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PubMed1989

[Flow cytometric analysis of cell proliferation kinetics using the anti-BrdUrd antibody].

There is a growing interest in analysis of cell kinetics in a field of clinical oncology. The development of a monoclonal antibody to bromodeoxyuridine (BrdUrd), a thymidine analogue, is epoch making in the research of cell kinetics, and flow cytometry (FCM) combined with the antibody facilitates clinical use of information concerning cell kinetics of tumor cells. In this review article, we show flow cytometric analysis of cell kinetics using the antibody. Two color analysis of DNA-BrdUrd makes it easy and rapid to obtain cell kinetic data of both tumor and non-tumor cells, without the use of DNA precursors labeled with isotopes. A fraction of labeled cells in the mid S phase (FLSm) method provides more detail analysis of the cell cycle, although it needs multiple samplings after pulse labeling with BrdUrd. Alternatively, a short term continuous labeling method can roughly estimate cell kinetic parameters but this method very simple. Furthermore, effects of anticancer drugs and radiation to human malignant tumors can be rapidly evaluated with the above methods. The protocol of cancer treatment will be determined based on cell kinetic data from an individual patient.

Antibodies, Monoclonal↗

PubMed1993

An analysis of adequacy of dialysis in a selected population on CAPD for over 3 years: the influence of urea and creatinine kinetics.

Adequacy of dialysis is addressed to minimize the negative consequences of underdialysis. The results of the NCSD demonstrated that urea kinetic modelling (UKM) is a useful tool in the measurement of adequacy of dialysis: Kt/V values of 1 are considered adequate to prevent complications when daily protein intake is appropriate. For CAPD no similar prospective studies exist, and consequently there is no information comparable to that for haemodialysis. Preliminary data have communicated controversial results about the usefulness of UKM for CAPD prescription. On the other hand creatinine kinetics has been proposed as an alternative to UKM to define adequacy of dialysis on CAPD. However, these results should be considered preliminary because of the small size of the series and the short observation period. The objective of this paper is to study the role of these two kinetic models in defining adequacy of dialysis in a long-term, selected CAPD population. We have studied 56 patients treated for at least 3 years on CAPD (3-11 years; mean observation period 5 years; 3996 patient-months). Urea kinetic model (UKM) (urea weekly (W) Kt/V and NPCR) and creatinine kinetics (Efficacy number (EN) and K) were determined annually. According to the present data we can conclude that Kt/V is an appropriate tool for defining adequacy in patients on CAPD. Values of weekly Kt/V greater than 1.7-1.8, with NPCR more than 1 g/kg/day, assure an adequate clinical outcome. Conversely, values on the Kt/V scale less than 1.7 entail a greater rate of complications. Our data also confirm that maintaining a serum albumin > 536-551 mumol/l (3.7-3.8 g/dl) is related to lower morbidity and mortality, and should be a major objective for CAPD patients. Consequently a relationship between dose of dialysis in terms of urea mass balance and protein nutrition can be established. After 5 years on CAPD, when all residual renal function is generally lost, Kt/V values in excess of 1.9 are difficult to achieve. Based on currently available information, we do agree with the interest of performing a prospective study on adequacy of dialysis based on the urea kinetic model in CAPD, which definitely defines its role. On the other hand, creatinine kinetics did not show sufficient discriminative capacity in terms of adequacy of dialysis. EN and Kt of creatinine values in the lower range (4.5-6 and less than 45-50 1/week respectively) were not necessarily associated with a poorer clinical outcome.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1999

Multisubstrate biodegradation kinetics of naphthalene, phenanthrene, and pyrene mixtures.

Biodegradation kinetics of naphthalene, phenanthrene and pyrene were studied in sole-substrate systems, and in binary and ternary mixtures to examine substrate interactions. The experiments were conducted in aerobic batch aqueous systems inoculated with a mixed culture that had been isolated from soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Monod kinetic parameters and yield coefficients for the individual compounds were estimated from substrate depletion and CO(2) evolution rate data in sole-substrate experiments. In all three binary mixture experiments, biodegradation kinetics were comparable to the sole-substrate kinetics. In the ternary mixture, biodegradation of naphthalene was inhibited and the biodegradation rates of phenanthrene and pyrene were enhanced. A multisubstrate form of the Monod kinetic model was found to adequately predict substrate interactions in the binary and ternary mixtures using only the parameters derived from sole-substrate experiments. Numerical simulations of biomass growth kinetics explain the observed range of behaviors in PAH mixtures. In general, the biodegradation rates of the more degradable and abundant compounds are reduced due to competitive inhibition, but enhanced biodegradation of the more recalcitrant PAHs occurs due to simultaneous biomass growth on multiple substrates. In PAH-contaminated environments, substrate interactions may be very large due to additive effects from the large number of compounds present.

Biodegradation, Environmental↗

PubMed2000

Biodegradation kinetics of benzene, toluene, and phenol as single and mixed substrates for Pseudomonas putida F1.

Although microbial growth on substrate mixtures is commonly encountered in bioremediation, wastewater treatment, and fermentation, mathematical modeling of mixed substrate kinetics has been limited. We report the kinetics of Pseudomonas putida F1 growing on benzene, toluene, phenol, and their mixtures, and compare mathematical models to describe these results. The three aromatics are each able to act as carbon and energy sources for this strain. Biodegradation rates were measured in batch cultivations following a protocol that eliminated mass transfer limitations for the volatile substrates and considered the culture history of the inoculum and the initial substrate to inoculum mass ratio. Toluene and benzene were better growth substrates than phenol, resulting in faster growth and higher yield coefficients. In the concentration ranges tested, toluene and benzene biodegradation kinetics were well described by the Monod model. The Monod model was also used to characterize phenol biodegradation by P. putida F1, although a small degree of substrate inhibition was noted. In mixture experiments, the rate of consumption of one substrate was found to be affected by the presence of the others, although the degree of influence varied widely. The substrates are catabolized by the same enzymatic pathway, but purely competitive enzyme kinetics did not capture the substrate interactions well. Toluene significantly inhibited the biodegradation rate of both of the other substrates, and benzene slowed the consumption of phenol (but not of toluene). Phenol had little effect on the biodegradation of either toluene or benzene. Of the models tested, a sum kinetics with interaction parameters (SKIP) model provided the best description of the paired substrate results. This model, with parameters determined from one- and two-substrate experiments, provided an excellent prediction of the biodegradation kinetics for the three-component mixture.

Algorithms↗

PubMed2006

Kinetics of pure cultures of hydrogen-oxidizing denitrifying bacteria and modeling of the interactions among them in mixed cultures.

In this study we report the isolation of four denitrifying bacteria from a batch reactor, where the progress of hydrogenotrophic denitrification was examined. Only three of the strains had the ability to use hydrogen as electron donor. In the present work, kinetic batch experiments were carried out in order to study the dynamic characteristics of pure and defined mixed cultures of hydrogen-oxidizing denitrifying bacteria, under anoxic conditions, in a defined synthetic medium, in the presence of nitrates. Kinetic models were developed and the kinetic parameters were determined from the batch experiments for each bacterium separately. The behavior of mixed cultures and the interactions between the bacteria were described using kinetic models based on the kinetic models developed for each bacterium separately and their predictions were compared with the results from mixed culture experiments. The mathematical models that were developed and validated in the present work are capable of describing the behavior of the bacteria in pure and mixed cultures, and in particular, the kinetics of nitrate and nitrite reduction and cell growth.

Anaerobiosis↗

PubMed1979

Acetaminophen kinetics in acutely poisoned patients.

A kinetic model of acetaminophen elimination over a wide dose range has been developed on the basis of (1) kinetic data from normal adults who received a usual dose (up to 2 gm) of the drug and (2) the composition of urinary metabolites of acetaminophen excreted within 24 hr by 29 patients who had ingested up to 26 gm of acetaminophen in suicide attempts (including 2 that were fatal and 5 with evidence of severe intoxication). The model consists of the following parallel pathways: conjugation with glucuronide by Michaelis-Menten kinetics, conjugation with sulfate by Michaelis-Menten kinetics, renal excretion of acetaminophen by apparent first-order kinetics, and formation of an oxidative metabolite (which is responsible for the hepatotoxicity of acetaminophen) by apparent first-order kinetics. There is good agreement between the model-predicted and actual urinary excretion of individual acetaminophen metabolites for doses of 0.8 to 26 gm and between model-predicted and actual plasma acetaminophen concentrations in both the low (normal subjects) and high (intoxicated subjects) concentration ranges. Computer simulations indicate that unsaturation of acetaminophen sulfate formation, previously shown to be feasible in vivo, should decrease the formation of the hepatotoxic metabolite. This prediction is consistent with experimental data obtained in preliminary studies on mice.

Acetaminophen↗

PubMed2006

Basics and applications of solid-state kinetics: a pharmaceutical perspective.

Most solid-state kinetic principles were derived from those for homogenous phases in the past century. Rate laws describing solid-state degradation are more complex than those in homogenous phases. Solid-state kinetic reactions can be mechanistically classified as nucleation, geometrical contraction, diffusion, and reaction order models. Experimentally, solid-state kinetics is studied either isothermally or nonisothermally. Many mathematical methods have been developed to interpret experimental data for both heating protocols. These methods generally fall into one of two categories: model-fitting and model-free. Controversies have arisen with regard to interpreting solid-state kinetic results, which include variable activation energy, calculation methods, and kinetic compensation effects. Solid-state kinetic studies have appeared in the pharmaceutical literature over many years; some of the more recent ones are discussed in this review.

Chemistry, Pharmaceutical↗

PubMed1998

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study.

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.

Animals↗

PubMed2003

Analysis of the differences in the folding kinetics of structurally homologous proteins based on predictions of the gross features of residue contacts.

It is a general notion that proteins with very similar three-dimensional structures would show very similar folding kinetics. However, recent studies reveal that the folding kinetic properties of some proteins contradict this thought (i.e., the members in a same protein family fold through different pathways). For example, it has been reported that some beta-proteins in the intracellular lipid-binding protein family fold through quite different pathways (Burns et al., Proteins 1998;33:107-118). Similar differences in folding kinetics are also observed in the members of the globin family (Nishimura et al., Nat Struct Biol 2000;7:679-686). In our study, we examine the possibility of predicting qualitative differences in folding kinetics of the intracellular lipid-binding proteins and two globin proteins (i.e., myoglobin and leghemoglobin). The problem is tackled by means of a contact map based on the average distance statistics between residues, the Average Distance Map (ADM), as constructed from sequence. The ADMs for the three proteins show overall similarity, but some local differences among maps are also observed. Our results demonstrate that some properties of the protein folding kinetics are consistent with local differences in the ADMs. We also discuss the general possibility of predicting folding kinetics from sequence information.

Carrier Proteins↗

PubMed2001

Enoyl-ACP reductase (FabI) of Haemophilus influenzae: steady-state kinetic mechanism and inhibition by triclosan and hexachlorophene.

Steady-state kinetics, equilibrium binding, and primary substrate kinetic isotope effect studies revealed that the reduction of crotonyl-CoA by NADH, catalyzed by Haemophilus influenzae enoyl-ACP reductase (FabI), follows a rapid equilibrium random kinetic mechanism with negative interaction among the substrates. Two biphenyl inhibitors, triclosan and hexachlorophene, were studied in the context of the kinetic mechanism. IC(50) values for triclosan in the presence and absence of NAD(+) were 0.1 +/- 0.02 and 2.4 +/- 0.02 microM, respectively, confirming previous observations that the E-NAD(+) complex binds triclosan more tightly than the free enzyme. Preincubation of the enzyme with triclosan and NADH suggested that the E-NADH complex is the active triclosan binding species as well. These results were reinforced by measurement of binding kinetic transients. Intrinsic protein fluorescence changes induced by binding of 20 microM triclosan to E, E-NADH, E-NAD(+), and E-crotonyl-CoA occur at rates of 0.0124 +/- 0.001, 0.0663 +/- 0.002, 0.412 +/- 0.01, and 0.0069 +/- 0.0001 s(-1), respectively. The rate of binding decreased with increasing crotonyl-CoA concentrations in the E-crotonyl-CoA complex, and the extrapolated rate at zero concentration of crotonyl-CoA corresponded to the rate observed for the binding to the free enzyme. This suggests that triclosan and the acyl substrate share a common binding site. Hexachlorophene inhibition, on the other hand, was NAD(+)- and time-independent; and the calculated IC(50) value was 2.5 +/- 0.4 microM. Steady-state inhibition patterns did not allow the mode of inhibition to be unambiguously determined, but binding kinetics suggested that free enzyme, E-NAD(+), and E-crotonyl-CoA have similar affinity for hexachlorophene, since the k(obs)s were in the same range of 20-24 s(-1). When the E-NADH complex was mixed with hexachlorophene ligand, concentration-independent fluorescence quenching at 480 nm was observed, suggesting at least partial competition between NADH and hexachlorophene for the same binding site. Mutual exclusivity studies, together with the above-discussed results, indicate that triclosan and hexachlorophene bind at different sites of H. influenzae FabI.

Binding Sites↗

PubMed1995

Quantitative fluorescence method for continuous measurement of DNA hybridization kinetics using a fluorescent intercalator.

We present a quantitative fluorescence method for continuous measurement of DNA or RNA hybridization (including renaturation) kinetics using a fluorescent DNA intercalator. The method has high sensitivity and can be used with reaction volumes as small as 1 microliter and amounts of DNA around 1 ng. The method is based on the observations that (i) for the usual hybridization conditions, intercalators such as ethidium bromide bind (intercalate) to double-stranded DNA (dsDNA) but not single-stranded DNA or RNA and (ii) there is a large increase in fluorescence intensity when intercalators such as ethidium bromide bind to dsDNA. In this application, the intercalator can be considered as a quantitative indicator of dsDNA concentration. When a small amount of intercalator is added to a hybridizing solution, the fluorescence intensity of the intercalators increases with increase in dsDNA. The hybridization reaction can thus be monitored by continuously recording fluorescence intensity vs time. Because the amount of intercalator bound to dsDNA is not necessarily proportional to dsDNA concentration, the time-dependent fluorescence intensity graph is not identical to the kinetic graph [dsDNA] vs t. However, the fluorescence intensity vs time graph can easily be converted to the true [dsDNA] vs t graph by means of an experimental calibration graph of fluorescence intensity vs [dsDNA]. This calibration graph is obtained in a separate experiment using samples containing known amounts of dsDNA in the ethidium bromide buffer used in the kinetic measurement. We present results of experimental tests of the intercalator technique using ethidium bromide as an intercalator and DNA from Escherichia coli and lambda-phage and Poly(I)-Poly(C) RNA hybrids. These DNA and RNA samples have Cot1/2 values that cover a range of 10(6). Our experimental results show that (i) the kinetics of hybridization are not significantly perturbed by the intercalator at concentrations where no more than 10% of the binding sites on DNA or RNA hybrids are occupied, (ii) the kinetic graphs obtained by the intercalator fluorescence method and corrected with the calibration graph agree with kinetic graphs obtained by optical absorbance measurements at 260 nm, and (iii) the intercalator technique can be used in the different salt environments often used to increase the velocity of the hybridization reaction and at the hybridization temperatures (35-75 degrees C) normally used to minimize nonspecific hybridization.

Bacteriophage lambda↗

PubMed1999

The use of chromogenic reference substrates for the kinetic analysis of penicillin acylases.

Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low K(m) values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower k(cat)/K(m) values, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis, and Kluyvera citrophila. The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.

Alcaligenes↗

PubMed2000

Kinetic modeling of ATP synthesis by ATP synthase and its mechanistic implications.

Based on the torsional mechanism of ATP synthesis by ATP synthase, a kinetic scheme has been developed in this work. The scheme considers adenine nucleotide transport, binding of substrates ADP and P(i), unbinding of product ATP, and ATP synthesis. This kinetic scheme has been analyzed mathematically, and a kinetic model has been obtained to explain the experimentally observed hyperbolic Michaellian dependence of the rate of ATP synthesis on the ADP concentration by ATP synthase under physiological steady-state operating conditions. The principal results of the kinetic model have been compared with the experimental data and an estimate of the enzymological kinetic parameters V(max), K(M), and K(I) has been determined. Mechanistic implications arising from further analysis of the kinetic model have been discussed. These biological implications provide deep insight into the sequence of events leading to ATP synthesis.

Adenosine Diphosphate↗

PubMed1995

Local perturbations by ligand binding of hydrogen deuterium exchange kinetics in a four-helix bundle protein, acyl coenzyme A binding protein (ACBP).

Amide hydrogen exchange kinetics of the individual amides in a four-helix bundle protein, acyl-coenzyme A binding protein, have been studied by nuclear magnetic resonance spectroscopy. The kinetics of amides with exchange rate constants in the range of 10(-25) to 10(-6.5) S-1 at pH 6.65 in free protein and the ligand-protein complex have been measured, and the effect of binding the ligand, palmitoyl-coenzyme A, on individual exchange rates has been analysed. Specific correlations between exchange kinetics and the structural properties of the individual amides known from the three-dimensional structure of acyl-coenzyme A binding protein have been examined. Furthermore, an analysis has been performed comparing the structural perturbations of the protein-ligand interactions known from the three dimensional structure of the complex of palmitoyl-coenzyme A and acyl-coenzyme A binding protein with the ligand-induced perturbations on the amides exchange kinetics. Finally, the ligand-induced perturbations on hydrogen exchange have been compared with those on 15N relaxation. The results suggest that hydrogen exchange kinetics in the individual sites of acyl-coenzyme A binding protein are primarily determined by local structural features; they show that ligand binding gives rise mainly to changes localized at the sites of interaction between protein and ligand; they imply that the perturbation of exchange kinetics caused by ligation can be either, as in one example a local stabilisation of the pre-exchange equilibrium induced by formation of a hydrogen bond, or as seen here in several examples a reduction of the dynamic processes that lead to the opening and closing processes of the pre-exchange equilibrium. The results seem not to indicate changes in the rate of the final chemical exchange step.

Amides↗

PubMed1998

Folding intermediates of wild-type and mutants of barnase. II. Correlation of changes in equilibrium amide exchange kinetics with the population of the folding intermediate.

There is an unanswered question from previous studies of 1H/2H-exchange of amide protons of barnase. Under certain conditions, there is a relatively abrupt change from EX2 towards EX1 kinetics as the temperature is slightly increased. The change in kinetics for different mutants is not directly related to their changes in stability. We have measured the stability of the folding intermediate of barnase (I) in 2H2O under a variety of conditions and calculated its population at different temperatures. The change in kinetics correlates with the change in the population of the folding intermediate. At higher temperatures and pH, the free energy of I becomes higher than that of the denatured state, D, and the kinetics becomes EX1. The data fit a simple kinetic scheme. Such changes in kinetics may be used to detect the presence of intermediates in the folding reaction at equilibrium in native conditions, but cannot distinguish whether they are on or off-pathway.

Amides↗

PubMed1998

Folding kinetics of the SH3 domain of PI3 kinase by real-time NMR combined with optical spectroscopy.

The refolding kinetics of the chemically denatured SH3 domain of phosphatidylinositol 3'-kinase (PI3-SH3) have been monitored by real-time one-dimensional 1H NMR coupled with a variety of other biophysical techniques. These experiments indicate that the refolding kinetics of PI3-SH3 are biphasic. The slow phase (27 (+/- 8)% amplitude) is due to a population of substantially unfolded molecules with an incorrectly configured cis proline residue. The fast phase (73 (+/- 8)% amplitude) corresponds to the folding of protein molecules with proline residues in a trans configuration in the unfolded state. NMR experiments indicate that the first species populated after the initiation of folding exhibit poor chemical shift dispersion and have spectra very similar to that of the denatured protein in 8 M guanidine hydrochloride. Linear combinations of the first spectrum and of the spectrum of the native protein accurately reconstruct all of the spectra acquired during refolding. Consistent with this, native side-chain and backbone H alpha atom packing (NMR), secondary structure (far-UV circular dichroism), burial of aromatic residues (near-UV circular dichroism), intrinsic fluorescence and peptide binding activity are all recovered with effectively identical kinetics. Equilibrium unfolding and folding/unfolding kinetics yield, within experimental error, identical values for the free energy of unfolding (delta Gu-H2O = 3.38 kcal mol-1) and for the slope of the free energy of unfolding versus denaturant concentration (meq = 2.33 kcal mol-1 M-1). Together, these data provide strong evidence that PI3-SH3 folds without significant population of kinetic well-structured intermediates. That PI3-SH3 folds slowly (time constant 2.8 seconds in H2O at 20 degrees C) indicates that slow refolding is not always a consequence of kinetic traps but may be observed even when a protein appears to fold via a simple, two-state mechanism.

Amino Acid Sequence↗

PubMed1999

Hydrogen exchange kinetics of proteins in denaturants: a generalized two-process model.

The recent progress in measurements on the amide hydrogen exchange (HX) in proteins under varying denaturing conditions, both at equilibrium and in transient relaxation, necessitates the development of a unifying theory which quantitatively relates the HX rates to the conformational energetics of the proteins. We present here a comprehensive kinetic model for the site-specific HX of proteins under varying solvent denaturing conditions based on the two-state protein folding model. The generalized two-process model considers both conformational fluctuations and residual protections, respectively, within the folded and unfolded states of a protein, as well as a global kinetic folding-unfolding transition between the two states. The global transition can be either rapid or slow, depending on the solvent condition for the protein. This novel model is applicable to the traditional equilibrium HX measurements in both EX2 and EX1 regimes, and also the recently introduced transient pulse-labeling HX experiments. A set of simple analytical equations is provided for quantitative interpretation of experimental data. The model emphasizes the use of full time-course of bi-exponential HX kinetics, rather than fitting time-course data to single rate constants, to obtain quantitative information about fluctuating conformers within the folded and unfolded states of proteins. This HX kinetic model naturally unfolds into a simple two-state and two-stage kinetic interpretation for protein folding. It suggests that the various observed intermediates of a protein can be interpreted as dominant isomers of either the folded or the unfolded state under different solvent conditions. This simple, minimalist's view of protein folding is consistent with various recent experimental observations on folding kinetics by HX.

Hydrogen↗

PubMed1999

Pathway modulation, circular permutation and rapid RNA folding under kinetic control.

The thermodynamics and folding kinetics of a circularly permuted construct of the ribozyme from Bacillus subtilis RNase P are analyzed and compared with the folding properties of the wild-type ribozyme using optical spectroscopy and catalytic activity. The folding of the wild-type ribozyme is slow due to the rearrangement of kinetically trapped species containing misfolded structures. To test whether any misfolded structure arises from interactions between the two independently folding domains of the RNase P RNA, a circular permuted form was created where one of the two phosphodiester bonds connecting these domains is broken. This construct folds approximately 15-fold faster (t1/2 approximately nine seconds) than the wild-type ribozyme at 37 degreesC. While the complete folding of both domains is kinetically indistinguishable in the wild-type ribozyme, one domain folds much faster than the other domain in the circularly permuted construct. Hence, the major kinetic trap in the folding of the wild-type RNase P RNA involves interdomain interactions. This kinetic trap is avoidable at 37 degreesC in the circularly permuted RNA. However, at temperatures below 30 degreesC or when refolding begins from an equilibrium intermediate stabilized by submillimolar concentrations of Mg2+, a subpopulation containing an interdomain misfold still forms. These results indicate that the folding pathway of this large RNA is highly malleable and can be under kinetic control.

Bacillus subtilis↗