Kinetic study of protein unfolding and refolding using urea gradient electrophoresis

When total cytoplasmic RNA from mouse Friend cells is fractionated using oligo(dT)-cellulose or poly(U)-Sepharose chromatography, approximately 20% of the messenger RNA activity (as measured in the reticulocyte lysate cell-free system) remains in the unbound fraction, even though this contains < 0.5% of the poly(A) (as measured by titration with poly(U)). This RNA, operationally defined as poly(A)^?, is found almost entirely in polysome structures in vivo. Its major translation products, as shown by one-dimensional sodium dodecyl sulphate-containing gels, are the histones and actin. Two-dimensional gels (isoelectric focusing: sodium dodecyl sulphate/gel electrophoresis) show that, with the exception of the mRNAs coding for histones, poly(A)^? mRNA encodes similar proteins to poly(A)⁺ mRNA, though in very different abundances. This is directly confirmed by the arrest of the translation of the abundant poly(A)^? mRNAs after hybridization with a complementary DNA transcribed from poly(A)⁺ RNA.RNA sequences which are rare in the poly(A)⁺ RNA are also found in poly(A)^? RNA, as shown by hybridizing a cDNA transcribed from poly(A)⁺ RNA to total and poly(A)^? polysomal RNA. That this does not simply represent a flow-through of poly(A)⁺ RNA is indicated by (i) the lack of poly(A) by hybridizing to poly(U) in this fraction, (ii) the fact that further passage through poly(U)-Sepharose does not remove the hybridizing sequences, (iii) the very different quantitative distribution of proteins encoded by poly(A)⁺ and poly(A)^? RNAs. We also think that it does not result from removal of poly(A) from polyadenylated RNAs during extraction because RNAs prepared using the minimum of manipulations give similar results. The distribution of both total mRNA and α and β globin mRNAs between poly(A)⁺ and poly(A)^? RNA does not change significantly during the dimethyl sulphoxide-induced differentiation of Friend cells.     

The nature of the rate-limiting steps in the refolding of the cofactor-dependent protein aspartate aminotransferase

The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied following unfolding in 6 m guanidine hydrochloride for different periods of time. Whereas reactivation of equilibrium-unfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k approximately 0.75 min(-1) at 20 degrees C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stopped-flow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5'-phosphate (PLP) to the polypeptide is much slower (k approximately 0.08 min(-1)). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.     

The nature of the rate-limiting step in aniline hydroxylation involving cytochrome p-450 rat liver microsomes.

The kinetics of aniline hydroxylation was studied with: (1) rat liver microsomes involving NADPH and O2 (system 1), (2) hepatic microsomes and tert-butylhydroperoxide (system 2) and (3) microsomes and cumyl hydroperoxide (system 3) at 15--37 degrees C. The reactions were characterized by the values of the aniline oxidation rate constants, k2 = V/E0, where E0 is the initial concentration of cytochrome P-450: K 1/2 = 1.60 - 10(8) EXP (-13 400/RT) sec-1, k 2/2 = 1.66 - 10(9) exp (-14 500/RT) sec-1, k 3/2 = 6.83 - 10(9) exp (-15 300/RT) sec-1. The values of delta H0 and delta S0, were calculated and compared for the three systems. The evidence suggests that oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned systems. The nature of aniline binding to cytochrome P-450 and that of the hydroxylating agent have been discussed.     

Kinetic investigations of the rate-limiting step in human 12- and 15-lipoxygenase

Mammalian lipoxygenases have been implicated in several inflammatory disorders; however, the details of the kinetic mechanism are still not well understood. In this paper, human platelet 12-lipoxygenase (12-hLO) and human reticulocyte 15-lipoxygenase-1 (15-hLO) were tested with arachidonic acid (AA) and linoleic acid (LA), respectively, under a variety of changing experimental conditions, such as temperature, dissolved oxygen concentration, and viscosity. The data that are presented show that 12-hLO and 15-hLO have slower rates of product release (k(cat)) than soybean lipoxygenase-1 (sLO-1), but similar or better rates of substrate capture for the fatty acid (k(cat)/K(M)) or molecular oxygen [k(cat)/K(M(O)2)]. The primary, kinetic isotope effect (KIE) for 15-hLO with LA was determined to be temperature-independent and large ((D)k(cat) = 40 +/- 8), over the range of 10-35 degrees C, indicating that C-H bond cleavage is the sole rate-limiting step and proceeds through a tunneling mechanism. The (D)k(cat)/K(M) for 15-hLO, however, was temperature-dependent, consistent with our previous results [Lewis, E. R., Johansen, E., and Holman, T. R. (1999) J. Am. Chem. Soc. 121, 1395-1396], indicating multiple rate-limiting steps. This was confirmed by a temperature-dependent, k(cat)/K(M) solvent isotope effect (SIE), which indicated a hydrogen bond rearrangement step at low temperatures, similar to that of sLO-1 [Glickman, M. H., and Klinman, J. P. (1995) Biochemistry 34, 14077-14092]. The KIE could not be determined for 12-hLO due to its inability to efficiently catalyze LA, but the k(cat)/K(M) SIE was temperature-independent, indicating distinct rate-limiting steps from both 15-hLO and sLO-1.     

Structural studies on acid unfolding and refolding of recombinant human interferon gamma

Interferon gamma is distinguished from other types of interferons in its instability upon acid treatment, as demonstrated by a loss of antiviral activity. Acid unfolding and refolding experiments were performed with recombinant DNA derived human interferon gamma. When the protein was subjected to unfolding and refolding, the refolded protein showed two peaks (peaks I and II) in gel filtration which have been shown to differ in size, structure, and antiviral activity. When the smaller, peak II, form was unfolded by dialysis against 0.01 M HCl containing 0.1 M NaCl (pH 2) and refolded by dialysis against various solvents at neutral pH, it re-formed as peak II but also generated peak I, and the ratio of the two forms was dependent on protein concentration and solvent conditions. Higher protein concentrations and higher ionic strength led to a greater ratio of peak I to peak II. Phosphate buffers caused precipitation of peak I. Since peak II is 4-8 times more active than peak I in the antiviral bioassay, generation of peak I by acid treatment of peak II should lead to a decrease in antiviral activity.     

Kinetic investigation of unfolding and partial refolding of a crab satellite (dA-dT)n.

Crab (dA-dT)n was isolated from the testes of Cancer borealis by a procedure involving separation of DNA and segregation of the satellite fraction by Hg2+ binding/Cs2SO4 density gradient ultracentrifugation. The titration of crab (dA-dT)n samples at 10 degrees indicated a sharp absorbance change at pH 11.98 in agreement with the pHm value observed for synthetic poly(dA-dT) under identical conditions. The reversal of the titration, however, resulted only in about 50% recovery of the original absorbance (at 260 nm) in marked contrast to the complete reversibility of the synthetic material. pH-jump experiments were carried out for the purpose of characterizing the rates and mechanisms of conformational transitions brought about by changes in the solution environment. It was found that the disintegration of the putative native structure of crab (dA-dT)n starts with a very fast reaction (occurring within the 6-msec deadtime of the instrument and comprising 65% of the total absorbance change) and it is completed via a slower first-order reaction (k = 66 sec minus 1). It is postulated that the first process is due to the rapid untwisting of end regions and, perhaps, some short hairpin-like helical branches present on the macromolecules. The second reaction is believed to be the end-to-end type unwinding of the double-helical backbone of crab (dA-dT)n. In the presence of low concentration (3 mug/ml) of Hg2+ ions the overall rate of disintegration process decreased drastically. pH jumps from pH values above pHm to values below were used to study the rates of absorbance changes corresponding to the refolding of the strands of denatured crab (DA-dT)n. A concentration independent process consisting of two phases was observed. The first phase was a gradual nonexponential process spanning the first second of the reaction, and the other, a very slow first-order process characterized by the rate constant value of 0.053 sec minus 1. It is proposed that the first part of the process (involving about 24% of nucleotide residues) is an intramolecular formation of helical hairpins (frequently interrupted by mismatching bases) and the second part is a manifestation of some association of the extant unpaired bases during the folding of the branched structure. Refolded crab (dA-dT)n samples when subjected again to pH greater than pHm in the stopped-flow apparatus displayed not the disintegration pattern of the native crab (dA-dT)n but rather that of synthetic poly(dA-dT. The marked facility of crab (dA-dT)n macromolecules for rapid conformational transitions induced by slight changes in the solution environment might be relevant to the biological function of this DNA.     

The rate-limiting step in the actomyosin adenosinetriphosphatase cycle

We have previously shown that myosin does not have to detach from actin during each cycle of ATP hydrolysis. In the present study, using the A-1 isoenzyme of myosin subfragment 1, we have investigated the nature of the rate-limiting steps in the ATPase cycle. Our results show that, at 15 degrees C, at very low ionic strength, KATPase determined from the double-reciprocal plot of ATPase activity vs. actin concentration is more than 6-fold stronger than KBINDING determined by directly measuring the binding of A-1 myosin subfragment 1 to actin during steady-state ATP hydrolysis. Computer modeling shows that this large difference between KATPase and KBINDING is not compatible with Pi release being the rate-limiting step in the ATPase cycle. If Pi release is not rate limiting, it is possible that the ATP hydrolysis step, itself, is rate limiting. However, this predicts that, at high actin concentration, the value of the initial Pi burst should be close to zero. Therefore, we measured the magnitude of the initial Pi burst in the presence of actin, using both direct measurement and measurement of relative fluorescence magnitude. Our results suggest that the magnitude of the initial Pi burst in the presence of actin is considerably higher than would be expected if the ATP hydrolysis step were the rate-limiting step in the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism spectroscopy of the intermediates that precede the rate-limiting step of the refolding pathway of bovine pancreatic trypsin inhibitor. Relationship of conformation and the refolding pathway

Circular dichroism spectra of the partially folded trapped intermediates were measured in order to aid in the elucidation of the conformational forces which determine a nonrandom, nonsequential pathway of disulfide bond formation upon refolding of bovine pancreatic trypsin inhibitor. Whatever conformation was responsible for the kinetic rates of the intermediates should be stabilized by the presence of their trapped disulfide bonds. The near-ultraviolet spectra provide considerable information about the environments of the aromatic and disulfide side chains. The predominant single-disulfide intermediate has significant nonrandom conformation not present in the fully reduced protein, with aromatic rings and the disulfide bond in stabilized asymmetric environments. Forming either of the two nonnative, but kinetically important, second disulfides in this intermediate does not produce unequivocably different conformations. Forming a second native, but kinetically unproductive, disulfide produces a substantial decrease in randomness, which may hinder formation of the third disulfide. The largest conformational changes occur upon disulfide rearrangement to the stable, correctly refolded, two- and three-disulfide species. Interpretation of the far-ultraviolet spectra in terms of the secondary structure of the intermediates is uncertain, due to the atypical spectra of the folded forms of the protein. Consequently, we are unable to determine unambiguously the secondary structure of the intermediates. However, all the spectra show that nonrandom conformations of the polypeptide chain gradually appear as disulfide bond formation progresses, as expected from the nonrandom pathway of the latter.     

NMR studies on the equilibrium unfolding of ketosteroid isomerase by urea

Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of (1)H-(15)N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the beta5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the beta6-strand. The results thus suggest that the interface region primarily around the beta5- and beta6-strands could play an important role as the starting positions in the unfolding process of KSI.     

The nature of the rate-limiting step in aniline hydroxylation involving cytochrome P-450 from rat liver microsomes

The kinetics of aniline hydroxylation with: 1) rat liver microsomes involving NADPH and O2 (System I); 2) hepatic microsomes and tertiary butylhydroperoxide (System II) and 3) microsomes and cumyl hydroperoxide (System III) within 15--37 degrees C has been studied. The reactions were characterized by the values of the aniline oxidation rate constants, k2=v/[E]0, where [E]0 is the initial concentration of cytochrome P--450: k1 2=1,60.10(8) exp (--13400/RT) sec-1., k2 2=1,66.10(9) exp (--14500/RT) sec-1., k3 2=6,83.10(9) exp (--15300/RT) sec-1. The values of delta H* and delta S* were calculated and compared for these three systems. A conclusion is drawn that the act of oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned system.     

Kinetic studies of the oxidation of glutathione in protein refolding buffer

Here, we examined the change of glutathione (GSH) under different conditions and determined the appropriate kinetic schemes to describe its change of concentration. GSH was continuously oxidized into glutathione disulfide (GSSG) as incubation period increased at the temperatures ranging from 10 to 50 °C while certain oxidation by-products were also observed at the later stage of reaction at higher temperatures (70–90 °C). The addition of 0.3 mM GSSG in 3 mM GSH solution delayed the onset of GSH oxidation without significantly changing the rate of GSH oxidation. Our results also revealed that GSH oxidation could be facilitated upon the addition of copper (II) ion whereas GSH oxidation was found to be decelerated when EDTA was present. In kinetic analysis, the reaction orders of GSH oxidation under various conditions were determined. Moreover, the temperature dependence of the rate of GSH oxidation was modeled by Arrhenius and Eyring equations and resultant activation parameters were also presented.     

The unfolding and refolding of cytoplasmic aspartate aminotransferase from pig heart.

The unfolding of cytoplasmic aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from protein fluorescence, c.d. and thiol-group reactivity indicated that the enzyme was unfolded in 6 M-GdnHCl. Spectroscopic studies showed that this unfolding was accompanied by dissociation of the pyridoxal 5'-phosphate cofactor. On dilution of the GdnHCl, re-activation of the enzyme occurred in reasonable yield, provided that dithiothreitol and pyridoxal 5'-phosphate were present. The regain of activity obeyed second-order kinetics. In the absence of added dithiothreitol and pyridoxal 5'-phosphate, substantial formation of high-Mr aggregates occurred.     

Thermodynamics of the denaturation of pepsinogen by urea.

The denaturation of swine pepsinogen has been studied as a function of urea concentration, pH, and temperature. The unfolding of the protein by urea has been found to be fully reversible under different conditions of pH, temperature, and denaturant concentration. Kinetic experiments have shown that the transition shows two-state behavior at 25 degrees C in the pH range 6-8 covered in this study. Analysis of the equilibrium data obtained at 25 degrees C according to Tanford (Tanford, C. (1970), Adv. Protein Chem. 24, 1) and Pace (Pace, N.C. (1975), Crit. Rev. Biochem. 3, 1) leads to the conclusion that the free energy of stabilization of native pepsinogen, relative to the denatured state, under physiological conditions, is only 6-12 kcal mol-1. The temperature dependence of the equilibrium constant for the unfolding of pepsinogen by urea in the range 20-50 degrees C at pH 8.0 can be described by assigning the following values of thermodynamic parameters for the denaturation at 25 degrees C: deltaH=31.5 kcal mol-1; deltaS=105 cal deg-1 mol-1; and deltaCp=5215 cal deg-1 mol-1.     

The isocitrate dehydrogenase phosphorylation cycle. Identification of the primary rate-limiting step

In Escherichia coli, the branch point between the Krebs cycle and the glyoxylate bypass is regulated by the phosphorylation of isocitrate dehydrogenase (IDH). Phosphorylation inactivates IDH, forcing isocitrate through the bypass. This bypass is essential for growth on acetate but does not serve a useful function when alternative carbon sources, such as glucose or pyruvate, are also present. When pyruvate or glucose is added to a culture growing on acetate, the cells responded by dephosphorylating IDH and thus inhibiting the flow of isocitrate through the glyoxylate bypass. In an effort to identify the primary rate-limiting step in the response of IDH phosphorylation to alternative carbon sources, we have examined the response rates of congenic strains of E. coli which express different levels of IDH kinase/phosphatase, the bifunctional protein which catalyzes this phosphorylation cycle. The rate of the pyruvate-induced dephosphorylation of IDH was proportional to the level of IDH kinase/phosphatase, indicating that IDH kinase/phosphatase was primarily rate-limiting for dephosphorylation. However, the identity of the primary rate-limiting step appears to depend on the stimulus, since the rate of dephosphorylation of IDH in response to glucose was independent of the level of IDH kinase/phosphatase.     

The unfolding and attempted refolding of mitochondrial aspartate aminotransferase from pig heart.

The role of prostaglandins in the regulation of muscle protein breakdown is controversial. We examined the influence of arachidonic acid (5 microM), prostaglandin E2 (PGE2) (2.8 microM) and the prostaglandin-synthesis inhibitor indomethacin (3 microM) on total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscles incubated under different conditions in vitro. In other experiments, the effects of indomethacin, administered in vivo to septic rats (3 mg/kg, injected subcutaneously twice after induction of sepsis by caecal ligation and puncture) on plasma levels and muscle release of PGE2 and on total and myofibrillar protein breakdown rates were determined. Total and myofibrillar proteolysis was assessed by measuring production by incubated muscles of tyrosine and 3-methylhistidine respectively. Arachidonic acid or PGE2 added during incubation of muscles from normal rats did not affect total or myofibrillar protein degradation under a variety of different conditions in vitro. Indomethacin inhibited muscle PGE2 production by incubated muscles from septic rats, but did not lower proteolytic rates. Administration in vivo of indomethacin did not affect total or myofibrillar muscle protein breakdown, despite effective plasma levels of indomethacin with decreased plasma PGE2 levels and inhibition of muscle PGE2 release. The present results suggest that protein breakdown in skeletal muscle of normal or septic rats is not regulated by PGE2 or other prostaglandins.     

The rate-limiting step of the protamine-induced adenosine triphosphatase activity of adenosine triphosphate-G-actin.

The release of Pi from the Pi-G-actin-ADP complex is the rate-limiting step in the ATPase activity that is shown by ATP-G-actin in the presence of protamine.     

Studies on the irreversible step of pepsinogen activation

The bond cleavage step of pepsinogen activation has been investigated in a kinetic study in which the denatured products of short-term acidifications were separated on SDS-polyacrylamide gels and the peptide products were quantitated by densitometry. Although several peptide products were observed, under the conditions of the experiments (pH values between 2.0 and 2.8, 22 degrees C), the only one that was a product of an initial bond cleavage was the 44-residue peptide, which upon removal from pepsinogen yields pepsin. The rate constant for this bond cleavage is 0.015 s-1 at pH 2.4, which is the same as that at which the alkali-stable potential activity of pepsinogen had been found to convert to the alkali-labile activity of pepsin. When the conversion of zymogen to enzyme was followed by the change in fluorescence of adsorbed 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS), the rate of change in TNS fluorescence was the same as the conversion to alkali lability. However, pepstatin blocked the bond cleavage of pepsinogen to pepsin, but it permitted the fluorescence change to proceed. In fact, it accelerated the apparent rate of change of TNS fluorescence by shifting the pKa of an essential conjugate acid from 1.7 to 2.6. The conversion to alkali lability, therefore, may be considered to be a composite of a relatively slow conformational change (at the measured rate), followed immediately by a relatively fast bond cleavage.     

Kinetics of unfolding and refolding of proteins. I. Mathematical analysis

A mathematical method for the phenomenological analysis of unimolecular reaction kinetics is presented. Starting from the solution to the differential equations of the system, equations have been developed to relate the unknown parameters inherent to any reaction mechanism, such as the microscopic rate constants for individual reaction steps, to the experimental quantities obtained from the rate of change of average physical properties of the system. Provided that kinetic measurements can be made in both forward and reverse directions, all unknown parameters can be evaluated for many simple mechanisms, and for some of them characteristic relations between observable quantities can be established, which can serve as criteria for immediate rejection. The results will be applied in the following papers to experimental studies of denaturation and renaturation of several proteins.