Biomedical subjects

C Frieden

Publications and source records attributed to C Frieden.

At least 55 records · Page 3Linked to original sources

PubMed1992

Dynamic NMR spectral analysis and protein folding: identification of a highly populated folding intermediate of rat intestinal fatty acid-binding protein by 19F NMR.

The folding of intestinal fatty-acid binding protein has been monitored by 19F NMR after incorporation of 6-fluorotryptophan into the protein. The two resonances resulting from the two tryptophans of this protein showed different dependencies on denaturant concentration. One of the resonances was in slow chemical exchange between two resonance frequencies, native and completely unfolded. The changes for this resonance occurred over a denaturant concentration range identical to that monitored by circular dichroism or fluorescence during unfolding. The other resonance continued to show changes at concentrations of denaturant well above that needed to complete the unfolding transition as monitored by optical techniques. Site directed mutagenesis showed that tryptophan-82 was the residue responsible for the unexpected behavior. We conclude, based on complete line-shape analysis, that there are significant concentrations of one or more intermediates in equilibrium with the native and unfolded forms. The structure of the intermediate(s) is more similar to the completely unfolded form of the protein than to the native structure, since little if any secondary structure is present. Further, these structure(s) persist at high denaturant concentrations and may represent local initiating sites in the folding of this beta-sheet protein.

Amino Acid Sequence↗

PubMed1992

The rate of formation of transition-state analogues in the active site of adenosine deaminase is encounter-controlled: implications for the mechanism.

We have previously shown that purine riboside, when bound to adenosine deaminase, forms a complex in which C-6 of the purine is tetrahedral [Kurz, L. C., & Frieden, C. (1987) Biochemistry 26, 8450]. We now report the rates of formation of enzyme-inhibitor complexes of two types, those which do and those which do not form such tetrahedral intermediates. In both cases, the rates are encounter-controlled since the progress curves for formation of the complexes are well-described by a simple second-order approach to equilibrium and the rate constants show an inverse solvent viscosity dependence. Assuming that the formation of the intermediate-analogue complex is preceded by an initial ground-state analogue complex, the lifetime of that ground-state complex must be less than approximately 20 microseconds. All of the enzyme-inhibitor complexes studied share three characteristics: (1) the complexes generate large UV-difference spectra; (2) a substantial solvent isotope effect is found on the enzyme's affinity for the inhibitors; and (3) a new signal appears in the CD spectra of the complexes. Two of the nucleosides studied, 1-deazapurine riboside and 1-deaza-adenosine, form complexes which appear to mimic a ground-state rather than a reactive intermediate when bound to adenosine deaminase. We find that the values for the association rate constants for those inhibitors which form intermediate analogues are very similar to that for adenosine. The presence of a significant solvent isotope effect on the affinity of all inhibitors is attributable in part to a large transfer isotope effect on the free ligand and in part to an effect on the bound ligand. This complicates use of the solvent isotope effect in applications of the multiple isotope method for estimating intrinsic isotope effects and commitment factors.

Adenosine↗

PubMed1992

Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.

Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316).

Actins↗

PubMed1991

Effects of point mutations in a hinge region on the stability, folding, and enzymatic activity of Escherichia coli dihydrofolate reductase.

The role of a hinge region in the folding, stability, and activity of Escherichia coli dihydrofolate reductase was investigated with three site-directed mutants at valine-88, the central residue of the hinge. The three mutants, V88A and V88I and a valine-88 deletion, were created to perturb the packing of hydrophobic residues in the interior of a loose turn formed by residues 85-91. Deleting the valine-88 residue destabilized the protein by 2.93 +/- 0.6 kcal/mol as determined by equilibrium unfolding transitions in urea monitored by circular dichroism at 20 degrees C. Substitution of alanine for valine-88 stabilized the protein by -0.20 +/- 0.02 kcal/mol, and the isoleucine substitution was mildly destabilizing by 1.73 +/- 0.2 kcal/mol. Although there was no clear correlation between side-chain volume and stability, these results suggest that side-chain interactions in the interior of the turn influence the folding and stability of dihydrofolate reductase. The specific activity of the valine deletion mutant was approximately twice that of the wild-type protein while the specific activities of the V88A and V88I proteins were only slightly greater than the wild type. The full time courses of the reactions catalyzed by the mutants were almost identical with that for the wild type, indicating no major changes in the kinetic mechanism. Additionally, the rate constants associated with interconversion between various forms of the apoenzyme were identical for the mutant and wild-type enzymes. The rate constants for refolding transitions were examined by dilution of urea-inactivated protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1991

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.

Academic Medical Centers↗

PubMed1990

Folding of a predominantly beta-structure protein: rat intestinal fatty acid binding protein.

The equilibrium and kinetic properties of the unfolding-refolding transitions of Escherichia coli derived rat intestinal fatty acid binding protein have been examined using several different denaturants. This protein, which contains 2 tryptophans but no prolines or cysteines, has a predominantly beta-structure: its 10 antiparallel beta-strands are organized into 2 orthogonal sheets surrounding a large solvent-filled internal cavity. For urea and guanidine hydrochloride, the completely reversible transition was monitored by circular dichroism, absorbance, and fluorescence spectroscopy. Each of these data sets was best fit by a simple, two-state model involving only native and unfolded forms. However, linear extrapolation to determine the free energy of folding in the absence of denaturant resulted in different values for the free energy of folding depending upon which denaturant was used. When fluorescence was used to monitor the transition, the extrapolated free energy estimates for the two denaturants were markedly different: 10.03 +/- 0.24 kcal mol-1 for urea versus 5.22 +/- 0.33 kcal mol-1 for guanidine hydrochloride. The midpoints of these transitions were 5.51 and 1.36 M, respectively. The transition caused by either denaturant as monitored by circular dichroism and absorbance spectroscopy was virtually coincident with that monitored by fluorescence, further supporting the assignment of a two-state model for the equilibrium results. The addition of a 2-fold molar excess of ligand (oleate) increased the extrapolated estimates approximately 2.5 kcal mol-1 for both denaturants.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Effect of filamin and controlled linear shear on the microheterogeneity of F-actin/gelsolin gels.

We have previously established [Cortese and Frieden, J. Cell Biol. 107:1477-1487, 1988] that actin gels formed under shear are microheterogeneous. In this study, the effect of cross-linking (by chicken gizzard filamin), severing (by plasma gelsolin), and shear on actin microheterogeneity are investigated using fluorescence photobleaching recovery and video microscopy. We find that filamin and shear form microheterogeneous F-actin:gelsolin gels by different mechanisms. Bundling of actin:gelsolin filaments by filamin can be explained by an increase in the apparent length of the filaments due to interfilament binding, resulting in a decrease of the polymer number concentration at which filaments organize into anisotropic phases. Some intrafilament binding of filamin to actin filaments may also be present, and those filaments coated with filamin immobilize more slowly than actin under the same polymerization conditions. The length of F-actin/gelsolin filaments seems to be a major factor in controlling the extent of bundling relative to network formation. In contrast, the effect of shear on the microheterogeneity of actin:gelsolin filaments is consistent with our previous proposal that shear aligns actin filaments, allowing filament-filament interactions and phase formation to occur. Short filaments are unable to organize into branched actin networks, but they can create large aggregates under low shear. Longer actin filaments will exist as networks with variable levels of branching and are less sensitive to shear. The effect of the intensity of a shear field on the spatial distribution of actin may involve a progressively more random orientation of actin molecules and bundles. A regular pattern develops across the sample at low shear rates (0.04-1.39 s-1), and becomes very irregular at higher shear rates (greater than 10 s-1). We suggest here that actin-binding proteins and shear can control the transition between isotropic networks and anisotropic phases by their effect on apparent length and local filament concentration, and also that this transition can have substantial effects on the resistance of cells to mechanical stress.

Actin Cytoskeleton↗

PubMed1990

Refolding of Escherichia coli dihydrofolate reductase: sequential formation of substrate binding sites.

The kinetics of refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3) have been examined upon dilution of unfolded enzyme in 4.5 M urea to 1.29 M urea in 0.02 M phosphate buffer (pH 7.2) at 10 degrees C. Changes in the intrinsic protein fluorescence on refolding are characterized by four phases. Based on changes in the amplitudes of these phases, as a consequence of quenching of the intrinsic fluorescence by ligands, it is possible to determine the step at which a ligand binds during the refolding process. The results show that either NADP or NADPH binds to the last species formed in a sequence involving three intermediates between the unfolded and native states. Dihydrofolate, on the other hand, binds during the formation of the second observed intermediate. When refolding is performed in the presence of methotrexate, an analogue of dihydrofolate, and NADPH, NADPH binds, as determined from changes in NADPH fluorescence, to the third observed intermediate rather than the last (fourth) species formed. Measurements of the recovery of enzymatic activity during refolding suggest that dihydrofolate also induces NADPH binding prior to the final observed folding phase. These results define more closely the formation of structural domains during the folding of dihydrofolate reductase.

Binding Sites↗

PubMed1989

Analysis of progress curves by simulations generated by numerical integration.

A highly flexible computer program written in FORTRAN is presented which fits computer-generated simulations to experimental progress-curve data by an iterative non-linear weighted least-squares procedure. This fitting procedure allows kinetic rate constants to be determined from the experimental progress curves. Although the numerical integration of the rate equations by a previously described method [Barshop, Wrenn & Frieden (1983) Anal. Biochem. 130, 134-145] is used here to generate predicted curves, any routine capable of the integration of a set of differential equations can be used. The fitting program described is designed to be widely applicable, easy to learn and convenient to use. The use, behaviour and power of the program is explored by using simulated test data.

Computer Simulation↗

PubMed1989

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.

Kinetics↗

PubMed1989

Actin polymerization induces a shape change in actin-containing vesicles.

We have encapsulated actin filaments in the presence and absence of various actin-binding proteins into lipid vesicles. These vesicles are approximately the same size as animal cells and can be characterized by the same optical microscopic and mechanical techniques used to study cells. We demonstrate that the initially spherical vesicles can be forced into asymmetric, irregular shapes by polymerization of the actin that they contain. Deformation of the vesicles requires that the actin filaments be on average at least approximately 0.5 micron long as shown by the effects of gelsolin, an actin filament-nucleating protein. Filamin, a filament-crosslinking protein, caused the surfaces of the vesicles to have a smoother appearance. Heterogeneous distribution of actin filaments within the vesicles is caused by interfilament interactions and modulated by gelsolin and filamin. The vesicles provide a model system to study control of cell shape and cytoskeletal organization, membrane-cytoskeleton interactions, and cytomechanics.

Actins↗

PubMed1989

Protein fragments as probes in the study of protein folding mechanisms: differential effects of dihydrofolate reductase fragments on the refolding of the intact protein.

We describe an approach for investigating the protein folding process, using protein fragments as inhibitory probes of the refolding protein. The refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3), reversibly unfolded in 7 M urea, was monitored by the reappearance of enzyme activity after diluting the unfolded enzyme into low urea concentrations (less than or equal to 2 M) in the presence of substrates. Of eight protein fragments produced by limited proteolysis of the 159-residue enzyme, three isolated peptides--Ser-49/Glu-90, Ile-91/Glu-154, and Gln-102/Glu-154--were evaluated for their effects on the recovery of the refolding protein's enzymatic activity. By this criterion, 13 microM peptide Gln-102/Glu-154 inhibits the refolding of 0.015 microM enzyme by approximately 80%, while the related peptide, Ile-91/Glu-154, and peptide Ser-49/Glu-90 at the same concentration inhibit the recoverable activity of the refolding enzyme by less than or equal to 20%. None of these three peptides has any significant effect on the activity of the folded enzyme. Our results indicate that peptides may inhibit refolding differentially and that these effects may be extremely sensitive to fragment sequence and composition. We suggest that peptide specificity in the inhibition of protein folding may be exploited as a structural probe of protein folding mechanisms.

Electrophoresis, Polyacrylamide Gel↗

PubMed1988

pH-induced changes in G-actin conformation and metal affinity.

Metal-induced conformational changes in actin at 20 degrees C have been investigated as a function of pH using actin labeled at Cys-374 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. At pH 8, the addition of a high Ca2+ concentration (2 mM) to G-actin gives an instantaneous fluorescence increase while the addition of a high Mg2+ concentration gives both an instantaneous and a slow fluorescence increase. The instantaneous increase is interpreted as divalent cation binding to low-affinity, relatively nonspecific sites, while the slow response is attributed to Mg2+ binding to specific sites of moderate affinity [Zimmerle, C.T., Patane, K., & Frieden, C. (1987) Biochemistry 26, 6545-6552]. The magnitudes of both the instantaneous and slow fluorescence increases associated with Mg2+ addition to G-actin are shown here to decrease as the pH is lowered while the fluorescence of labeled G-actin in the presence of low or moderate Ca2+ concentrations (less than 200 microM) increases. The pH-dependent data suggest that protonation of a single class of residues with an approximate pK of 6.8 alters the immediate environment of the label differently depending upon the cation bound at the moderate-affinity site. The pH-dependent changes in the magnitude of the slow fluorescence response upon Mg2+ addition to Ca2+-actin are not associated with changes in the Mg2+ affinity at the moderate-affinity site but result from protonation altering the fluorescence response to Mg2+ binding. Protonation of this same class of residues is proposed to induce an actin conformation similar to that induced by cation binding at the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1988

Effect of pH on the mechanism of actin polymerization.

The effect of pH on the Mg2+-induced polymerization of rabbit skeletal muscle G-actin at 20 degrees C was examined. Polymerization data were obtained at various initial concentrations of Mg2+, Ca2+, and G-actin between pH 6 and 7.5. The data were found to fit a kinetic mechanism for actin polymerization previously proposed at pH 8 in which Mg2+ binding at a moderate-affinity site on actin induces an isomerization of the protein enabling more favorable nucleation [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886]. The data also suggest the formation of actin dimers induced by Mg2+ binding is over 2 orders of magnitude more favorable at pH 6 than at pH 8. Little effect on trimer formation is found over this pH range. In addition, the conformation induced by nonspecific binding of metal to low-affinity sites becomes more favorable as the pH is lowered. The critical concentration for filament formation is also decreased at lower pH. The kinetic data do not support fragmentation occurring under any of the conditions examined. Furthermore, as Mg2+ exchange for Ca2+ at a high-affinity site (Kd less than 10(-9) M) fails to alter significantly the polymerization kinetics, Ca2+ release from this site appears unnecessary for either the nucleation or the elongation of actin filaments.

Actins↗

PubMed1988

Mechanism for nucleotide exchange in monomeric actin.

Rabbit skeletal muscle G-actin has been treated to obtain ADP, 1,N6-ethenoadenosine diphosphate (epsilon-ADP), or 1,N6-ethenoadenosine triphosphate (epsilon-ATP) at the nucleotide binding site and either Mg2+ or Ca2+ at high- and moderate-affinity metal binding sites. Apparent rates or rate constants for the displacement of the actin-bound nucleotides by epsilon-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20 degrees C of the fluorescence difference between bound and free epsilon-ATP or epsilon-ADP. In the presence of Ca2+, displacement of ADP by epsilon-ATP or epsilon-ADP by ATP is a biphasic process, but in the presence of low (less than 10 microM) Mg2+ concentrations, it is a slow first-order process. At high levels of Mg2+ (greater than 50 microM), low ADP concentrations displace epsilon-ATP from G-actin as a consequence of Mg2+ binding to moderate-affinity sites on the actin. Displacement of epsilon-ATP by ATP in the presence of either Ca2+ or Mg2+ is slow at low ATP concentrations, but the rate is increased by high ATP concentrations. Using ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we find that nucleotide exchange is affected differently by the removal of Ca2+ from the high-affinity site compared to Ca2+ removal from moderate-affinity sites. A mechanism for the displacement reaction is proposed in which there are two forms of an actin-ADP complex and metal binding influences the ratio of these forms as well as the binding of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1988

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.

Animals↗

PubMed1988

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.

Actin Cytoskeleton↗

PubMed1988

Construction of a fol mutant strain of Escherichia coli for use in dihydrofolate reductase mutagenesis experiments.

A strain of Escherichia coli with the fol gene deleted and a kan gene inserted in its place was created for use in cloning and isolation of mutant dihydrofolate reductase. Southern blot analysis and dihydrofolate reductase enzyme assays confirmed the delta fol::kan genotype. A thyA mutation accompanied the fol deletion and is required for survival of a dihydrofolate reductase-deficient strain.

Chromosome Deletion↗