding mechanism

Escherichia coli RNase H folds through a partially folded kinetic intermediate that mirrors a rarely populated, partially unfolded form detectable by native-state hydrogen exchange under equilibrium conditions. Residue 53 is at the interface of two helices known to be structured in this intermediate. Kinetic refolding studies on mutant proteins varying in size and hydrophobicity at residue 53 support a contribution of hydrophobicity to the stabilities of the kinetic intermediate and the transition state. Packing interactions also play a significant role in the stability of these two states, though they play a much larger role in the native-state stability. One dramatic mutation, I53D, results in the conversion from a three-state to a two-state folding mechanism, which is explained most easily through a simple destabilization of the kinetic intermediate such that it is no longer stable with respect to the unfolded state. These results demonstrate that interactions that stabilize an intermediate can accelerate folding if these same interactions are present in the transition state. Our results are consistent with a hierarchical model of folding, where the intermediate consists of native-like interactions, is on-pathway, and is productive for folding.