Equilibrium and kinetics of the folding and unfolding of canine milk lysozyme

The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form of the protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populated as an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from the kinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding with the same molten globule intermediate. Although the observed kinetic refolding curves of both forms were single-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and the burst-phase intermediates of both forms were identical to each other with respect to their stability, indicating that the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the molten globule state observed at equilibrium. The phi-value analysis, based on the effect of Ca2+ on the folding and unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transition state of folding. A comparison of the result with that previously reported for alpha-lactalbumin indicated that the folding initiation site is different between canine milk lysozyme and alpha-lactalbumin, and hence, the folding pathways must be different between the two proteins. These results thus provide an example of the phenomenon wherein proteins that are very homologous to each other take different folding pathways. It is also shown that the native state of the apo form is composed of at least two species that interconvert.     

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.     

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.     

Characterization of the critical state in protein folding. Effects of guanidine hydrochloride and specific Ca2+ binding on the folding kinetics of alpha-lactalbumin

The reversible unfolding and refolding kinetics of alpha-lactalbumin induced by concentration jump of guanidine hydrochloride were measured at pH 7.0 and 25 degrees C using tryptophan absorption at 292 nm, with varying concentrations of the denaturant and free Ca2+. The refolding reaction of alpha-lactalbumin from the fully unfolded (D) state occurs through the two stages: (1) instantaneous formation of a compact intermediate (the A state) that has a native-like secondary structure; (2) tight packing of the preformed secondary structure segments to lead finally to the native structure, this stage being the rate-determining step of the reaction and associated with acquisition of the specific structure necessary for strong Ca2+ binding. Under strongly native conditions, the observed kinetics of refolding is also complicated by the presence of a slow-folding species (10%) in the unfolded state. Considering these facts, the microscopic rate constants in folding and unfolding directions have been evaluated from the observed kinetics and from the equilibrium constants of the transitions among the native (N), A and D states. Close linear relationships have been found in the plots of the activation free energies, obtained from the microscopic rate constants, against the denaturant concentration. They are similar to the linear relationship between the free energy of unfolding and the denaturant concentration. It was demonstrated that the slope of the plots should be approximately proportional to a change in accessible surface area of the protein during the respective activation process, and that only a third of the difference in accessible surface area between A and N is buried in the critical activated state of folding. However, the selective effect of Ca2+ binding on the folding rate constant has been observed also, demonstrating that the specific Ca2+-binding substructure in the N state is already organized in the activated state. Thus, only a part of the protein molecule involving the Ca2+-binding region is organized in the activated state, with the other part of the molecule being left less organized, suggesting that the second stage of folding may be a sequential growing process of organized assemblage of the performed secondary structure segments.     

Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.     

Differential salt-induced stabilization of structure in the initial folding intermediate ensemble of barstar

The effects of two salts, KCl and MgCl(2), on the stability and folding kinetics of barstar have been studied at pH 8. Equilibrium urea unfolding curves were used to show that the free energy of unfolding, deltaG(UN), of barstar increased from a value of 4.7 kcalmol(-1) in the absence of salt to a value of 6.9 kcalmol(-1) in the presence of 1M KCl or 1M MgCl(2). For both salts, deltaG(UN) increases linearly with an increase in concentration of salt from 0M to 1M, suggesting that stabilization of the native state occurs primarily through a Hofmeister effect. Refolding kinetics were studied in detail in the presence of 1M KCl as well as in the presence of 1M MgCl(2), and it is shown that the basic folding mechanism is not altered upon addition of salt. The major effects on the refolding kinetics can be attributed to the stabilization of the initial burst phase ensemble, I(E), by salt. Stabilization of structure in I(E) by KCl causes the fluorescence properties of I(E) to change, so that there is an initial burst phase change in fluorescence at 320 nm, during refolding. The structure in I(E) is stabilized by MgCl(2), but no burst phase change in fluorescence at 320 nm is observed during refolding. The fluorescence emission spectra of I(E) show that when refolding is initiated in 1M KCl, the three tryptophan residues in I(E) are less solvent exposed than when folding is initiated in 1M MgCl(2). Stabilization of I(E) leads to an acceleration in the rate of the fast observable phase of folding by both salts, suggesting that structure of the transition state resembles that of I(E). The stabilization of I(E) by salts can be accounted for largely by the same mechanism that accounts for the stabilization of the native state of the protein, namely through the Hofmeister effect. The salts do not affect the rates of the slower phases of folding, indicating that the late intermediate ensemble, I(L), is not stabilized by salts. Stabilization of the native state results in deceleration of the fast unfolding rate, which has virtually no dependence on the concentration of KCl or MgCl(2) at high concentrations. The observation that the salt-induced stabilization of structure in I(E) is accompanied by an acceleration in the fast folding rate, suggests that I(E) is likely to be a productive on-pathway intermediate.     

Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9

The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and Im9 have been characterised at pH 7.0 and 10 degrees C. These proteins are 60 % identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms. The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s-1, an unfolding rate of 0. 01 s-1, and a highly compact transition state that has approximately 95 % of the native surface area buried). By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10 degrees C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10 degrees C) the intermediate populated during Im7 refolding is significantly stabilised (KUI=125). Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (DeltaDeltaG 9.3 kJ/mol) and the DeltaG and m values determined accord with those obtained from the fit to the kinetic data. The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general.     

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.     

Kinetics of folding and unfolding of goat alpha-lactalbumin

The equilibrium unfolding and the kinetic folding and unfolding of goat alpha-lactalbumin (GLA) were studied by near- and far-ultraviolet circular dichroism (CD) and by stopped-flow fluorescence spectroscopy. Specifically, the influence of environmental conditions such as pH and Ca2+ binding was examined. Compared to the apo-form, the Ca2+-bound form was found to be strongly stabilized in equilibrium conditions at pH 7.5 and 25 degrees C. The kinetics of the refolding of apo-GLA show a major change of fluorescence intensity during the experimental dead-time, but this unresolved effect is strongly diminished in holo-GLA. In both cases, however, the chevron plots can adequately be fitted to a three-state model. Moreover, double-mix stopped-flow experiments showed that the native state (N) is reached through one major pathway without the occurrence of alternative tracks. In contrast to the homologous bovine alpha-lactalbumin (BLA), the compactness of GLA is strongly influenced by the presence of Ca2+ ions. Unlike the two-state transition observed in guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation experiments at higher pH, an equilibrium intermediate state (I) is involved in denaturation at pH 4.5. In the latter case, analysis of the kinetic data makes clear that the intermediate and the unfolded states (U) show practically no Gibbs free energy difference and that they are in rapid equilibrium with each other. A possible explanation for these variations in stability and in folding characteristics with pH could be the degree of protonation of His107 that directly influences non-native interactions. Variation of environmental conditions and even small differences in sequence, therefore, can result in important effects on thermodynamic and folding parameters.     

Aggregation as the basis for complex behaviour of cutinase in different denaturants

We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equilibrium and kinetic data, and the intermediate accumulating at low denaturant concentrations is off-pathway. However, in GdmCl, refolding rates, and consequently the stability of the native state, vary significantly depending on whether refolding takes place from the pH- or GdmCl-denatured state, possibly due to transient formation of aggregates during folding from the GdmCl-denatured state. In GdmSCN, stability is reduced by several kcal/mol with significant aggregation in the unfolding transition region. The basis for the large variation in folding behaviour may be the denaturants' differential ability to support formation of exposed hydrophobic regions and consequent changes in aggregative properties during refolding.     

Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway.The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).     

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.     

Three-state protein folding: experimental determination of free-energy profile

When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three-state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea-dependent rates of the I<-->N and U<-->N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration.     

Formation of on- and off-pathway intermediates in the folding kinetics of Azotobacter vinelandii apoflavodoxin

The folding kinetics of the 179-residue Azotobacter vinelandii apoflavodoxin, which has an alpha-beta parallel topology, have been followed by stopped-flow experiments monitored by fluorescence intensity and anisotropy. Single-jump and interrupted refolding experiments show that the refolding kinetics involve four processes yielding native molecules. Interrupted unfolding experiments show that the two slowest folding processes are due to Xaa-Pro peptide bond isomerization in unfolded apoflavodoxin. The denaturant dependence of the folding kinetics is complex. Under strongly unfolding conditions (>2.5 M GuHCl), single exponential kinetics are observed. The slope of the chevron plot changes between 3 and 5 M denaturant, and no additional unfolding process is observed. This reveals the presence of two consecutive transition states on a linear pathway that surround a high-energy on-pathway intermediate. Under refolding conditions, two processes are observed for the folding of apoflavodoxin molecules with native Xaa-Pro peptide bond conformations, which implies the population of an intermediate. The slowest of these two processes becomes faster with increasing denaturant concentration, meaning that an unfolding step is rate-limiting for folding of the majority of apoflavodoxin molecules. It is shown that the intermediate that populates during refolding is off-pathway. The experimental data obtained on apoflavodoxin folding are consistent with the linear folding mechanism I(off) <==> U <==> I(on) <== > N, the off-pathway intermediate being the molten globule one that also populates during equilibrium denaturation of apoflavodoxin. The presence of such on-pathway and off-pathway intermediates in the folding kinetics of alpha-beta parallel proteins is apparently governed by protein topology.     

Unassisted refolding of urea unfolded rhodanese

In vitro refolding after urea unfolding of the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) normally requires the assistance of detergents or chaperonin proteins. No efficient, unassisted, reversible unfolding/folding transition has been demonstrated to date. The detergents or the chaperonin proteins have been proposed to stabilize folding intermediates that kinetically limit folding by aggregating. Based on this hypothesis, we have investigated a number of experimental conditions and have developed a protocol for refolding, without assistants, that gives evidence of a reversible unfolding transition and leads to greater than 80% recovery of native enzyme. In addition to low protein concentration (10 micrograms/ml), low temperatures are required to maximize refolding. Otherwise optimal conditions give less than 10% refolding at 37 degrees C, whereas at 10 degrees C the recovery approaches 80%. The unfolding/refolding phases of the transition curves are most similar in the region of the transition, and refolding yields are significantly reduced when unfolded rhodanese is diluted to low urea concentrations, rather than to concentrations near the transition region. This is consistent with the formation of "sticky" intermediates that can remain soluble close to the transition region. Apparently, nonnative structures, e.g. aggregates, can form rapidly at low denaturant concentrations, and their subsequent conversion to the native structure is slow.     

Relationship between kinetic and equilibrium folding intermediates of creatine kinase

Creatine kinase (CK) is a dimeric enzyme important in ATP regeneration in cells where energy demands are high. The folding of CK under equilibrium and transient conditions has been studied in detail and is found to be complex. At equilibrium in 0.8 M GuHCl, 90% of CK molecules are in the form of a partially structured, monomeric intermediate. We exploit this property to measure kinetics of refolding and unfolding to and from this equilibrium intermediate (EI), using far-UV circular dichroism and intrinsic fluorescence as structural probes. We are thus able to compare the properties of EI and the kinetic intermediate formed during the burst phase in refolding. Native CK and EI unfold with rate constants in seconds and milliseconds, respectively. As is observed for refolding of fully-denatured CK, refolding from EI to the native state shows a burst phase followed by two exponential phases. The burst phase refolding intermediate is inferred to have more structure and greater stability than the equilibrium intermediate. When refolding from the fully-denatured state in 0.8 M GuHCl, the equilibrium intermediate is formed within the dead-time of mixing in the stopped-flow apparatus. The equilibrium intermediate may thus represent a kinetic intermediate formed early during folding.     

Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy

The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein. The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS)     

荧光研究表明去辅基天青蛋白突变体M121L至少存在两种不同的构象

以往对绿脓杆菌去辅基天青蛋白变性机制的研究认为它经历了一个复杂的反应过程,相比之下,锌离子替代的天青蛋白的变性符合简单的二态模型。以脲为变性剂对去辅基天青蛋白突变体M121L的变性过程进行了研究。结果表明,虽然稳态条件下突变体的变性／复性符合二态模型,但其动力学过程复杂,并可用溶液中存在着两种可以相互转化的构象的变性／复性来解释。天然态N1去折叠的速度快,其重折叠的速度也快,N1的折叠机制可用存在着折叠途径上的快速折叠中间体模型来描述;天然态N2的去折叠速度慢,其重折叠主要是首先生成天然态N1,然后再缓慢地转化成N2。添加Zn^2 能够把两种构象整合成一种构象,相应地,Zn^2 替代的天青蛋白突变体的变性过程简化为单指数过程。对该突变体的研究加深了对天青蛋白去折叠机制的理解。     

The mechanism of stabilization of the structure of nuclease-T by binding of ligands.

The rate of unfolding of Nuclease-T at pH 8,20 degrees was determined as a function of concentration of the ligands deoxythymidine 3',5'-diphosphate (pdTp) and Ca2+ on the basis of the rate of exchange between free fragment, Nuclease-T(50-149) and labeled fragment, Nuclease-T-(50-149) incorporated in the structure of nuclease-T (Taniuchi, H. (1973) J. Biol. Chem. 248, 5164-5174). The rate constant of unfolding of unliganded Nuclease-T' was 4.6 times 10-4s-1. Those of Nuclease-T' bound with pdTp, with Ca2+, and with both pdtp and Ca2+ were 9.0 times 10-5, 1.6 times 10-4, and 2.2 times 10-5s-1, respectively. The association constants of pdTp and Ca2+ with Nuclease-T' were found to be 1.0 times 10-4 and 2.0 times 10-2 m-1, respectively. Those of pdTp with Nuclease-T' plus Ca2+ and of Ca2+ with Nuclease-T' plus pdTp were 4 times 10-5 and 1.4 times 10-4M-1, respectively. The calculation of free energy change on the basis of the association constants shows that the magnitude of negative free energy change involved in the binding of either of the two ligands increases by approximately 2 kcal when the other ligand is already bound. There is a correlation between the free energy change and the specifically coupled with the cooperative interacions operating throught the three-dimensional structure resulting in strengthening of the interactions throughtout the structure, including those with the ligands, without a large change in conformation.