Microheterogeneity of actin gels formed under controlled linear shear.

The diffusion coefficients and fluorescence polarization properties of actin subjected to a known shear have been determined both during and after polymerization, using a modification of a cone-plate Wells-Brookfield rheometer that allows monitoring of samples with an epifluorescence microscope. Fluorescence polarization and fluorescence photobleaching recovery experiments using rhodamine-labeled actin as a tracer showed that under conditions of low shear (shear rates of 0.05 s-1), a spatial heterogeneity of polymerized actin was observed with respect to fluorescence intensity and the diffusion coefficients with actin mobility becoming quite variable in different regions of the sample. In addition, complex changes in fluorescence polarization were noted after stopping the shear. Actin filaments of controlled length were obtained using plasma gelsolin (gelsolin/actin molar ratios of 1:50 to 1:300). At ratios of 1:50, neither spatial heterogeneity nor changes in polarization were observed on subjecting the polymerized actin to shear. At ratios of approximately 1:100, a decrease on the intensity of fluorescence polarization occurs on stopping the shear. Longer filaments exhibit spatial micro-heterogeneity and complex changes in fluorescence polarization. In addition, at ratios of 1:100 or 1:300, the diffusion coefficient decreases as the total applied shear increased. This behavior is interpreted as bundling of filaments aligned under shear. We also find that the F-actin translational diffusion coefficients decrease as the total applied shear increases (shear rates between 0.05 and 12.66 s-1), as expected for a cumulative process. When chicken gizzard filamin was added to gelsolin-actin filaments (at filamin/actin molar ratios of 1:300 to 1:10), a similar decrease in the diffusion coefficients was observed for unsheared samples. Spatial microheterogeneity might be related to the effects of the shear field in the alignment of filaments, and the balance between a three-dimensional network and a microheterogeneous system (containing bundles or anisotropic phases) appears related to both shear and the presence of actin-binding proteins.

Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle.

Fluorescent antibody staining experiments with both isolated myofibrils and muscle fibers grown in culture show that AMP deaminase is bound to the myofibril in the A band. The strongest staining occurs at each end of the A band. The approximate width of the fluorescent stripes and their relation to the A band remains constant as a function of sarcomere length. Removal of enzyme from the myofibrils leads to loss of staining, and readdition of purified enzyme restores the original staining pattern. A histoenzymatic method for the detection of AMP deaminase activity in cultured fibers gives comparable localization. The results are consistent with the previous observation (Ashby, B. and C. Frieden. 1977.J. Biol. Chem. 252:1869--1872) that AMP deaminase forms a tight complex in solution with subfragment-2 (S-2) of myosin or with heavy meromyosin (HMM).

Localization and mobility of gelsolin in cells.

To investigate the physiologic role of gelsolin in cells, we have studied the location and mobility of gelsolin in a mouse fibroblast cell line (C3H). Gelsolin was localized by immunofluorescence of fixed and permeabilized cells and by fluorescent analog cytochemistry of living cells and cells that were fixed and/or permeabilized. Overall, the images show that in living cells gelsolin has a diffuse cytoplasmic distribution, but in fixed cells a minor fraction is associated with regions of the cell that are rich in actin filaments. The latter fraction is more prominent after permeabilization of the fixed cells because some diffuse gelsolin is not fixed and is therefore lost during permeabilization, confirmed by immunoblots. To determine quantitatively whether gelsolin is bound to actin filaments in living cells, we measured the mobility of microinjected fluorescent gelsolin by fluorescence photobleaching recovery. Gelsolin is fully mobile with a diffusion coefficient similar to that of control proteins. As a positive control, fluorescent phalloidin, which binds actin filaments, is totally immobile. These results are supported by immunoblots on cells permeabilized with detergent. All the endogenous gelsolin is extracted, and the half-time for the extraction is approximately 5 s, which is about the rate predicted for diffusion. Therefore, gelsolin is not tightly bound to actin filaments in cells. The most likely interpretation of the difference between living and fixed cells is that fixation traps a fraction of gelsolin that is associated with actin filaments in short-lived complexes.

Microinjection of gelsolin into living cells.

Gelsolins are actin-binding proteins that cap, nucleate, and sever actin filaments. Microinjection of cytoplasmic or plasma gelsolin into living fibroblasts and macrophages did not affect the shape, actin distribution, deformability, or ruffling activity of the cells. Gelsolin requires calcium for activity, but the NH2-terminal half is active without calcium. Microinjection of this proteolytic fragment had marked effects: the cells rounded up, stopped ruffling, became soft, and stress fibers disappeared. These changes are similar to those seen with cytochalasin, which also caps barbed ends of actin filaments. Attempts to raise the cytoplasmic calcium concentration and thereby activate the injected gelsolin were unsuccessful, but the increases in calcium concentration were minimal or transient and may not have been sufficient. Our interpretation of these results is that at the low calcium concentrations normally found in cells, gelsolin does not express the activities observed in vitro at higher calcium concentrations. We presume that gelsolin may be active at certain times or places if the calcium concentration is elevated to a sufficient level, but we cannot exclude the existence of another molecule that inhibits gelsolin. Microinjection of a 1:1 gelsolin/actin complex had no effect on the cells. This complex is stable in the absence of calcium and has capping activity but no severing and less nucleation activity as compared with either gelsolin in calcium or the NH2-terminal fragment. The NH2-terminal fragment-actin complex also has capping and nucleating activity but no severing activity. On microinjection it had the same effects as the fragment alone. The basis for the difference between the two complexes is unknown. The native molecular weight of rabbit plasma gelsolin is 82,500, and the extinction coefficient at 280 nm is 1.68 cm2/mg. A new simple procedure for purification of plasma gelsolin is described.

The regulation of protein polymerization.

Reversible polymerization reactions are essential for many cellular functions. This review briefly discusses some of the mechanisms for controlling the rate and extent of these processes.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Career choices of graduates from Washington University's Medical Scientist Training Program.

The Medical Scientist Training Program at Washington University has been in existence for 21 years; by 1990, 148 students had completed the program leading to the combined M.D./Ph.D. degree. Of these graduates, most (95%) chose to enter residency programs rather than postdoctoral fellowships. Of the 72 who had completed their residencies or postdoctoral training by 1990, the vast majority (89%) reported that they were returning to academic institutions or the National Institutes of Health. Those in academic institutions were primarily in clinical departments, with medicine being the first choice. Most said they were devoting much of their time to basic research. These graduates were progressing rapidly into tenured positions and appear to have been well funded.