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1.
Sasahara K  Demura M  Nitta K 《Proteins》2002,49(4):472-482
The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.  相似文献   

2.
The kinetics of protein folding for horse ferricytochrome c was investigated by stopped-flow methods, using far-UV circular dichroism (CD), near-UV CD, and tryptophan fluorescence to probe the formation of secondary structure and tertiary interactions. In the far-UV region of the CD spectrum (222 nm), 44% of the total change associated with refolding occurs within the dead time of the stopped-flow experiment, indicating that a significant amount of helical secondary structure is formed in less than 4 ms. The remaining changes in the ellipticity at 222 nm occur in two kinetic phases with time constants of about 40 ms and 0.7 s, respectively. In contrast, there is no evidence for rapid changes in the ellipticity at 289 nm: an aromatic CD band, which is indicative of the formation of a tightly packed core, only begins to appear in a 400-ms step and is completed in a final 10-s phase. The fluorescence of a single tryptophan at position 59, which becomes quenched upon folding via nonradiative energy transfer to the heme group, provides complementary information on the condensation of the polypeptide chain during refolding. The fluorescence-detected stopped-flow folding kinetics of ferricytochrome c exhibits a 35% decrease in fluorescence during the dead time, suggesting that a substantial decrease in the average tryptophan-heme distance occurs on a submillisecond time scale. The subsequent fluorescence changes exhibit two prominent phases with time constants of about 20 and 300 ms, followed by a minor 5-s phase.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
R67 dihydrofolate reductase (DHFR) is a homotetrameric enzyme. Its subunit has a core structure consisting of five antiparallel beta-strands that form a compact beta-barrel. Our interest was to describe the molecular mechanism of the complete folding pathway of this beta-sheet protein, focusing on how the oligomerization steps are coordinated with the formation of secondary and tertiary structures all along the folding process. The folding kinetics of R67 dihydrofolate reductase into dimers at pH 5.0 were first examined by intrinsic tryptophan fluorescence, fluorescence energy transfer, and circular dichroism spectroscopy. The process was shown to consist of at least four steps, including a burst, a rapid, a medium, and a slow phase. Measurements of the ellipticity at 222 nm indicated that about 50% of the total change associated with refolding occurred during the 4 ms dead time of the stopped-flow instrument, indicating a substantial burst of secondary structure. The bimolecular association step was detected using fluorescence energy transfer and corresponded to the rapid phase. The slow phase was attributed to a rate-limiting isomerization of peptidyl-prolyl bonds involving 15% of the unfolded population. A complete folding pathway from the unfolded monomer to the native tetramer was proposed and an original model based upon the existence of early partially folded monomeric intermediates, rapidly stabilized in a dimeric form able to self-associate into the native homotetramer was formulated. The rate constants of these various steps were determined by fitting the kinetic traces to this model and supported our mechanistic assumptions.  相似文献   

4.
Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.  相似文献   

5.
To probe the role of individual disulfide bonds in the folding kinetics of hen lysozyme, the variants with two mutations, C30A,C115A, C64A,C80A, and C76A,C94A, were constructed. The corresponding proteins, each lacking one disulfide bond, were produced in Escherichia coli as inclusion bodies and solubilized, purified, and renatured/oxidized using original protocols. Their enzymatic, spectral, and hydrodynamic characteristics confirmed that their conformations were very similar to that of native wild-type (WT) lysozyme. Stopped-flow studies on the renaturation of these guanidine-unfolded proteins with their three disulfides intact showed that, for the three variants, the native far-UV ellipticity was regained in a burst phase within the 4-ms instrument dead-time. The transient overshoots of far-UV ellipticity and tryptophan fluorescence that follow the burst phase, as well as the kinetics of transient 8-anilino-1-naphthalene-sulfonic acid (ANS) binding, were diversely affected depending on the variant. Together with previous reports on the folding kinetics of WT lysozyme carboxymethylated on cysteines 6 and 127, detailed analysis of the kinetics showed that (1) none of the disulfide bonds were indispensable for the rapid formation (<4 ms) of the native-like secondary structure; (2) the two intra-alpha-domain disulfides (C6-C127 and C30-C115) must be simultaneously present to generate the trapped intermediate responsible for the slow folding population observed in WT lysozyme; and (3) the intra-beta-domain (C64-C80) and the inter-alphabeta-domains (C76-C94) disulfides do not affect the kinetics of formation of the trapped intermediate but are involved in its stability.  相似文献   

6.
The kinetics of Ca2+ dissociation from fluo-3 was measured using stopped flow fluorimetry. Analysis of dissociation revealed, in contrast to other commonly used fluorescent Ca2+ indicators, a biexponential behaviour with two distinct dissociation rates of 550 s-1 and 200 s-1 at physiological pH and room temperature. The dissociation rate constant of the fast phase increases to 700 s-1 at physiological temperature, whereas that of the slow phase does not change markedly. While the rate constants do not depend on pH between 6.6 and 7.8, the dissociation turns out to be monoexponential at pH 5.86. The association rate of Ca2+ to fluo-3 could not be measured within the mixing dead time and is estimated to be above 10(9) M-1 s-1. Since the rate constants of fluo-3 are larger than those of other fluorescent Ca2+ indicators, fluo-3 is well suited for investigations of Ca2+ oscillations in biological systems.  相似文献   

7.
Cytochrome c-554 of the ammonia-oxidizing chemolithoautotropic bacteria is thought to mediate electron transfer from hydroxylamine oxidoreductase to a terminal oxidase and/or to ammonia monooxygenase. The cytochrome has four c hemes which interact magnetically and have the same redox potential. We report that the kinetics of reduction of ferric cytochrome c-554 by dithionite or the oxidation of ferrous cytochrome c-554 by O2 or H2O2 are complex and multiphasic. Transient rapid-scan difference spectra indicate discrete maxima at approximately 418 nm, 425 nm and 432 nm. Absorbance changes at all three difference maxima appear to occur in all kinetic phases, although not in equal amounts for each wavelength. Reduction by 20 mM dithionite was biphasic. At pH 7.5 the first phase, which involved approximately 50% of the total absorbance change, had a rate constant (20 degrees C) of 140 s-1 and energy of activation of 20 kJ X mol-1. The slow phase had a rate constant 0.43 s-1 and a relatively high energy of activation, 87 kJ X mol-1, suggesting that a change in protein configuration accompanied the reaction. As the pH of the solution increased, the rate constant for both phases decreased and the fraction of absorbance change in the rapid phase increased. Oxidation of ferrous cytochrome c-554 by O2 involved a discrete rapid phase with a rate constant of 14 s-1, accounting for 6% of the absorbance. The remainder of the reaction was multiphasic with rate constants in the range 0.1-0.01 s-1. With H2O2 as the oxidant, the rapid phase involved 39% of the change in absorbance with a rate constant of 19 s-1. The remainder of the reoxidation was multiphasic with rate constants ranging over 0.4-0.01 s-1.  相似文献   

8.
Refolding kinetics of two homologous proteins, lysozyme and alpha-lactalbumin, were studied by following the time-dependent changes in the circular dichroism spectra in the aromatic and the peptide regions. The refolding was initiated by 20-fold dilution of the protein solutions originally unfolded at 6 M guanidine hydrochloride, at pH 1.5 for lysozyme and pH 7.0 for alpha-lactalbumin at 4.5 degrees C. In the aromatic region, almost full changes in ellipticity that were expected from the equilibrium differences in the spectra between the native and unfolded proteins were observed kinetically. The major fast phase of lysozyme folding has a decay time of 15 s. The decay time of alpha-lactalbumin depends on the presence or absence of bound Ca2+: 10 s for the holoprotein and 100 s for the apoprotein. In the peptide region, however, most of the ellipticity changes of the two proteins occur within the dead time (less than 3 s) of the present measurements. This demonstrates existence of an early folding intermediate which is still unfolded when measured by the aromatic bands but has folded secondary structure as measured by the peptide bands. Extrapolation of the ellipticity changes to zero time at various wavelengths gives a spectrum of the folding intermediate. Curve fitting of the peptide spectra to estimate the secondary structure fractions has shown that the two proteins assume a similar structure at an early stage of folding and that the intermediate has a structure similar to that of partially unfolded species produced by heat and, for alpha-lactalbumin, also by acid and a moderate concentration of guanidine hydrochloride.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

9.
Lignin peroxidase compound III. Mechanism of formation and decomposition   总被引:9,自引:0,他引:9  
Lignin peroxidase compound III (LiPIII) was prepared via three procedures: (a) ferrous LiP + O2 (LiPIIIa), (b) ferric LiP + O2-. (LiPIIIb), and (c) LiP compound II + excess H2O2 followed by treatment with catalase (LiPIIIc). LiPIIIa, b, and c each have a Soret maximum at approximately 414 nm and visible bands at 543 and 578 nm. LiPIIIa, b, and c each slowly reverted to native ferric LiP, releasing stoichiometric amounts of O2-. in the process. Electronic absorption spectra of LiPIII reversion to the native enzyme displayed isosbestic points in the visible region at 470, 525, and 597 nm, suggesting a single-step reversion with no intermediates. The LiPIII reversion reactions obeyed first-order kinetics with rate constants of approximately 1.0 X 10(-3) s-1. In the presence of excess peroxide, at pH 3.0, native LiP, LiPII, and LiPIIIa, b, and c are all converted to a unique oxidized species (LiPIII*) with a spectrum displaying visible bands at 543 and 578 nm, but with a Soret maximum at 419 nm, red-shifted 5 nm from that of LiPIII. LiPIII* is bleached and inactivated in the presence of excess H2O2 via a biphasic process. The fast first phase of this bleaching reaction obeys second-order kinetics, with a rate constant of 1.7 X 10(1) M-1 s-1. Addition of veratryl alcohol to LiPIII* results in its rapid reversion to the native enzyme, via an apparent one-step reaction that obeys second-order kinetics with a rate constant of 3.5 X 10(1) M-1 s-1. Stoichiometric amounts of O2-. are released during this reaction. When this reaction was run under conditions that prevented further reactions, HPLC analysis of the products demonstrated that veratryl alcohol was not oxidized. These results suggest that the binding of veratryl alcohol to LiPIII* displaces O2-., thus returning the enzyme to its native state. In contrast, the addition of veratryl alcohol to LiPIII did not affect the rate of spontaneous reversion of LiPIII to the native enzyme.  相似文献   

10.
Sasahara K  Demura M  Nitta K 《Biochemistry》2000,39(21):6475-6482
Equilibrium unfolding of hen egg white lysozyme as a function of GdnCl concentration at pH 0.9 was studied over a temperature range 268.2-303.2 K by means of CD spectroscopy. As monitored by far- and near-UV CD at 222 and 289 nm, the lack of coincidence between two unfolding transition curves was observed, which suggests the existence of a third conformational species in addition to native and unfolded states. The three-state model, in which a stable intermediate is populated, was employed to estimate the thermodynamic parameters for the GdnCl-induced unfolding. It was found that the transition from the native to intermediate states proceeds with significant changes in enthalpy and entropy due to an extremely cooperative process, while the transition from the intermediate to unfolded states shows a low cooperativity with small enthalpy and entropy changes. These results indicate that the highest energy barrier for the GdnCl-induced unfolding of hen lysozyme is located in the process from the native state to the intermediate state, and this process is largely responsible for the cooperativity of protein unfolding.  相似文献   

11.
M Oliveberg  B G Malmstr?m 《Biochemistry》1991,30(29):7053-7057
Internal electron-transfer reactions in cytochrome oxidase following flash photolysis of the CO compounds of the enzyme reduced to different degrees (2-4 electron equiv) have been followed at 445, 605, and 830 nm. Apart from CO dissociation and recombination, two kinetic phases are seen both at 445 and at 605 nm with rate constants of 2 x 10(5) and 1.3 x 10(4) s-1, respectively; at 605 nm, an additional phase with a rate constant of 400 s-1 is resolved. At 830 nm, only the second reaction phase (rate constant of 1.3 x 10(4) s-1) is observed. The amplitude of the first phase is largest with the two-electron-reduced enzyme, whereas that of the second phase is maximal at the three-electron-reduction level. Neither phase shows any marked pH dependence. The reaction in the first phase has a free energy of activation of 41 kJ mol-1 and an entropy of activation of -14 JK-1 mol-1. Analysis suggests that the two rapid reaction phases represent internal electron redistributions between the bimetallic site and cytochrome a, and between cytochrome a and CuA, respectively. The slow phase (400 s-1) probably involves a structural rearrangement.  相似文献   

12.
The reduction kinetics of NADPH:cytochrome P-450 reductase have been investigated by the laser flash photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Transients observed at 470 nm at neutral pH indicated that the oxidized reductase was reduced via second-order kinetics with a rate constant of 6.8 X 10(7) M-1 s-1. The second-order rate constant corresponding to the formation of the protein-bound semiquinone (measured at 585 nm) was essentially the same as that obtained at 470 nm (7.1 X 10(7) M-1 s-1). Subsequent to this rapid formation of protein-bound semiquinone, a partial exponential decay was observed at 585 nm. The rate of this decay remained invariant with protein concentration between pH 5.0 and 7.0, and a first-order rate constant of 70 s-1 was obtained for this process. This is assigned to intramolecular electron transfer from FADH. to FMN. Prior reduction of the enzyme to the one-electron level led to a decrease in both the second-order rate constant for reduction (2 X 10(7) M-1 s-1) and the first-order intraflavin electron transfer rate constant (15 s-1). The protein-bound FAD moiety of FMN-depleted reductase was reduced by 5-dRfH. with a second-order rate constant that was identical with that observed with the native enzyme (6.9 X 10(7) M-1 s-1). However, with this species no significant decay of the FAD semiquinone was observed at 585 nm following its rapid formation, consistent with the above assignment of this kinetic process.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

13.
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)  相似文献   

14.
Methanol-induced conformational transitions of hen egg white lysozyme were investigated with a combined use of far- and near-UV CD and NMR spectroscopies, ANS binding and small-angle X-ray scattering. Addition of methanol induced no global change in the native conformation itself, but induced a transition from the native state to the denatured state which was highly cooperative, as shown by the coincidence of transition curves monitored by the far- and near-UV CD spectroscopy, by isodichroic points in the far- and near-UV CD spectra and by the concomitant disappearance of individual 1H NMR signals of the native state. The ANS binding experiments could detect no intermediate conformer similar to the molten globule state in the process of the methanol denaturation. However, at high concentration of methanol, e.g., 60% (v/v) methanol/water, a highly helical state (H) was realized. The H state had a helical content much higher than the native state, monitored by far-UV CD spectroscopy, and had no specific tertiary structure, monitored both by near-UV CD and NMR spectroscopy. The radius of gyration in the H state, 24.9 angstroms, was significantly larger than that in the native state (15.7 angstroms). The Kratky plot for the H state did not show a clear peak and was quite similar to that for the urea-denatured state, indicating a complete lack of globularity. Thus we conclude that the H state has a considerably expanded, flexible broken rod-like conformation which is clearly distinguishable from the "molten globule" state. The stability of both N and H states depends on pH and methanol concentration. Thus a phase diagram involving N and H was constructed.  相似文献   

15.
Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.  相似文献   

16.
Slow dissociation of ATP from the calcium ATPase   总被引:1,自引:0,他引:1  
The acyl-phosphate intermediate of the sarcoplasmic reticulum calcium ATPase reaction, formed in a brief incubation of vesicular enzyme with 5 microM [gamma-32P]ATP and calcium, reacts biphasically with added ADP (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4). Both the burst size and the rate constant for the slow phase increase with increasing ADP concentration in the way that is expected if the burst represents very rapid formation of an equilibrium amount of enzyme-bound ATP and the slow phase represents rate-limiting dissociation of ATP. Also consistent with this interpretation are the slow labeling of phosphoenzyme under conditions in which unlabeled ATP must dissociate first and the observation of a burst of ATP formation on ADP addition to phosphoenzyme. Values of the equilibrium constants for ADP dissociation from phosphoenzyme (0.75 mM), for ATP formation on the enzyme (2.3), and for the ATP dissociation rate constant (37 s-1) were obtained from a quantitative analysis of the data.  相似文献   

17.
S Hahm  B Durham  F Millett 《Biochemistry》1992,31(13):3472-3477
The reactions of yeast cytochrome c peroxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) [Ru(II)] were studied by flash photolysis. All of the derivatives formed complexes with cytochrome c peroxidase compound I (CMPI) at low ionic strength (2 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in electron transfer to the ferric heme group in cytochrome c, followed by electron transfer to the radical site in CMPI. This reaction was biphasic and the rate constants were independent of CMPI concentration, indicating that both phases represented intracomplex electron transfer from the cytochrome c heme to the radical site in CMPI. The rate constants of the fast phase were 5200, 19,000, 55,000, and 14,300 s-1 for the derivatives modified at lysines 13, 25, 27, and 72, respectively. The rate constants of the slow phase were 260, 520, 200, and 350 s-1 for the same derivatives. These results suggest that there are two binding orientations for cytochrome c on CMPI. The binding orientation responsible for the fast phase involves a geometry that supports rapid electron transfer, while that for the slow phase allows only slow electron transfer. Increasing the ionic strength up to 40 mM increased the rate constant of the slow phase and decreased that of the fast phase. A single intracomplex electron transfer phase with a rate constant of 2800 s-1 was observed for the lysine 72 derivative at this ionic strength. When a series of light flashes was used to titrate CMPI to CMPII, the reaction between the cytochrome c derivative and the Fe(IV) site in CMPII was observed. The rate constants for this reaction were 110, 250, 350, and 140 s-1 for the above derivatives measured in low ionic strength buffer.  相似文献   

18.
Kinetics of disulfide reduction in alpha-lactalbumin by dithiothreitol are investigated by measuring time-dependent changes in absorption at 310 nm and in CD ellipticity at 270 nm (pH 8.5 or 7.0, and 25 degrees C). When the disulfide-intact protein is folded, the kinetics are biphasic. The disulfide bond between the half-cystines-6 and -120 is reduced in the fast phase, and the other three disulfide bonds are reduced in the slow phase. The apparent rate constants of the two phases are both proportional to the concentration of dithiothreitol, indicating that both phases are expressed by bimolecular reactions. However, detailed molecular mechanisms that determine the reaction rates are markedly different between the two phases. The slow phase shows a sigmoidal increase in the reaction rate with increasing concentration of a denaturant, urea, and is also accelerated by destabilization of the native state on removal of the bound Ca2+ ion in the protein. The disulfide bonds are apparently protected against the reducing agent in the native structure. The fast phase reaction rate is, however, decreased with an increase in the concentration of urea, and the disulfide bond shows extraordinary superreactivity in native conditions. It is 140 times more reactive than normal disulfides in the fully accessible state, and three-disulfide alpha-lactalbumin produced by the fast phase assumes nativelike structure under a strongly native condition. As ionic strength does not affect the superreactivity of this disulfide bond, electrostatic contributions to the reactivity must be negligible. Inspection of the disulfide bond geometry based on the refined X-ray coordinates of baboon alpha-lactalbumin [Acharya et al. (1989) J. Mol. Biol. 208, 99-127] and comparison of the geometry with those in five other proteins clearly demonstrate that the superreactivity arises from the geometric strain imposed on this disulfide bond by the native structure folding. Relationships of the disulfide strain energy to the protein stability and the disulfide reactivity are discussed.  相似文献   

19.
Sasahara K  Nitta K 《Proteins》2006,63(1):127-135
The equilibrium and kinetics of folding of hen egg-white lysozyme were studied by means of CD spectroscopy in the presence of varying concentrations of ethanol under acidic condition. The equilibrium transition curves of guanidine hydrochloride-induced unfolding in 13 and 26% (v/v) ethanol have shown that the unfolding significantly deviates from a two-state mechanism. The kinetics of denaturant-induced refolding and unfolding of hen egg-white lysozyme were investigated by stopped-flow CD at three ethanol concentrations: 0, 13, and 26% (v/v). Immediately after dilution of the denaturant, the refolding curves showed a biphasic time course in the far-UV region, with a burst phase with a significant secondary structure and a slower observable phase. However, when monitored by the near-UV CD, the burst phase was not observed and all refolding kinetics were monophasic. To clarify the effect of nonnative secondary structure induced by the addition of ethanol on the folding/unfolding kinetics, the kinetic m values were estimated from the chevron plots obtained for the three ethanol concentrations. The data indicated that the folding/unfolding kinetics of hen lysozyme in the presence of varying concentrations of ethanol under acidic condition is explained by a model with both on-pathway and off-pathway intermediates of protein folding.  相似文献   

20.
The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of the native protein, but devoid of enzyme activity. The fluorescence emission spectrum of this intermediate was determined. Only the slow phase was detected for the unfolding process; it was not attributable to proline isomerization. Several models were assumed, and simulated kinetics derived from these models were compared with the experimental results. A plausible one accounting for most of the data is proposed.  相似文献   

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