EPR-detected folding kinetics of externally located cysteine-directed spin-labeled mutants of iso-1-cytochrome c.

We report the application of our newly developed dielectric resonator-based flow and stopped-flow kinetic EPR systematically to probe protein folding in yeast iso-1-cytochrome c at cysteine-directed spin-labeled locations. The locations studied have not been previously directly probed by other techniques, and we observe them on a time scale stretching from 50 micros to seconds. On the basis of crystal structure and homology information, the following mutation-tolerant, externally located cysteine labeling sites were chosen (in helices, T8C, E66C, and N92C; in loops, E21C, V28C, H39C, D50C, and K79C), and labeling at these sites was not destabilizing. Dilution of denaturant was used to induce folding and thereby to cause a change in the spin label EPR signal as folding altered the motion of the spin label. Under folding conditions, including the presence of imidazole to eliminate kinetic trapping due to heme misligation, a phase of folding on the 20-30 ms time scale was found. This phase occurred not only at the T8C and N92C labeling sites in the N- and C-terminal helices, where such a phase has been associated with folding in these helices, but overall at labeling sites throughout the protein. In the absence of imidazole the 20-30 ms phase disappeared, and another phase having the time scale of 1 s appeared throughout the protein. There was evidence under all conditions for a burst phase on a scale of less than several milliseconds which occurred at labeling positions V28C, H39C, D50C, E66C, and K79C in the middle of the protein sequence. At spin-labeled D50C rapid-mix flow EPR indicated a very short approximately 50 micros phase possibly associated with the prefolding or compaction of the loop to which D50 belongs. Spin labels have been criticized as perturbing the phenomena which they measure, but our spin labeling strategy has reported common kinetic themes and not perturbed, disconnected kinetic events.     

Ultrafast events in the folding of ferrocytochrome c

Laser flash photolysis and stopped-flow methods have been used to study the dynamic events in the micro- to millisecond time bin in the refolding of horse ferrocytochrome c in the full range of guanidine hydrochloride concentration at pH 12.8 (+/-0.1), 22 degrees C. Under the absolute refolding condition, the earliest relaxation time of the unfolded protein chain is less than 1 micros. The chain then undergoes diffusive dynamics-mediated contraction and expansion, in which intrapolypeptide ligands make transient contacts with the heme iron, giving rise to two distinct kinetic phases of approximately 0.4 and approximately 3 micros. Under moderate to absolute refolding conditions, the rates of these processes show little dependence on the denaturant concentration, indicating the absence of structural element in the incipient or the relaxed state. Chain expansion and contraction events continue until the polypeptide finds a stable and supportive transition state. The crossing of this transition barrier, which rate-limits the folding of alkaline ferrocytochrome c, is characterized by a stopped-flow measured time constant of approximately 3 ms in aqueous solvent. Observed kinetics thus implicate no submillisecond folding structure. The folding kinetics is effectively two state in which the unfolded polypeptide first relaxes to an unstructured chain and then crosses over a late rate-limiting barrier to achieve the native conformation. The experimentally observed rates as a function of guanidine hydrochloride concentration have been simulated by numerically calculated microscopic rates of a simple kinetic model that captures the essential features of folding.     

Multiple folding pathways for the P4-P6 RNA domain

We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with approximately 1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-yield automated synthesis of oligoribonucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridine residue. P4-P6 forms its higher-order tertiary structure rapidly, with k(obs) = 15-31 s(-1) (t(1/2) approximately 20-50 ms) at 35 degrees C and [Mg(2+)] approximately 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 degrees C, demonstrating a large activation enthalpy DeltaH(double dagger) approximately 26 kcal/mol; the activation entropy DeltaS(double dagger) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) approximately 1 s) folding component is also observed. The folding kinetics are both ionic strength- and temperature-dependent; the slow phase vanishes upon increasing [Na(+)] in the cacodylate buffer, and the kinetics switch completely from fast at 30 degrees C to slow at 40 degrees C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radical cleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiary interactions are present even without Mg(2+) in the initial state. The fast kinetic phase reflects folding that is facilitated by these interactions, whereas the slow kinetics are observed when these interactions are disrupted at lower ionic strength and higher temperature.     

The conformational transition of horse heart porphyrin c

The heme iron of horse heart cytochrome c was selectively removed using anhydrous HF. The product, porphyrin c, exhibits the viscosity, far ultraviolet circular dichroic, and fluorescence properties characteristic for native cytochrome c. However, porphyrin c is more susceptible to denaturation by guanidine hydrochloride and by heat than is the parent cytochrome. All of the conformational parameters of porphyrin c exhibit a common reversible transition centered at 0.95 m guanidine hydrochloride at 23 degrees C and pH 7.0. Guanidine denatured porphyrin c refolds in two kinetic phases having time constants of 20 and 200 ms as detected by stopped flow absorbance or fluorescence measurement, with about 80% of the observed change in the faster phase. The kinetics of porphyrin c refolding are not significantly altered by increasing the viscosity of the refolding solvent 15-fold by addition of sucrose. We suggest that the folding of guanidine denatured cytochrome c is not a diffusion-limited process and that the requirement for protein axial ligation elicits the slow (s) kinetic phase observed in the refolding of cytochrome c.     

Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin

The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements. Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone. This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant. The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region. The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C. The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25. This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant. Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein. We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76.     

Refolding of beta-lactoglobulin studied by stopped-flow circular dichroism at subzero temperatures

Refolding of bovine beta-lactoglobulin was studied by stopped-flow circular dichroism at subzero temperatures. In ethylene glycol 45%-buffer 55% at -15 degrees C, the isomerization rate from the kinetic intermediate rich in alpha-helix to the native state is approximately 300-fold slower than that at 4 degrees C in the absence of ethylene glycol, whereas the initial folding is completed within the dead time of the stopped-flow apparatus (10 ms). At -28 degrees C, we observed at least three phases; the fastest process, accompanied by an increase of alpha-helix content, is completed within the dead time of the stopped-flow apparatus (10 ms), the second phase, accompanied by an increase of alpha-helix content with the rate of 2 s(-1), and the third phase, accompanied by a decrease of alpha-helix content. This last phase, corresponding to the isomerization process at -15 degrees C described above, was so slow that we could not monitor any changes within 4 h. Based on the findings above, we propose that rapid alpha-helix formation and their concurrent collapse are common even in proteins rich in beta-structure in their native forms.     

Kinetics of the lamellar-inverse hexagonal phase transition determined by time-resolved X-ray diffraction.

The kinetics of the lamellar (L alpha)-inverse hexagonal (HII) phase transition in diacylphosphatidylethanolamine (PE)--water systems were probed with time-resolved X-ray diffraction. Transition kinetics in the fast time regime (approximately 100 ms) were studied by initiating large temperature jumps (up to 30 degrees C) with a 50-ms electrical current pulse passed through a lipid-salt water dispersion, resulting in ohmic heating of the sample. Diffraction with a time resolution to 10 ms was acquired at the National Synchrotron Light Source. The time constant for the phase transition for 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was on the order of 100 ms for the largest temperature jumps recorded. Faster transition behavior was found for a 1,2-dielaidoyl-sn-glycero-3-PE mixture. The HII lattice parameters for both systems were seen to swell from an initial value commensurate with the lamellar lattice to the final equilibrium value. The rate of swelling was seen to be independent of the magnitude of the temperature jump. For small temperature jumps (less than 10 degrees C), the phase transition kinetics slow dramatically, and transition studies can readily be performed on a conventional rotating anode X-ray source. At 4 degrees C, a DOPE sample was observed to slowly convert to the hexagonal phase over the course of a week, with the decay in the lamellar intensity fitting a power law behavior over four decades of time. This power law behavior is shown to have interesting consequences to the determination of the phase transition temperature of lipid-water dispersions by conventional methods such as calorimetry.     

The folding of staphylococcal nuclease in the presence of methanol or guanidine thiocyanate

The effect of methanol on the folding of staphylococcal nuclease has been investigated. Equilibrium thermal unfolding transitions were monitored by fluorescence emission. The transition was very sensitive to the presence of methanol (at pH 7.0), the Tm decreased from above 50 degrees C for aqueous solution to below 0 degree C for 70% methanol. The transitions were fully reversible and conformed to two-state behavior. A linear relationship was observed between the hydrophobicity of the solvent and both the Tm and the change in delta G for unfolding. The effect of pH on the transition in 50% methanol at 0 degree C was essentially the same as for aqueous solution, with a cooperative transition in the vicinity of apparent pH (pH*) 4. The unfolding transition was determined as a function of guanidine thiocyanate in aqueous and 50% methanol solvents. The midpoints of the transitions were 0.30 and 0.20 M, respectively, at 2.1 degrees C. The kinetics of folding at 0 degree C were compared in aqueous, 50% methanol and 0.30 M guanidine thiocyanate solvents, by monitoring changes in the tryptophan fluorescence intensity. Triphasic kinetics for refolding in both aqueous and 50% methanol solutions were observed in stopped-flow experiments. In both solvent systems the slowest phase is ascribed to proline isomerization. The kinetics of refolding were monitored at subzero temperatures in 50% methanol at pH* 7.0 in manual mixing experiments. Biphasic kinetics were observed at temperatures between 0 and -35 degrees C. A third, faster phase, was inferred from the missing amplitude. The energies of activation were 20.0 and 17.2 kcal mol-1, respectively, for the two slower phases. At -33.8 degrees C, the observed pseudo first-order rate constants were 1.2 x 10(-3) and 2.1 x 10(-5) s-1. At temperatures above -35 degrees C, the sum of the observed amplitudes was essentially constant at 70-75% of the expected total amplitude. At lower temperatures the amplitude of the refolding reaction decreased, and the native state was not formed (unless the temperature was increased), due to the formation of a trapped intermediate state. This intermediate has circular dichroism and fluorescence properties consistent with a compact state with some molten globule characteristics.     

Kinetics of Pi-Pi exchange in rat liver mitochondria. Rapid filtration experiments in the millisecond time range

Phosphate-phosphate exchange through the inorganic phosphate (Pi) carrier of rat liver mitochondria was investigated by a new rapid filtration technique, which does not require the use of transport inhibitors to stop the reaction and offers high time resolution (starting from 10 ms), thus allowing kinetic measurements on a fine time scale even at room temperature. At approximately 22 degrees C, isotopic equilibrium of [32P]Pi is achieved within 0.8-2.5 s--depending on the Pi concentration--and an initial linear phase, lasting for 400-500 ms, is observed. Complete inhibition of Pi exchange by an excess (33 nmol/mg) of mersalyl, a well-known organomercurial inhibitor, required 200 ms, pointing to the insufficiency of this reagent for effective inhibitor stop. On the other hand, investigation of the effect of mersalyl (allowed to react with mitochondria for at least 20 s) on the initial rate of Pi exchange supports earlier observations on the protective effect of this inhibitor; i.e., up to 3 nmol of mersalyl/mg of protein does not decrease the transport rate whereas these low concentrations protect approximately 50% of the transport capacity from irreversible inactivation by N-ethylmaleimide. In nonrespiring mitochondria, at pH 7.3, Pi exchange exhibited a Km of 1.6 mM and a Vmax of 3.0 mumol min-1 (mg of mitochondrial protein)-1. The increase of the membrane potential without any concomitant change of delta pH had no significant influence on the kinetic parameters. The maximal velocity of Pi transport is significantly higher than the maximal velocity of all the other components of oxidative phosphorylation at comparable temperatures. The possible physiological significance of this excess capacity is discussed.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

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.     

New structural insights into the refolding of ribonuclease T1 as seen by time-resolved Fourier-transform infrared spectroscopy

To get new structural insights into different phases of the renaturation of ribonuclease T1 (RNase T1), the refolding of the thermally unfolded protein was initiated by rapid temperature jumps and detected by time-resolved Fourier-transform infrared spectroscopy. The characteristic spectral changes monitoring the formation of secondary structure and tertiary contacts were followed on a time scale of 10(-3) to 10(3) seconds permitting the characterization of medium and slow folding reactions. Additionally, structural information on the folding events that occurred within the experimental dead time was indirectly accessed by comparative analysis of kinetic and steady-state refolding data. At slightly destabilizing refolding temperatures of 45 degrees C, which is close to the unfolding transition region, no specific secondary or tertiary structure is formed within 180 ms. After this delay all infrared markers bands diagnostic for individual structural elements indicate a strongly cooperative and relatively fast folding, which is not complicated by the accumulation of intermediates. At strongly native folding temperatures of 20 degrees C, a folding species of RNase T1 is detected within the dead time, which already possesses significant amounts of antiparallel beta-sheets, turn structures, and to some degree tertiary contacts. The early formed secondary structure is supposed to comprise the core region of the five-stranded beta-sheet. Despite these nativelike characteristics the subsequent refolding events are strongly heterogeneous and slow. The refolding under strongly native conditions is completed by an extremely slow formation or rearrangement of a locally restricted beta-sheet region accompanied by the further consolidation of turns and denser backbone packing. It is proposed that these late events comprise the final packing of strand 1 (residues 40-42) of the five-stranded beta-sheet against the rest of this beta-sheet system within an otherwise nativelike environment. This conclusion was supported by the comparison of refolding of RNase T1 and its variant W59Y RNase T1 that enabled the assignment of these very late events to the trans-->cis isomerization reaction of the prolyl peptide bond preceding Pro-39.     

Energetics of folding subtilisin BPN'.

Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. Here we document for the first time the in vitro folding of the mature form of subtilisin. Subtilisin was modified by site-directed mutagenesis to be proteolytically inactive, allowing the impediments to folding to be systematically examined. First, the thermodynamics and kinetics of calcium binding to the high-affinity calcium A-site have been measured by microcalorimetry and fluorescence spectroscopy. Binding is an enthalpically driven process with an association constant (Ka) equal to 7 x 10(6) M-1. Furthermore, the kinetic barrier to calcium removal from the A-site (23 kcal/mol) is substantially larger than the standard free energy of binding (9.3 kcal/mol). The kinetics of calcium dissociation from subtilisin (e.g., in excess EDTA) are accordingly very slow (t1/2 = 1.3 h at 25 degrees C). Second, to measure the kinetics of subtilisin folding independent of calcium binding, the high-affinity calcium binding site was deleted from the protein. At low ionic strength (I = 0.01) refolding of this mutant requires several days. The folding rate is accelerated almost 100-fold by a 10-fold increase in ionic strength, indicating that part of the free energy of activation may be electrostatic. At relatively high ionic strength (I = 0.5) refolding of the mutant subtilisin is complete in less than 1 h at 25 degrees C. We suggest that part of the electrostatic contribution to the activation free energy for folding subtilisin is related to the highly charged region of the protein comprising the weak ion binding site (site B).(ABSTRACT TRUNCATED AT 250 WORDS)     

Folding of staphylococcal nuclease A studied by equilibrium and kinetic circular dichroism spectra

The urea-induced unfolding of staphylococcal nuclease A has been studied by circular dichroism both at equilibrium and by the kinetics of unfolding and refolding (pH 7.0 and 4.5 degrees C), as a function of Ca2+ and thymidine 3',5'-diphosphate (pdTp) concentration. The results are as follows. (1) The unfolding transition is shifted to higher concentrations of urea by Ca2+ and pdTp, and the presence of both ligands further stabilizes the protein. (2) In the first stage of kinetic refolding, the peptide ellipticity changes rapidly within the dead time of stopped-flow measurement (15 ms), indicating accumulation of a transient intermediate. This intermediate is remarkably less stable than those of other globular proteins previously studied. (3) Dependence of the folding and unfolding rate constants on urea concentration indicates that the critical activated state of folding ("transition state") has considerable structural organization. The transition state does not, however, have the capacity to bind Ca2+ and pdTp, as indicated by the effects of these ligands on the unfolding rate constant. (4) There are at least four different phases in the refolding kinetics in native conditions below 1 M urea. In the absence of pdTp, there are two phases in unfolding, while in the presence of pdTp the unfolding kinetics show a single phase. Some characteristics of the transient intermediate and of the transition state for folding are discussed.     

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.     

Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies.

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.     

Cytochrome P-450cam and putidaredoxin interaction during electron transfer.

Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s-1 at 25 degrees C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 645 nm absorbance band of high-spin Fe(III) cytochrome P-450cam; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin Fe(III) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the EPR signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P-450. The mechanism of the loss of the EPR signal of cytochrome P-450 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation.     

Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate

The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.     

The initial inward current in spherical clusters of chick embryonic heart cells

The rapid inward sodium current in spherical clusters of 11-d-old embryonic chick heart cells, ranging in size between 65 and 90 micron diameter, was studied using the two-microelectrode voltage-clamp technique. Using these preparations, it was possible to resolve the activation phase of the rapid inward current for potentials negative to -25 mV at 37 degrees C. The rapid inward current exhibited a voltage and time dependence similar to that observed in other excitable tissues. It was initiated at potential steps more positive than -45 mV. The magnitude of the current reached its maximum value at a potential of approximately -20 mV. The measured reversal potential was that predicted by the Nernst equation for sodium ions. The falling phase of the current followed a single exponential time-course with a time constant of inactivation, tau h, ranging between 2.14 ms at -40 mV and 0.18 ms at -5 mV. The time constant of inactivation, tau h, determined by a single voltage-step protocol was compared to the constant, tau c, determined by a double voltage-step protocol and no significant different between the two constants of inactivation was found. Furthermore, the time constants of inactivation and reactivation at the same potential in the same preparation were similar. The results of this study demonstrate that the sodium current of heart cells recorded at 37 degrees C can be described by Hodgkin-Huxley kinetics with speeds approximately four times faster than the squid giant axon at 15 degrees C.     

Highly nonexponential kinetics in the early-phase refolding of proteins at low temperatures

Theory and simulations predict that the folding kinetics of protein-like heteropolymers become nonexponential and glassy (i.e., controlled by escape from different low-energy misfolded states) at low temperatures, but there was little experimental evidence for such behavior of proteins. We have developed a stopped-flow instrument working reliably down to -40 degrees C with high mixing capability and applied it to study the refolding kinetics of horse cytochrome c (cyt c) and hen egg white lysozyme at temperatures below 0 degrees C in the presence of antifreeze NaCl, LiCl, or ethylene glycol and above 0 degrees C in the presence and absence of antifreeze. The refolding was initiated by rapid dilution of the guanidine hydrochloride unfolded proteins, and the kinetics were monitored by intrinsic tryptophan fluorescence. Highly nonexponential kinetics extended over 3 decades in time (0.01-10 s) were observed in the early phases of the refolding of cyt c and lysozyme in the temperature range of -35 to 5 degrees C. These results are in agreement with the theoretical prediction, suggesting that the folding energy landscapes of these proteins are rugged in the upper portions.