The structure of nonvertebrate actin: implications for the ATP hydrolytic mechanism.

The structures of Saccharomyces cerevisiae, Dictyostelium, and Caenorhabditis elegans actin bound to gelsolin segment-1 have been solved and refined at resolutions between 1.9 and 1.75 A. These structures reveal several features relevant to the ATP hydrolytic mechanism, including identification of the nucleophilic water and the roles of Gln-137 and His-161 in positioning and activating the catalytic water, respectively. The involvement of these residues in the catalytic mechanism is consistent with yeast genetics studies. This work highlights both structural and mechanistic similarities with the small and trimeric G proteins and restricts the types of mechanisms responsible for the considerable enhancement of ATP hydrolysis associated with actin polymerization. The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins.

Intestinal fatty acid binding protein: the folding mechanism as determined by NMR studies.

The intestinal fatty acid binding protein is composed of two beta-sheets surrounding a large interior cavity. There is a small helical domain associated with the portal for entry of the ligand into the cavity. Denaturation of the protein has been monitored in a residue-specific manner by collecting a series of two-dimensional (1)H-(15)N heteronuclear single-quantum coherence (HSQC) NMR spectra from 0 to 6.5 M urea under equilibrium conditions. In addition, rates for hydrogen-deuterium exchange have been measured as a function of denaturant concentration. Residual, native-like structure persists around hydrophobic clusters at very high urea concentrations. This residual structure (reflecting only about 2-7% persistence of native-like structure) involves the turns between beta-strands and between the two short helices. If this persistence is assumed to reflect transient native-like structure in these regions of the polypeptide chain, these sites may serve as nucleation sites for folding. The data obtained at different urea concentrations are then analyzed on the basis of peak intensities relative to the intensities in the absence of urea reflecting the extent of secondary structure formation. At urea concentrations somewhat below 6.5 M, specific hydrophobic residues in the C-terminal beta-sheet interact and two strands, the D and E strands in the N-terminal beta-sheet, are stabilized. These latter strands surround one of the turns showing residual structure. With decreasing urea concentrations, the remaining strands are stabilized in a specific order. The early strand stabilization appears to trigger the formation of the remainder of the C-terminal beta-sheet. At low urea concentrations, hydrogen bonds are formed. A pathway is proposed on the basis of the data describing the early, intermediate, and late folding steps for this almost all beta-sheet protein. The data also show that there are regions of the protein which appear to act in a concerted manner at intermediate steps in refolding.

PapD-like chaperones provide the missing information for folding of pilin proteins.

A fundamental question in molecular biology is how proteins fold into domains that can serve as assembly modules for building up large macromolecular structures. The biogenesis of pili on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding via small chaperones, secretion, and assembly. The results presented here support the hypothesis that pilus subunit folding and biogenesis proceed via mechanisms termed donor strand complementation and donor strand exchange. Here we show that the steric information necessary for pilus subunit folding is not contained in one polypeptide sequence. Rather, the missing information is transiently donated by a strand of a small chaperone to allow folding. Providing the missing information for folding, via a 13-amino acid peptide extension to the C-terminal end of a pilus subunit, resulted in the production of a protein that no longer required the chaperone to fold. This mechanism of small periplasmic chaperone function described here deviates from classical hsp60 chaperone-assisted folding.

Purification and polymerization properties of two lethal yeast actin mutants.

The budding yeast Saccharomyces cerevisiae contains a single actin gene and the gene product, actin, is essential for growth. Two mutants of yeast actin that do not support yeast growth were prepared from yeast by coexpressing the mutant and a 6-histidine-tagged wild-type actin followed by separation of the wild-type and mutant actin using Ni-NTA chromatography as described elsewhere [Buzan, J., Du, J., Karpova, T., and Frieden, C. (1999) Proc. Natl. Acad. Sci. USA 96, 2823-2827]. The mutations, in muscle actin numbering, were at positions 334 (Glu334Lys) and 168 (Gly168Arg) and were chosen based on phenotypic changes observed in the behavior of actin mutants of Caenorhabditis elegans. Glu334 is located on the surface of actin between subdomains 1 and 3 while Gly168 is located in a region near actin-actin contacts in the actin filament. The Glu334Lys mutant polymerized slightly faster than wild-type yeast actin, suggesting that loss of interactions with some actin binding protein, rather than loss of actin-actin contacts, was responsible for its inability to support yeast growth. The Gly168Arg mutant polymerized at a rate similar to wild-type but the extent was considerably less, kinetic characteristics suggesting a high critical concentration (ca. 4 microM) without a large change in the ability to form nuclei for the nucleation-elongation process.

Cooperative effects of potassium, magnesium, and magnesium-ADP on the release of Escherichia coli dihydrofolate reductase from the chaperonin GroEL.

Previous investigation has shown that at 22 degrees C and in the presence of the chaperonin GroEL, the slowest step in the refolding of Escherichia coli dihydrofolate reductase (EcDHFR) reflects release of a late folding intermediate from the cavity of GroEL (Clark AC, Frieden C, 1997, J Mol Biol 268:512-525). In this paper, we investigate the effects of potassium, magnesium, and MgADP on the release of the EcDHFR late folding intermediate from GroEL. The data demonstrate that GroEL consists of at least two conformational states, with apparent rate constants for EcDHFR release that differ by four- to fivefold. In the absence of potassium, magnesium, and ADP, approximately 80-90% of GroEL resides in the form with the faster rate of release. Magnesium and potassium both shift the distribution of GroEL forms toward the form with the slower release rate, though cooperativity for the magnesium-induced transition is observed only in the presence of potassium. MgADP at low concentrations (0-50 microM) shifts the distribution of GroEL forms toward the form with the faster release rate, and this effect is also potassium dependent. Nearly identical results were obtained with a GroEL mutant that forms only a single ring, demonstrating that these effects occur within a single toroid of GroEL. In the presence of saturating magnesium, potassium, and MgADP, the apparent rate constant for the release of EcDHFR from wild-type GroEL at 22 degrees C reaches a limiting value of 0.014 s(-1). For the single ring mutant of GroEL, the rate of EcDHFR release under the same conditions reaches a limiting value of 0.024 s(-1), suggesting that inter-ring negative cooperativity exists for MgADP-induced substrate release. The data suggest that MgADP preferentially binds to one conformation of GroEL, that with the faster apparent rate constant for EcDHFR release, and induces a conformational change leading to more rapid release of substrate protein.

Histidine-tagged wild-type yeast actin: its properties and use in an approach for obtaining yeast actin mutants.

Wild-type and an N-terminal 6-histidine-tagged actin have each been expressed by using a yeast strain that contains the actin gene on a plasmid and not on the chromosome. Yeast strains have also been constructed that use two plasmids, one expressing the wild-type protein and the other the 6-histidine-tagged protein. Yeast cells can be grown with either plasmid alone or with both plasmids together and appear to be normal in that the growth rates of all the yeast strains are quite similar, as is the morphology of the yeast cells. The polymerization properties of the 6-histidine-tagged actin appear almost identical to wild-type actin expressed from the chromosome. When the wild-type and 6-histidine-tagged actin are coexpressed, they can be purified by standard techniques and then separated using nickel-nitrilotriacetate chromatography. The method can be used to prepare actin mutants including those that are nonfunctional or might not support yeast growth for other reasons.

Native Escherichia coli and murine dihydrofolate reductases contain late-folding non-native structures.

We have examined the equilibrium and kinetic folding properties of two structurally homologous dihydrofolate reductases, Escherichia coli DHFR (EcDHFR) and murine DHFR (MuDHFR), as a function of temperature and ligand concentration. Conformational heterogeneity in native DHFR is well documented, and the results demonstrate that the non-native form(s) represents late intermediate(s) in the folding process. We have measured the concentrations of native and non-native forms and the rate constants for their interconversion over a temperature range of 3 degreesC to 49 degreesC, allowing characterization of the thermodynamic as well as the kinetic properties of the final folding step(s) relative to the overall folding reaction. Differences in ligand binding suggest that the intermediate structures for these two proteins may be different during refolding.

The chaperonin GroEL binds to late-folding non-native conformations present in native Escherichia coli and murine dihydrofolate reductases.

Dihydrofolate reductases from mouse (MuDHFR) or Escherichia coli (EcDHFR) are shown to refold via several intermediate forms, each of which can bind to the chaperonin GroEL. When stable complexes with GroEL are formed, they consist of late-folding intermediates. In addition, we find that late-folding intermediates that are present in the native enzyme bind to GroEL. For the E. coli and murine proteins, the extent of protein bound increases as the temperature is increased from 8 degreesC to 42 degreesC, at which temperature either protein is completely bound as the last (EcDHFR) or the last two (MuDHFR) folding intermediate(s). Thus for EcDHFR, the binding is transient at low temperature (<30 degreesC) and stable at high temperature (>35 degreesC). For MuDHFR, complex formation appears less temperature dependent. In general, the data demonstrate that the overall binding free energy for the interaction of GroEL with native DHFR is the sum of the free energy for the first step in DHFR unfolding, which is unfavorable, and the free energy of binding the non-native conformation, which is favorable. For EcDHFR, this results in an overall binding free energy that is unfavorable below 30 degreesC. Over the temperature range of 8 degreesC to 42 degreesC, GroEL binds MuDHFR more tightly than EcDHFR, due partially to a small free energy difference between two pre-existing non-native conformations of MuDHFR, resulting in binding to more than one folding intermediate.

The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes.

Fatty acid binding proteins (FABPs) exhibit a beta-barrel topology, comprising 10 antiparallel beta-sheets capped by two short alpha-helical segments. Previous studies suggested that fatty acid transfer from several FABPs occurs during interaction between the protein and the acceptor membrane, and that the helical domain of the FABPs plays an important role in this process. In this study, we employed a helix-less variant of intestinal FABP (IFABP-HL) and examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from this protein to model membranes in comparison to the wild type (wIFABP). In marked contrast to wIFABP, IFABP-HL does not show significant modification of the AOFA transfer rate as a function of either the concentration or the composition of the acceptor membranes. These results suggest that the transfer of fatty acids from IFABP-HL occurs by an aqueous diffusion-mediated process, i.e., in the absence of the helical domain, effective collisional transfer of fatty acids to membranes does not occur. Binding of wIFABP and IFABP-HL to membranes was directly analyzed by using a cytochrome c competition assay, and it was shown that IFABP-HL was 80% less efficient in preventing cytochrome c from binding to membranes than the native IFABP. Collectively, these results indicate that the alpha-helical region of IFABP is involved in membrane interactions and thus plays a critical role in the collisional mechanism of fatty acid transfer from IFABP to phospholipid membranes.

Kinetic studies on the effect of yeast cofilin on yeast actin polymerization.

The effect of yeast cofilin on the kinetics of polymerization of yeast actin has been examined at 20 degrees C at both pH 8.0 and 6.6. In the absence of cofilin, the kinetic data may be described by a simple nucleation-elongation mechanism. Kinetic data in the presence of cofilin suggests a complex dependence on the cofilin concentration. At low cofilin-to-actin ratios, cofilin increases the rate of polymerization in a way best fit by assuming filament fragmentation. The apparent fragmentation rate constants increase with increasing cofilin concentration leveling off above a cofilin-to-actin ratio of 1:8 and are independent of pH. At higher cofilin-to-actin ratios, a nonpolymerizable cofilin-G-actin complex forms resulting in a decreased rate of polymerization. The data from fluorescence photobleaching recovery experiments at low cofilin-to-actin ratios are consistent with the presence of severed filaments at both pH 8 and 6.6. However, at pH 8 and a cofilin-to-actin ratio of 1:16, about 40-50% of the total actin is present as G-actin after polymerization while at pH 6.6 little or no G-actin is present at the same cofilin-to-actin ratio. The results suggest some cooperativity with respect to cofilin binding to filamentous actin which may be pH dependent.

The three-dimensional structure of a helix-less variant of intestinal fatty acid-binding protein.

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic 15.1-kDa protein that appears to function in the intracellular transport and metabolic trafficking of fatty acids. It binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two five-stranded antiparallel beta-sheets and a helix-turn-helix domain. To investigate the role of the helical domain, we engineered a variant of I-FABP by deleting 17 contiguous residues and inserting a Ser-Gly linker (Kim K et al., 1996, Biochemistry 35:7553-7558). This variant, termed delta17-SG, was remarkably stable, exhibited a high beta-sheet content and was able to bind fatty acids with some features characteristic of the wild-type protein. In the present study, we determined the structure of the delta17-SG/palmitate complex at atomic resolution using triple-resonance 3D NMR methods. Sequence-specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 25 degrees C and used to define the consensus 1H/13C chemical shift-derived secondary structure. Subsequently, an iterative protocol was used to identify 2,544 NOE-derived interproton distance restraints and to calculate its tertiary structure using a unique distance geometry/simulated annealing algorithm. In spite of the sizable deletion, the delta17-SG structure exhibits a backbone conformation that is nearly superimposable with the beta-sheet domain of the wild-type protein. The selective deletion of the alpha-helical domain creates a very large opening that connects the interior ligand-binding cavity with exterior solvent. Unlike wild-type I-FABP, fatty acid dissociation from delta17-SG is structurally and kinetically unimpeded, and a protein conformational transition is not required. The delta17-SG variant of I-FABP is the only wild-type or engineered member of the intracellular lipid-binding protein family whose structure lacks alpha-helices. Thus, delta17-SG I-FABP constitutes a unique model system for investigating the role of the helical domain in ligand-protein recognition, protein stability and folding, lipid transfer mechanisms, and cellular function.

Refolding of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate reductase in the presence of ligand: a stopped-flow NMR spectroscopy study.

Escherichia coli dihydrofolate reductase contains five tryptophan residues that are spatially distributed throughout the protein and located in different secondary structural elements. When these tryptophans are replaced with [6-19F]tryptophan, distinct native and unfolded resonances can be resolved in the 1-D 19F NMR spectra. Using site-directed mutagenesis, these resonances have been assigned to individual tryptophans [Hoeltzli, S. D., and Frieden, C. (1994) Biochemistry 33, 5502-5509], allowing both the native and unfolded environments of each tryptophan to be monitored during the refolding process. We have previously used these assignments and stopped-flow NMR to investigate the behavior of specific regions of the protein during refolding of apo dihydrofolate reductase from urea in real time. These studies now have been extended to investigate the real time behavior of specific regions of the protein during refolding of dihydrofolate reductase in the presence of either NADP+ or dihydrofolate. As observed for the apoprotein, in the presence of either ligand, unfolded resonance intensities present at the first observed time point (1.5 s) disappear in two phases similar to those monitored by either stopped-flow fluorescence or circular dichroism spectroscopy. The existence of unfolded resonances which disappear slowly indicates that an equilibrium exists between the unfolded side chain environment and one or more intermediates, and that formation of at least one intermediate is cooperative. The results of this study are consistent with previous fluorescence studies demonstrating that dihydrofolate binds at an earlier step in the folding process than does NADP+ [Frieden, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4413-4416] and provide a structural interpretation of the previous results. In the apoprotein as well as in the presence of either ligand, the protein folds via at least one cooperatively formed, solvent-protected intermediate which contains secondary structure. In the presence of NADP+, a stable native-like side chain environment forms in the regions around tryptophans 30, 133, and 47 in an intermediate which cannot bind NADP+ tightly. Native side chain environment forms in the regions around tryptophans 22 and 74 only in the structure which is able to bind NADP+ tightly. In the presence of dihydrofolate, stable native-like side chain environment forms cooperatively in the regions around each tryptophan in a non-native intermediate which must undergo a conformational change prior to binding NADP+. The presence of ligands influences the processes which occur during the folding of dihydrofolate reductase, and the ligand may in effect serve as part of the hydrophobic core.

Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein.

We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.

Protein folding: how the mechanism of GroEL action is defined by kinetics.

We propose a mechanism for the role of the bacterial chaperonin GroEL in folding proteins. The principal assumptions of the mechanism are (i) that many unfolded proteins bind to GroEL because GroEL preferentially binds small unstructured regions of the substrate protein, (ii) that substrate protein within the cavity of GroEL folds by the same kinetic mechanism and rate processes as in bulk solution, (iii) that stable or transient complexes with GroEL during the folding process are defined by a kinetic partitioning between formation and dissociation of the complex and the rate of folding and unfolding of the protein, and (iv) that dissociation from the complex in early stages of folding may lead to aggregation but dissociation at a late stage leads to correct folding. The experimental conditions for refolding may play a role in defining the function of GroEL in the folding pathway. We propose that the role of GroES and MgATP, either binding or hydrolysis, is to regulate the association and dissociation processes rather than affecting the rate of folding.

GroEL-mediated folding of structurally homologous dihydrofolate reductases.

Using stopped-flow fluorescence techniques, we have examined both the refolding and unfolding reactions of four structurally homologous dihydrofolate reductases (murine DHFR, wild-type E. coli DHFR, and two E. coli DHFR mutants) in the presence and absence of the molecular chaperonin GroEL. We show that GroEL binds the unfolded conformation of each DHFR with second order rate constants greater than 3 x 10(7) M(-1)s(-1) at 22 degrees C. Once bound to GroEL, the proteins refold with rate constants similar to those for folding in the absence of GroEL. The overall rate of formation of native enzyme is decreased by the stability of the complex between GroEL and the last folding intermediate. For wild-type E. coli DHFR, complex formation is transient while for the others, a stable complex is formed. The stable complexes are the same regardless of whether they are formed from the unfolded or folded DHFR. When complex formation is initiated from the native conformation, GroEL binds to a pre-existing non-native conformation, presumably a late folding intermediate, rather than to the native state, thus shifting the conformational equilibrium toward the non-native species by mass action. The model presented here for the interaction of these four proteins with GroEL quantitatively describes the difference between the formation of a transient complex and a stable complex as defined by the rate constants for release and rebinding to GroEL relative to the rate constant for the last folding step. Due to this kinetic partitioning, three different mechanisms can be proposed for the formation of stable complexes between GroEL and either murine DHFR or the two E. coli DHFR mutants. These data show that productive folding of GroEL-bound proteins can occur in the absence of nucleotides or the co-chaperonin GroES and suggest that transient complex formation may be the functional role of GroEL under normal conditions.

Intestinal fatty acid binding protein: a specific residue in one turn appears to stabilize the native structure and be responsible for slow refolding.

The intestinal fatty acid binding protein is one of a class of proteins that are primarily beta-sheet and contain a large interior cavity into which ligands bind. A highly conserved region of the protein exists between two adjacent antiparallel strands (denoted as D and E in the structure) that are not within hydrogen bonding distance. A series of single, double, and triple mutations have been constructed in the turn between these two strands. In the wild-type protein, this region has the sequence Leu 64/Gly 65/Val 66. Replacing Leu 64 with either Ala or Gly decreases the stability and the midpoint of the denaturation curve somewhat, whereas mutations at Gly 65 affect the stability slightly, but the protein folds at a rate similar to wild-type and binds oleate. Val 66 appears not to play an important role in maintaining stability. All double or triple mutations that include mutation of Leu 64 result in a large and almost identical loss of stability from the wild-type. As an example of the triple mutants, we investigated the properties of the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. As measured by the change in intrinsic fluorescence, this mutant (and similar triple mutants lacking leucine at position 64) folds much more rapidly than wild-type. The mutant, and others that lack Leu 64, have far-UV CD spectra similar to wild-type, but a different near-UV CD spectrum. The folded form of the protein binds oleate, although less tightly than wild-type. Hydrogen/deuterium exchange studies using electrospray mass spectrometry indicate many more rapidly exchangeable amide protons in the Leu 64 Ser/Gly 65 Ala/Val 66 Asn mutant. We propose that there is a loss of defined structure in the region of the protein near the turn defined by the D and E strands and that the interaction of Leu 64 with other hydrophobic residues located nearby may be responsible for (1) the slow step in the refolding process and (2) the final stabilization of the structure. We suggest the possibility that this region of the protein may be involved in both an early and late step in refolding.

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].