Comparison of three different fluorescent visualization strategies for detecting Escherichia coli ATP synthase subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The correlation between protein molecular weight and the number of lysine or basic amino acid residues was found to be high for broad range molecular weight standards, subunits of Escherichia coli F1F0-ATP synthase and the translated open reading frame of E. coli. A relatively poor correlation between protein molecular weight and the number of cysteine residues was observed in all cases. The ability of amine-reactive, thiol-reactive and basic amino acid-binding fluorophores to detect the eight subunits of F1F0-ATP synthase complex was assessed using 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), monobromobimane (MBB) and SYPRO Ruby protein gel stain, respectively. Though experimentally none of the fluorophores provided accurate estimates of the subunit stoichiometry of this complex, MDPF and SYPRO Ruby protein gel stain were capable of semiquantitative detection of every subunit. MBB, however, failed to detect subunits a, b and c of the hydrophobic F0 complex, as well as subunit epsilon of the F1 complex. All three fluorescent detection procedures permitted subsequent identification of representative subunits by peptide mass profiling using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The use of thiol-reactive fluorophores for the global analysis of protein expression profiles does not appear to be advisable as a significant number of proteins have few or no cysteine residues, thus escaping detection.

Fluorescence-based analyses of the effects of full-length recombinant TAF130p on the interaction of TATA box-binding protein with TATA box DNA.

We have used a combination of fluorescence anisotropy spectroscopy and fluorescence-based native gel electrophoresis methods to examine the effects of the transcription factor IID-specific subunit TAF130p (TAF145p) upon the TATA box DNA binding properties of TATA box-binding protein (TBP). Purified full-length recombinant TAF130p decreases TBP-TATA DNA complex formation at equilibrium by competing directly with DNA for binding to TBP. Interestingly, we have found that full-length TAF130p is capable of binding multiple molecules of TBP with nanomolar binding affinity. The biological implications of these findings are discussed.

Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor.

We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.

Accessory factors facilitate the binding of glucocorticoid receptor to the phosphoenolpyruvate carboxykinase gene promoter.

Glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene requires a glucocorticoid response unit (GRU) comprised of two non-consensus glucocorticoid receptor (GR) binding sites, GR1 and GR2, and at least three accessory factor elements (gAF1-3). DNA-binding accessory proteins are commonly required for the regulation of genes whose products play an important role in metabolism, development, and a variety of defense responses, but little is known about why they are necessary. Quantitative, real time homogenous assays of cooperative protein-DNA interactions in complex media (e.g. nuclear extracts) have not previously been reported. Here we perform quantitative, real time equilibrium and stopped-flow fluorescence anisotropy measurements of protein-DNA interactions in nuclear extracts to demonstrate that GR binds to the GR1-GR2 elements poorly as compared with a palindromic or consensus glucocorticoid response element (GRE). Inclusion of either the gAF1 or gAF2 element with GR1-GR2, however, creates a high affinity binding environment for GR. GR can undergo multiple rounds of binding and dissociation to the palindromic GRE in less than 100 ms at nanomolar concentrations. The dissociation rate of GR is differentially slowed by the gAF1 or gAF2 elements that bind two functionally distinct accessory factors, COUP-TF/HNF4 and HNF3, respectively.

Conformational equilibrium of the reactive center loop of antithrombin examined by steady state and time-resolved fluorescence measurements: consequences for the mechanism of factor Xa inhibition by antithrombin-heparin complexes.

Activation of antithrombin by high-affinity heparin as an inhibitor of factor Xa has been ascribed to an allosteric switch between two conformations of the reactive center loop. However, we have previously shown that other, weaker binding, charged polysaccharides can give intermediate degrees of activation [Gettins, P. G. W., et al. (1993) Biochemistry 32, 8385-8389]. To examine whether such intermediate activation results from different reactive center loop conformations or, more simply, from a different equilibrium constant between the same two extreme conformations, we have used NBD covalently bound at the P1 position of an engineered R393C variant of antithrombin as a fluorescent reporter group and measured fluorescence lifetimes of the label in free antithrombin as well as in antithrombin saturated with long-chain high-affinity heparin, high-affinity heparin pentasaccharide, long-chain low-affinity heparin, and dextran sulfate. Steady state emission spectra, anisotropies, and dynamic quenching measurements were also recorded. We found that the large steady state fluorescence enhancements produced by binding of activators resulted from relief of a static quench of fluorescence of NBD in approximately 50% of the labeled antithrombin molecules rather than from any large change in lifetimes, and that similar lifetimes were found for NBD in all activated antithrombin-oligosaccharide complexes. Similar anisotropies and positions of the NBD emission maxima were also found in the absence and presence of activators. In addition, NBD was accessible to quenching agents in both the absence and presence of activators, with an at most 2-fold increase in quenching constants between these two extremes. The simplest interpretation of the partial static quench in the absence of activators, the different degrees of enhancement by different antithrombin activators, and the similar fluorescence properties and quenching behavior of the different states is that there are two distinct types of conformational equilibrium involving three distinct states of antithrombin, which we designate A, A', and B. A and A' represent low-affinity or inactive states of approximately equal energy, both having the hinge residues inserted into beta-sheet A. A is fluorescent, while A' is statically quenched. State B represents the activated loop-expelled conformation in which none of the NBD fluorophores are statically quenched, as a result of the loop, including the P1-NBD, moving away from the body of the antithrombin. Different activators are able to shift the equilibrium to the high-activity (B) state to different extents and hence give different degrees of measured activity, and different degrees of relief of static quench. The similar properties and accessibility of the NBD in the A and B conformations also indicate that the P1 side chain is not buried in the low-activity A conformation, suggesting that an earlier proposal that activation involves exposure of the P1 side chain cannot be the explanation for activation. As an alternative explanation, heparin activation may give access to an exosite on antithrombin for binding to factor Xa and hence be the principal basis for enhancement of the rate of inhibition.

Molecular mechanism and energetics of clamp assembly in Escherichia coli. The role of ATP hydrolysis when gamma complex loads beta on DNA.

Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex. The gamma complex requires ATP to load beta onto DNA. A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydrolysis (green fluorescence; phosphate binding protein assay) were simultaneously measured with millisecond timing resolution. The two temporally correlated stopped-flow signals revealed that a preassembled beta. gamma complex composite rapidly binds primer/template DNA in an ATP hydrolysis independent step. Once bound, two molecules of ATP are rapidly hydrolyzed (approximately 34 s(-1)). Following hydrolysis, gamma complex dissociates from the DNA ( approximately 22 s(-1)). Once dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only approximately 3 s(-1). Two single-site beta dimer interface mutants were examined which had impaired steady-state rates of ATP hydrolysis. The pre-steady-state correlated kinetics of these mutants revealed a pattern essentially identical to wild type. The anisotropy data showed that these mutants decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.

A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends. DNA triggers a change in binding specificity of the gamma complex clamp loader.

The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, gamma complex has a much higher affinity for single-stranded DNA (K(d) in the nM range) than for a primed template (K(d) in the microM range) under steady-state conditions. However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nM for the initial binding of gamma complex to DNA and a dissociation constant of 5.7 microM for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPgammaS show that ATP binding converts gamma complex from a low affinity "inactive" to high affinity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of gamma complex provides a two-tiered mechanism enabling gamma complex to recognize primed template sites and load beta, while preventing gamma complex from competing with DNA polymerase III core for binding a newly loaded beta.DNA complex.

DNA bending by EcoRI DNA methyltransferase accelerates base flipping but compromises specificity.

EcoRI DNA methyltransferase was previously shown to bend its cognate DNA sequence by 52 degrees and stabilize the target adenine in an extrahelical orientation. We describe the characterization of an EcoRI DNA methyltransferase mutant in which histidine 235 was selectively replaced with asparagine. Steady-state kinetic and thermodynamic parameters for the H235N mutant revealed only minor functional consequences: DNA binding affinity (KDDNA) was reduced 10-fold, and kcat was decreased 30%. However, in direct contrast to the wild type enzyme, DNA bending within the mutant enzyme-DNA complexes was not observed by scanning force microscopy. The bending-deficient mutant showed enhanced discrimination against the methylation at nontarget sequence DNA. This enhancement of enzyme discrimination was accompanied by a change in the rate-limiting catalytic step. No presteady-state burst of product formation was observed, indicating that the chemistry step (or prior event) had become rate-limiting for methylation. Direct observation of the base flipping transition showed that the lack of burst kinetics was entirely due to slower base flipping. The combined data show that DNA bending contributes to the correct assembly of the enzyme-DNA complex to accelerate base flipping and that slowing the rate of this precatalytic isomerization can enhance specificity.

Measurement of the absolute temporal coupling between DNA binding and base flipping.

The absolute temporal couplings between DNA binding and base flipping were examined for the EcoRI DNA methyltransferase. The binding event (monitored using rhodamine-x fluorescence anisotropy) was monophasic with a second-order on-rate of 1.1 x 10(7) M-1 s-1

Time-resolved fluorescence anisotropy study of the refolding reaction of the alpha-subunit of tryptophan synthase reveals nonmonotonic behavior of the rotational correlation time.

Time-resolved fluorescence anisotropy of a bound extrinsic probe was studied in an effort to characterize dynamic properties of the transient partially folded forms that appear during the folding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli. Previous studies have shown that alphaTS, a single structural domain, can be cleaved into autonomously folding amino- and carboxy-folding units comprising residues 1-188 and 189-268, respectively [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. By use of a double-kinetic approach [Jones, B. E., Beechem, J. M., and Matthews, C. R. (1995) Biochemistry 34, 1867-1877], the rotational correlation time of 1-anilino-8-naphthalene sulfonate bound to nonpolar surfaces of folding intermediates was measured by time-correlated single photon counting at varying time delays following initiation of folding from the urea-denatured form by stopped-flow techniques. Comparison of the rotational correlation times for the full-length alphaTS and the amino-terminal fragment suggests that folding of the amino-terminal fragment and carboxy-terminal fragment is coordinated, not autonomous, on the milliseconds to seconds time scale. If a spherical shape is assumed, the apparent hydrodynamic radius of alphaTS after 5 ms is 26.8 A. The radius increases to 28.5 A by 1 s before decreasing to the radius for native alphaTS, 24.7 A, on a longer time scale (>25 s). Viewed within the context of the kinetic folding model of alphaTS [Bilsel, O., Zitzewitz, J. A., Bowers, K. E. , and Matthews, C. R. (1999) Biochemistry 38, 1018-1029], the initial collapse reflects the formation of an off-pathway burst-phase intermediate in which at least part of the carboxy folding unit interacts with the amino folding unit. The subsequent increase in rotational correlation time corresponds to the formation of an on-pathway intermediate that leads to the native conformation. The apparent increase in the radius for the on-pathway intermediate may reflect a change in the interaction of the two-folding units, thereby forming a direct precursor for the alpha/beta barrel structure.

Pre-steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia coli DNA polymerase III onto DNA.

The beta protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the gamma complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of beta onto DNA. Two beta dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), have been used to show that ATP binding is required for gamma complex and beta to associate with DNA, but that a gamma complex-catalyzed ATP hydrolysis is required for gamma complex to release the beta.DNA complex and complete the reaction. In the presence of ATP and gamma complex, the beta mutants associate with DNA as efficiently as wild type beta. However, completion of the reaction is much slower with the beta mutants because of decreased ATP hydrolysis by the gamma complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPgammaS in reactions using wild type beta. Thus, the assembly of beta around DNA is coupled tightly to the ATPase activity of the gamma complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.

Deconvolution of the fluorescence emission spectrum of human antithrombin and identification of the tryptophan residues that are responsive to heparin binding.

Heparin causes an allosterically transmitted conformational change in the reactive center loop of antithrombin and a 40% enhancement of tryptophan fluorescence. We have expressed four human antithrombins containing single Trp --> Phe mutations and determined that the fluorescence of antithrombin is a linear combination of the four tryptophans. The contributions to the spectrum of native antithrombin at 340 nm were 8% for Trp-49, 10% for Trp-189, 19% for Trp-225, and 63% for Trp-307. Trp-225 and Trp-307 accounted for the majority of the heparin-induced fluorescence enhancement, contributing 37 and 36%, respectively. Trp-49 and Trp-225 underwent spectral shifts of 15 nm to blue and 5 nm to red, respectively, in the antithrombin-heparin complex. The blue shift for Trp-49 is consistent with partial burial by contact with heparin, whereas the red shift for Trp-225 and large enhancement probably result from increased solvent access upon heparin-induced displacement of the contact residue Ser-380. The enhancement for Trp-307 may result from the heparin-induced movement of helix H seen in the crystal structure. The time-resolved fluorescence properties of individual tryptophans of wild-type antithrombin were also determined using the four variants and showed that Trp-225 and Trp-307 experienced the largest change in lifetime upon heparin binding, providing support for the steady-state fluorescence deconvolution.

Exonuclease-polymerase active site partitioning of primer-template DNA strands and equilibrium Mg2+ binding properties of bacteriophage T4 DNA polymerase.

The binding of bacteriophage T4 DNA polymerase (T4 pol) to primer-template DNA with 2-aminopurine (2AP) located at the primer terminus results in the formation of a hyperfluorescent 2AP state. Changes in this hyperfluorescent state were utilized to investigate the fractional concentration of primer-templates bound at the exonuclease and statically quenched polymerase sites. In the absence of Mg2+, a hydrophobic exonuclease site dominates over the polymerase site for possession of the primer terminus. The fractional concentration of primer termini in the exonuclease site was found to be 64 and 84% for correct (AP-T) and mismatched (AP-C) primer-templates, respectively. Exonuclease-deficient mutants, polymerase-switching mutants, and nucleoside triphosphates all shift this equilibrium toward the polymerase site. Synthesis of stereospecific hydrolysis resistant phosphorothioate 2AP-labeled DNA allowed Mg2+ ion binding titrations to be performed in the presence of bound DNA without the complication of the excision reaction. High- and low-affinity Mg2+ binding sites were observed in the presence of bound double-stranded (ds) DNA, with dissociation constants in the micromolar (WT Kd = 5.1 microM) and millimolar (WT Kd = 2.5 mM) concentration ranges. Mg2+ binding was found to be a key "conformational switch" for T4 pol. As the high-affinity Mg2+ binding sites are filled, the primer terminus migrates from the exonuclease site to a highly based stacked polymerase active site. Filling the low-affinity Mg2+ sites further shifts the primer terminus into the polymerase site. As the low-affinity Mg2+ sites are filled, T4 pol "loosens its grip" on the primer terminus, as shown by a large amplitude increase in the nanosecond rotational mobility of 2AP within the bound T4 complex. The hyperfluorescent exonuclease site is spatially localized to 2AP positioned on the primer end. The penultimate (n - 1) position, as well as n - 2 and n - 5 positions, reveals no detectable fluorescent enhancement upon binding. The observed position-dependent fluorescence data, when combined with time-resolved total-intensity and anisotropy data, suggest that the creation of the hyperfluorescent state is caused by phenylalanine 120 (F120) of T4 pol intercalating into 2AP primers much like that observed for phenylalanine 123 of RB69 DNA polymerase intercalating into deoxythymidine primers [Wang, J., et al. (1997) Cell 89, 1087-1099]. As Mg2+ binds in the exonuclease site of T4 pol, the primer terminus appears to be "pulled backward" into the active site, decreasing the concentration of F120-intercalated primer termini, and bringing the exonuclease active site residues closer to the primer terminus scissile phosphate bond.

Stopped-flow fluorescence study of precatalytic primer strand base-unstacking transitions in the exonuclease cleft of bacteriophage T4 DNA polymerase.

DNA polymerases are complex enzymes which bind primer-template DNA and subsequently either extend or excise the terminal nucleotide on the primer strand. In this study, a stopped-flow fluorescence anisotropy binding assay is combined with real-time measurements of a fluorescent adenine analogue (2-aminopurine) located at the 3'-primer terminus. Using this combined approach, the exact time course associated with protein binding, primer terminus unstacking, and base excision by the 3' --> 5' exonuclease of bacteriophage T4 (T4 pol) was examined. T4 pol binding and dissociation kinetics were found to obey simple kinetics, with identical on rates (kon = 4.6 x 10(8) M-1 s-1) and off rates (koff = 9.3 s-1) for both single-stranded primers and double-stranded primer-templates (at 100 microM Mg2+). Although the time course for T4 pol-DNA association and dissociation obeyed simple kinetics, at suboptimal Mg2+ concentrations (e.g., 100 microM), non-first-order sigmoidal kinetics were observed for the base-unstacking reaction of the primer terminus in double-stranded primer-templates. The observed sigmoidal kinetics for base unstacking demonstrate that T4 pol is a hysteretic enzyme [Frieden, C. (1970) J. Biol. Chem. 245, 5788-5799] and must exist in two DNA bound conformations which differ greatly in base-unstacking properties. A Mg2+-dependent time lag of 10 ms is observed between primer-template binding and the beginning of the unstacking transition, which is 50% complete at 22 +/- 1 ms after addition of 100 microM Mg2+. Following the hysteretic lag, a simple first-order primer terminus unstacking rate of 130 s-1 is resolved, which is protein and Mg2+ concentration-independent. For the processing of single-stranded primers, all kinetic complexity is lost, and T4 pol binding and primer end base-unstacking kinetics can be superimposed. These data reveal that the kinetic processing of double-stranded primer-template DNA by T4 pol is much more complex than that of single-stranded primers, and suggest that the intrinsic "switching rate" between the polymerase and exonuclease sites may be much faster than previously proposed.

Investigation of the binding of isoform-selective inhibitors to prostaglandin endoperoxide synthases using fluorescence spectroscopy.

Prostaglandin endoperoxide synthase (PGHS) is a heme protein that catalyzes the committed step in prostaglandin and thromboxane biosynthesis. Two isoforms of PGHS exist, a constitutive form termed PGHS-1 and an inducible form termed PGHS-2. We report here fluorescence resonance energy transfer analysis of isoform-selective inhibitors interacting with PGHS-1 and PGHS-2. By measuring fluorescence quenching due to the energy transfer of the inhibitor fluorescence to the heme prosthetic group of PGHS, we determined these inhibitors bind in the arachidonic acid substrate access channel with an R0 of 35 A for PGHS-1 with the PGHS-1 inhibitor and an R0 of 21 A for PGHS-2 with the PGHS-2 inhibitor. The observed fluorescence quenching is completely dynamic and dominated by quenching by the heme. Time-resolved results combined with molecular modeling determine the distance from the inhibitor to the heme moiety to be 20 A in PGHS-1 and 18 A in PGHS-2. Preliminary stopped-flow kinetic studies reveal that the rate of quenching is limited by a first-order protein transition, which is slow, and that bound inhibitor undergoes rapid exchange.

Direct real time observation of base flipping by the EcoRI DNA methyltransferase.

DNA methyltransferases are excellent prototypes for investigating DNA distortion and enzyme specificity because catalysis requires the extrahelical stabilization of the target base within the enzyme active site. The energetics and kinetics of base flipping by the EcoRI DNA methyltransferase were investigated by two methods. First, equilibrium dissociation constants (KDDNA) were determined for the binding of the methyltransferase to DNA containing abasic sites or base analogs incorporated at the target base. Consistent with a base flipping mechanism, tighter binding to oligonucleotides containing destabilized target base pairs was observed. Second, total intensity stopped flow fluorescence measurements of DNA containing 2-aminopurine allowed presteady-state real time observation of the base flipping transition. Following the rapid formation of an enzyme-DNA collision complex, a biphasic increase in total intensity was observed. The fast phase dominated the total intensity increase with a rate nearly identical to k(methylation) determined by rapid chemical quench-flow techniques (Reich, N. O., and Mashoon, N. (1993) J. Biol. Chem. 268, 9191-9193). The restacking of the extrahelical base also revealed biphasic kinetics with the recovered amplitudes from these off-rate experiments matching very closely to those observed during the base unstacking process. These results provide the first direct and continuous observation of base flipping and show that at least two distinct conformational transitions occurred at the flipped base subsequent to complex formation. Furthermore, our results suggest that the commitment to catalysis during the methylation of the target site is not determined at the level of the chemistry step but rather is mediated by prior intramolecular isomerization within the enzyme-DNA complex.

Design and characterization of a multisite fluorescence energy-transfer system for protein folding studies: a steady-state and time-resolved study of yeast phosphoglycerate kinase.

A multisite distance-based fluorescence resonance energy-transfer assay system was developed for the study of protein folding reactions. Single- and double-cysteine substitution mutagenesis was utilized to place sulfhydryl residues throughout the tertiary structure of the bidomain enzyme yeast phosphoglycerate kinase (PGK). These reactive cysteines were covalently modified with extrinsic donor [5-[[2-(2-iodoacetamido)ethyl]amino]-1-naphthalenesulfonic acid] and acceptor (5-iodoacetamidofluorescein) fluorescent labels. A detailed experimental strategy was followed, which revealed that, when these relatively large extrinsic fluorescent labels are covalently attached to properly selected solvent-exposed residues, they do not affect the intrinsic stability of the protein. The PGK crystal structure was combined with molecular dynamics simulations of the dyes built into the protein and time-resolved anisotropy experiments, in order to estimate a more realistic orientation factor, *, for each donor/acceptor pair. Time-resolved and steady-state fluorescence energy-transfer experiments revealed that this distance assay, spanning six different donor-acceptor distances, is linear and accurate (to within 10-20%) over the range of 30-70 A. This distance assay system for PGK allows for the measurement of long-range changes in intra- and interdomain spatial organization during protein folding reactions. The approach which we have developed can be applied to any protein system in which unique one- and two-site cysteine residues can be engineered into a protein. In the following paper [Lillo, M. P., et al. (1997) Biochemistry 36, 11273-11281], these multisite energy-transfer pairs are utilized for stopped-flow unfolding studies.

Real-time measurement of multiple intramolecular distances during protein folding reactions: a multisite stopped-flow fluorescence energy-transfer study of yeast phosphoglycerate kinase.

Understanding the set of rules which dictate how the primary amino acid sequence determines tertiary structure is an unsolved problem in biophysics. If it were possible to simultaneously measure all of the intramolecular distances in a protein (in real time) during a folding reaction, the "second" genetic code problem would be solved. Regrettably, no such technique currently exists. As a first step toward this goal, an optical distance assay system has been developed for a two-domain protein, yeast phosphoglycerate kinase (PGK), using Förster resonance energy transfer [Lillo, M. P., et al. (1997) Biochemistry 36, 11261-11272]. In this study, real-time stopped-flow distance changes are measured using six unique pairs of donor/acceptor fluorescent labels strategically placed throughout the tertiary structure of PGK. These multiple donor/acceptor sites were genetically engineered into PGK by cysteine substitution mutagenesis followed by extrinsic labeling with fluorescent probes, 5-[[[(2-iodoacetyl)amino]ethyl]amino]naphthalenesulfonic acid (as a donor) and 5-iodoacetamidofluorescein (acceptor). The unfolding of PGK is found to be a sequential multistep process (native --> I1 --> I2 --> unfolded) with rate constants of 0.30, 0.16, and 0.052 s-1, respectively (from native to unfolded). Unique to this unfolding study, six intramolecular distance vectors have been resolved for both the I1 and I2 states. With this distance information, it is shown that the transition from the native to I1 state can be modeled as a large hinge-bending motion, in which both domains "swing away" from each other by about 15 A. As the domains move apart, the carboxyl-terminal domain rotates almost 90 degrees about the hinge region connecting the two domains. It is also shown that the amino-terminal domain remains intact during the native --> I1 transition, consistent with our previous site-specific tryptophan fluorescence anisotropy stopped-flow study [Beechem, J. M., et al. (1995) Biochemistry 34, 13943-13948]. Future experiments are proposed which will attempt to resolve in detail the unfolding/refolding transitions in this protein with a resolution of approximately 5-10 A.

Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL.

The chaperonin GroEL is a double-ring structure with a central cavity in each ring that provides an environment for the efficient folding of proteins when capped by the co-chaperone GroES in the presence of adenine nucleotides. Productive folding of the substrate rhodanese has been observed in cis ternary complexes, where GroES and polypeptide are bound to the same ring, formed with either ATP, ADP or non-hydrolysable ATP analogues, suggesting that the specific requirement for ATP is confined to an action in the trans ring that evicts GroES and polypeptide from the cis side. We show here, however, that for the folding of malate dehydrogenase and Rubisco there is also an absolute requirement for ATP in the cis ring, as ADP and AMP-PNP are unable to promote folding. We investigated the specific roles of binding and hydrolysis of ATP in the cis and trans rings using mutant forms of GroEL that bind ATP but are defective in its hydrolysis. Binding of ATP and GroES in cis initiated productive folding inside a highly stable GroEL-ATP-GroES complex. To discharge GroES and polypeptide, ATP hydrolysis in the cis ring was required to form a GroEL-ADP-GroES complex with decreased stability, priming the cis complex for release by ATP binding (without hydrolysis) in the trans ring. These observations offer an explanation of why GroEL functions as a double-ring complex.