New PC versions of the kinetic-simulation and fitting programs, KINSIM and FITSIM.
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Computer Simulation↗
Biomedical subjects
C Frieden
Publications and source records attributed to C Frieden.
Real-time refolding studies of 6-19F-tryptophan labeled Escherichia coli dihydrofolate reductase using stopped-flow NMR spectroscopy.
Escherichia coli dihydrofolate reductase (ecDHFR, EC1.5.1.3) contains 5 tryptophan residues that have been replaced with 6-19F-tryptophan. Five native and four of the five unfolded tryptophan resonances can be resolved in the 1D 19F NMR spectra and have been assigned [Hoeltzli, S. D., & Frieden, C. (1994) Biochemistry 33, 5502-5509]. This resolution allows the behavior of the native and the unfolded resonances assigned to each individual tryptophan to be monitored during the unfolding or refolding process. We now use these assignments and stopped-flow NMR to investigate the real-time behavior of specific regions of the protein during refolding of DHFR after dilution from 4.6 to 2.3 M urea (midpoint of the transition = 3.8 M) at 5 degrees C. Approximately half of the intensity of each of the four unfolded resonances is present at the first measurable time point (1.5 s). Little native resonance intensity is detectable at this time. The remaining unfolded resonance intensities present then disappear in two phases, with rates similar to the two slowest phases observed by either stopped-flow fluorescence or circular dichroism spectroscopy upon refolding under the same conditions. Substantial total resonance intensity is missing during the first 20 s of the refolding process. The appearance of the majority of native resonance intensity (as assessed by the height of each of the five native tryptophan resonances) is slow and similar for all five tryptophans. In contrast, the largest amplitude changes observed by either stopped-flow far-UV circular dichroism spectroscopy or fluorescence spectroscopy, and the greatest loss of unfolded resonance intensity, occur much more rapidly. We conclude from these studies: (1) that, under these conditions, the unfolded state remains substantially populated after initiation of refolding; (2) that the early steps in refolding involve a solvent protected intermediate containing substantial secondary structure, but (3) that the stable native side chain interactions form slowly and are associated with the final rate-limiting phase of the refolding process. Preliminary analysis of the area of broadened native resonances suggests that these resonances may appear at different rates, indicating that some regions of the protein begin to sample a native-like side chain environment while side chain environment in other regions of the protein remains less ordered. The results of this study are consistent with the earlier studies demonstrating that mobility of side chains is an early step in unfolding [Hoeltzli, S. D., & Frieden, C. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318-9322] and that recovery of enzymatic activity occurs as a late step in the folding process [Frieden, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4413-4416].
Circular Dichroism↗
Intestinal fatty acid-binding protein: the structure and stability of a helix-less variant.
The structure of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) exhibits a beta-clam topology comprised of two five-stranded antiparallel beta-sheets surrounding a large solvent-filled cavity into which the ligand binds. It also contains two alpha-helices that span residues E15-A32 and join beta-strands A and B. This helical domain is conserved in all proteins of this family for which structures have been determined. In order to assess the structural and functional role of the helical domain, we engineered a variant of I-FABP by deleting residues 15-31 and inserting a Ser-Gly linker after residue 14. Circular dichroism measurements indicated that this I-FABP variant, termed delta 17-SG, has a high beta-sheet content similar to that of the wild-type protein. Two-dimensional NMR spectra of delta 17-SG revealed patterns similar to those observed for wild-type I-FABP, except for the selective absence of resonances and through-space interactions assigned to the helical domain. The delta 17-SG variant was less stable to denaturant than wild-type I-FABP, but the folding-unfolding transition was highly cooperative and reversible. Taking into account the lower stability, the refolding kinetics of delta 17-SG were essentially identical to those of wild-type. We conclude that delta 17-SG is a helix-less, essentially all-beta-sheet variant of I-FABP and that the helical domain is not a required element of the beta-clam topology of I-FABP. In addition, the helical domain does not appear to serve as a nucleation site for the refolding process. As shown in the accompanying paper [Cistola, D. P., Kim, K., Rogl, H., & Frieden, C. (1996) Biochemistry 35, 7559-7565], the helices may function to regulate the kinetics and energetics of ligand binding.
Amino Acid Sequence↗
Fatty acid interactions with a helix-less variant of intestinal fatty acid-binding protein.
Intestinal fatty acid-binding protein (I-FABP) binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two antiparallel beta-sheets. The structure also contains two short alpha-helices which form a cap over one end of the binding cavity adjacent to the methyl terminus of the fatty acid. In this study, we employed a helix-less variant of I-FABP known as delta 17-SG [Kim, K., Cistola, D.P.,& Frieden, C. (1996) Biochemistry 35, 7553-7558] to investigate the role of the helical region in maintaining the integrity of the binding cavity and mediating the acquisition of ligand. Fluorescence and NMR experiments were used to characterize the energetic, structural, and kinetic properties of fatty acid binding to this variant, and the results were compared and contrasted with those of wild-type I-FABP and a single-site mutant, R106T. Remarkably, oleate bound to delta 17-SG with a dissociation constant of 4.5 microM, a value comparable to that for R106T and approximately 20-100-fold higher than that for wild-type I-FABP. Heteronuclear two-dimensional NMR spectra for [2-13C]palmitate complexed with delta 17-SG revealed a pattern nearly identical to that observed for the wild-type protein, but distinct from that for R106T. In addition, the ionization behavior of bound [1-13C]palmitate and the nearest neighbor patterns for [2-13C]palmitate derived from 13C-filtered NOESY experiments were very similar for delta 17-SG and the wild-type protein. These results implied that the fatty acid-protein interactions characteristic of the carboxyl end of the fatty acid binding cavity in the wild-type protein were essentially intact in the helix-less variant. In contrast, 13C-filtered NOESY spectra of [16-13C]palmitate bound to delta 17-SG indicated that the fatty acid-protein interactions at the methyl end of the binding cavity were disrupted. As determined by stopped-flow fluorescence, the observed ligand association rates for both delta 17-SG and wild-type I-FABP increased with increasing oleate concentration, but only the wild-type protein exhibited a limiting value of 1000 s-1. This rate-limiting process was interpreted as a conformational change involving the helical region that allows the ligand access to the internal cavity. Simulation and fitting of the kinetic results yielded ligand association rates for delta 17-SG and wild-type I-FABP that were comparable. However, the dissociation rate for wild-type protein was 16-fold lower than that for delta 17-SG. We conclude that the alpha-helices of I_FABP are not required to maintain the integrity of the fatty acid binding cavity but may serve to regulate the affinity of fatty acid binding by selectively altering the dissociation rate constant. In this manner, conformational changes involving the alpha-helical domain may help control the transfer of fatty acids within the cell.
Amino Acid Sequence↗
Determination of regions in the dihydrofolate reductase structure that interact with the molecular chaperonin GroEL.
Dihydrofolate reductase (DHFR) from Escherichia coli does not interact with the molecular chaperonin GroEL regardless of whether the interaction is initiated from the native or the unfolded state. In contrast, murine DHFR shows a strong interaction with GroEL. Using the structure of human DHFR as a model for the murine protein, a superimposition of the two structures shows that there are three distinct external loops in the eukaryotic DHFR that are not present in the E. coli protein. Removal of one loop (residues 99-108) from the eukaryotic murine DHFR has no effect on the interaction with GroEL. On the basis of the differences in structures, we inserted either of two surface loops of murine DHFR into the corresponding regions of E. coli DHFR. In the first mutant (EcDHFR-i(9)36), residues 36 and 37 (L-N) of E. coli DHFR were replaced with the nine amino acid sequence T-T-S-S-V-E-G-K-Q. In the second mutant (EcDHFR-i(7)136), residues 136-139 (V-F-S-E) of E. coli DHFR were replaced with the seven amino acid sequence L-P-E-Y-P-G-V. Both E. coli DHFR mutants formed a complex with GroEL starting from either the native or the unfolded states of DHFR. The binding was specific since the presence of MgATP caused the release of the proteins from GroEL. As with murine DHFR, nonnative conformations of EcDHFR-i(9)36 and EcDHFR-i(7)136 are bound to GroEL. Fluorescence titration techniques were used to quantitate the interaction between GroEL and these proteins. A simple chromatographic procedure was developed to remove contaminating tryptophan containing peptides from GroEL samples. The mutant EcDHFR-i(7)136 binds to GroEL with a stoichiometry of 4-5 mol of DHFR per mol of GroEL tetradecamer, while murine DHFR binds to GroEL with a stoichiometry of 2 mol of DHFR per mol of GroEL tetradecamer. Both murine DHFR and EcDHFR-i(7)136 bind to GroEL very tightly, with equilibrium dissociation constants of less than 85 nM.
Amino Acid Sequence↗
Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.
Wild-type actin and a mutant actin were isolated from yeast (Saccharomyces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin. While similar to the properties of skeletal muscle actin, there are several important differences between the wild-type yeast and muscle actins. First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result from a difference in the steps involving formation of the nucleating species. Second, as measured with pyrene-labeled yeast actin, but not with intrinsic fluorescence, there is an overshoot in the fluorescence that has not been observed with skeletal muscle actin under the same conditions. Third, in order to simulate the polymerization process of wild-type yeast actin it is necessary to assume some fragmentation of the filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutant actin (R177A/D179A) has also been isolated and studied. The mutations are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elongation rates of the wild type and the mutant differ by only about 3-fold, and the slower polymerization of the mutant appears to result primarily from poorer nucleation.
Actins↗
Catalytic strategy of citrate synthase: subunit interactions revealed as a consequence of a single amino acid change in the oxaloacetate binding site.
The active site of pig heart citrate synthase contains a histidine residue (H320) which interacts with the carbonyl oxygen of oxaloacetate and is implicated in substrate activation through carbonyl bond polarization, a major catalytic strategy of the enzyme. We report here the effects on the catalytic mechanism of changing this important residue to glycine. H320G shows modest impairment in substrate Michaelis constants [(7-16)-fold] and a large decrease in catalysis (600-fold). For the native enzyme, the chemical intermediate, citryl-CoA, is both hydrolyzed and converted back to reactants, oxaloacetate and acetyl-CoA. In the mutant, citryl-CoA is only hydrolyzed, indicating a major defect in the condensation reaction. As monitored by the carbonyl carbon's chemical shift, the extent of oxaloacetate carbonyl polarization is decreased in all binary and ternary complexes. As indicated by the lack of rapid H320G--oxaloacetate catalysis of the exchange of the methyl protons of acetyl-CoA or the pro-S-methylene proton of propionyl-CoA, the activation of acetyl-CoA is also faulty. Reflecting this defect in acetyl-CoA activation, the carboxyl chemical shift of H320G-bound carboxymethyl-CoA (a transition-state analog of the neutral enol intermediate) fails to decrease on formation of the H3020G-oxaloacetate-carboxymethyl-CoA ternary complex. Progress curves and steady-state data with H320G using citryl-CoA as substrate show unusual properties: substrate inhibition and accelerating progress curves. Either one of two models with subunit cooperativity [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88; Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5, 365] quantitatively accounts for both the initial velocity data and the individual progress curves. The concentrations of all enzyme forms and complexes are assumed to rapidly reach their equilibrium values compared to the rate of substrate turnover. The native enzyme also behaves according to models for subunit cooperativity with citryl-CoA as substrate. However, the rates of formation/dissociation and reaction of complexes are kinetically significant. Comparisons of the values of kinetic constants between the native and mutants enzymes lead us to conclude that the mutant less readily undergoes a conformation change required for efficient activation of substrates.
Acetyl Coenzyme A↗
Stopped-flow NMR spectroscopy: real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase.
Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k approximately 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-flow circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that approximately 20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.
Circular Dichroism↗
An NADH-induced conformational change that mediates the sequential 3 beta-hydroxysteroid dehydrogenase/isomerase activities is supported by affinity labeling and the time-dependent activation of isomerase.
3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and steroid delta-isomerase were copurified as a single protein from human placental microsomes. Because NADH is an essential activator of isomerase (Kact = 2.4 microM, Vmax = 0.6 mumol/min/mg), the affinity alkylating nucleotide, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TADP), was synthesized. 8-BDB-TADP activates isomerase (Kact = 338 microM, Vmax = 2.1 mumol/min/mg) prior to inactivating the enzyme. The inactivation kinetics for isomerase fit the Kitz and Wilson model for time-dependent, irreversible inhibition by 8-BDB-TADP (KI = 314 microM, first order maximal rate constant kobs = 7.8 x 10(-3) s-1). NADH (50 microM) significantly protects isomerase from inactivation by 8-BDB-TADP (100 microM). The isomerase activity is inactivated more rapidly by 8-BDB-TADP as the concentration of the affinity alkylator increases from 67 microM (t1/2 = 8.4 min) to 500 microM (t1/2 = 2.4 min). In sharp contrast, the 3 beta-HSD activity is inactivated more slowly as the concentration of 8-BDB-TADP increases from 67 microM (t1/2 = 4.8 min) to 500 microM (t1/2 = 60.0 min). We hypothesized that the paradoxical kinetics of 3 beta-HSD inactivation is a consequence of the activation of isomerase by 8-BDB-TADP via a nucleotide-induced shift in enzyme conformation. Biophysical support for an NADH-induced conformational change was obtained using stopped-flow fluorescence spectroscopy. The binding of NADH (10 microM) quenches the intrinsic fluorescence of the enzyme protein in a time-dependent manner (rate constant kapp = 8.1 x 10(-3) s-1, t1/2 = 85 s). A time lag is also observed for the activation of isomerase by NADH. This combination of affinity labeling and biophysical data using nucleotide derivatives supports our model for the sequential reaction mechanism; the cofactor product of the 3 beta-HSD reaction, NADH, activates isomerase by inducing a conformational change in the single, bifunctional enzyme protein.
Adenosine Diphosphate↗
Intestinal fatty acid binding protein: folding of fluorescein-modified proteins.
The rat intestinal fatty acid binding protein is an almost all beta-sheet protein that encloses a large interior cavity into which the fatty acid ligand binds. The protein contains neither cysteine nor proline. In a previous report, six site-directed mutants were obtained, each having a single cysteine residue [Jiang, N., & Frieden, C., (1993) Biochemistry 32, 11015-11021] either in a turn or pointed into the cavity. In this report, each mutant has been unfolded in denaturant and modified with 5-iodoacetamido-fluorescein to introduce a large, bulky, and fluorescent group into the protein at a known position. In all cases, fluorescence changes indicated that the modified protein refolded, and circular dichroism measurements suggested that the refolded protein appeared to be mostly beta-sheet. Denaturation curves suggest that for two mutants intermediate structures exist at denaturant concentrations well below the midpoint of the unfolding curve. For each modified, folded protein, one- and two-dimensional 1H NMR spectra were accumulated and compared to the unmodified and wild-type proteins. While the spectra for the modified proteins showed a number of changes in chemical shifts, they were also consistent with folded proteins on the basis of the degree of chemical shift dispersion. Of the six modified mutant proteins, two appear to have the fluorescein group located in the cavity, but only one of these did not bind fatty acid. The remaining modified proteins are capable of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
19F NMR spectroscopy of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate reductase: equilibrium folding and ligand binding studies.
Escherichia coli dihydrofolate reductase contains five tryptophan residues distributed throughout its structure. In order to examine the regions of the protein surrounding these tryptophan residues, we have incorporated 6-fluorotryptophan into the protein. To assign the five resonances observed in the 19F NMR spectrum, five site-directed mutants of the enzyme were made, each with one tryptophan replaced by a phenylalanine. The 19F NMR spectra of the apoprotein, two binary complexes (with NADPH or methotrexate), and one ternary complex (with NADPH and methotrexate) were obtained. The chemical shifts of two of the tryptophan resonances (at positions 22 and 74) are particularly sensitive to ligand binding, while the remaining three (at positions 30, 47, and 133) change, but by less. Since several of the tryptophans are distant from the binding site, these results suggest that 19F NMR can detect ligand-induced changes that are propagated throughout the structure. In the apoprotein, the resonances of the tryptophans at positions 22 and 30 are broadened. In the binary complex with NADPH, the resonances of tryptophans 30 and 74 are broadened while that of tryptophan 22 almost disappears. The line broadening of the tryptophan 22 resonance may reflect motion in that part of the protein, since it is near a region that is disordered in the crystal structure of the apoprotein and its NADP+ complex. In contrast, in the ternary complex this region has a defined structure, and all resonances are of equal intensity and line width. The 19F NMR spectra of the apoprotein and the three ligand complexes were also examined as a function of urea concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
The tyrosine B10 hydroxyl is crucial for oxygen avidity of Ascaris hemoglobin.
The parasitic nematode Ascaris suum has a gene encoding a two-domain hemoglobin with remarkable oxygen avidity. The strong interaction with oxygen is a consequence of a particularly slow oxygen off-rate. The single polypeptide chain consists of two domains, each of which can be expressed separately in Escherichia coli as a globin-like protein exhibiting oxygen binding characteristics comparable with the native molecule. Site-directed mutagenesis was performed on the gene segment encoding domain one. The E7 position, involved in forming a hydrogen bond with the liganded oxygen in vertebrate globins, is a glutamine in both Ascaris domains. Conversion of this residue to leucine or alanine produced a hemoglobin variant with an oxygen off-rate 5- or 60-fold faster than that of unaltered domain one. Replacement of the tyrosine B10 with either phenylalanine or leucine (as found in vertebrate globins) yielded hemoglobin mutants with oxygen off-rates 280- or 570-fold faster, approaching rates found with vertebrate myoglobins. The data suggest that the distal glutamine hydrogen bonds with the liganded oxygen and that the tyrosine B10 hydroxyl contributes an additional hydrogen bond that appears substantially responsible for the extreme oxygen avidity of Ascaris hemoglobin.
Amino Acid Sequence↗
Numerical integration of rate equations by computer: an update.
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Computer Simulation↗
Analysis of kinetic data: practical applications of computer simulation and fitting programs.
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Computer Simulation↗
Intestinal fatty acid binding protein: characterization of mutant proteins containing inserted cysteine residues.
Site-directed mutagenesis was used to introduce cysteine residues into the rat intestinal fatty acid binding protein, an almost all beta-sheet protein that in the wild-type contains neither cysteine nor proline residues. Six mutants (I23C, S53C, V60C, L72C, L89C, and A104C) with a single cysteine residue substituted for a hydrophobic residue were characterized by their stability toward denaturants at pH 7.2 and 9.6, by their fluorescent properties, and by their reactivity toward the sulfhydryl modifying reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 4,4'-dipyridyl disulfide (4-PDS). In terms of protein stability, the substitutions were reasonably conservative with only two (V60C and L89C) being somewhat less stable than the wild-type. The mutant proteins differed considerably, however, in their reactivity toward the modifying reagents. One residue, Cys89, located in a hydrophobic core near a turn between two beta-strands, was unreactive, while two residues, Cys60 and Cys104, located in the middle of beta-strands in the cavity into which fatty acid binds, reacted only very slowly and were further protected by oleate. Cys53, located near a turn and partially buried, appeared to have an unusually low pK value. Two residues, Cys23 and Cys72, reacted more rapidly in the native protein than in the unfolded protein. Both residues are located near the portal for the fatty acid binding, and one, Cys72, was strongly protected from modification by the presence of oleate. Examination of the crystal structure indicates that Cys72 is not easily solvent-accessible. We conclude that this high reactivity for this residue may be a consequence of rapid conformational flexibility in this region of the structure.
Animals↗
Conditional dihydrofolate reductase deficiency due to transposon Tn5tac1 insertion downstream from the folA gene in Escherichia coli.
Transposon Tn5tac1 can generate conditional mutations by virtue of an outward-facing tac promoter, which is regulated by the lac repressor and isopropyl-beta-D-thiogalactopyranoside (IPTG). We report here on a Tn5tac1 insertion in Escherichia coli that results in a conditional (IPTG-elicited) folA mutant phenotype: During aerobic growth, IPTG caused decreased synthesis of dihydrofolate reductase (DHFR; encoded by the folA gene) and hypersensitivity to trimethoprim (a DHFR inhibitor); during anaerobic growth, IPTG elicited auxotrophy that was satisfied by thymine or glycine or threonine. The Tn5tac1 insertion was downstream from folA, with the tac promoter pointing into the gene (antisense direction). Complementation tests indicated that the conditional folA deficiency was a cis effect of transcription from the tac promoter, perhaps due to head-to-head collision between converging RNA polymerases.
Alleles↗
NMR and protein folding: equilibrium and stopped-flow studies.
NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen-deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use 19F-substituted amino acids to follow changes in side-chain environment upon folding. Current techniques of molecular biology allow assignments of 19F resonances to specific amino acids by site-directed mutagenesis. It is possible to follow changes and to analyze results from 19F spectra in real time using a stopped-flow device incorporated into the NMR spectrometer.
Flow Injection Analysis↗
Numerical integration of rate equations by computer.
This article describes the availability of a package of free, user-friendly computer programs (VAX, PC or Mac) that allow investigators to obtain insights into reaction mechanisms. The programs provide an easy way to use the full time-course of a reaction to evaluate kinetic mechanisms and determine rate constants for the transitions between the intermediates. While the programs can be used to examine any reaction mechanism, they are particularly useful for enzymatic reactions.
Enzymes↗