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C R Matthews

Publications and source records attributed to C R Matthews.

At least 55 records · Page 3Linked to original sources
PubMed1993

Luminescence studies with trp repressor and its single-tryptophan mutants.

Time-resolved and steady-state fluorescence, low-temperature phosphorescence, and optically detected magnetic resonance (ODMR) measurements have been made to resolve the luminescence contributions of the two intrinsic tryptophan residues in the subunits of trp aporepressor from Escherichia coli. Assignments of spectral information have been confirmed by use of the single-tryptophan mutants W19F and W99F. Solute fluorescence quenching studies show that both Trp19 and Trp99 are exposed to acrylamide and iodide, with Trp99 being the more exposed. Time-resolved and steady-state fluorescence measurements show Trp19 to have a bluer emission, a longer mean fluorescence decay time, a higher quantum yield, and essentially no independent rotational motion with respect to the protein. Trp99 is found to have a redder emission, a shorter mean fluorescence decay time, a lower quantum yield, and a significant degree of rotational freedom. Phosphorescence studies show a clear resolution of 0-0 vibronic transitions for each type of residue, with maxima at 407 and 415 nm that are assigned to Trp19 and Trp99, respectively. ODMR measurements show the zero-field splitting parameters to be quite characteristically different for each tryptophan residue. The existence of resonance energy transfer from Trp19 to Trp99, in the wild-type protein, is indicated by three types of data: comparison of the long-lived decay time (attributed to Trp19) in the absence (W99F) and presence (wild type) of the acceptor Trp99, comparison of the fluorescence quantum yield of the wild-type and mutant proteins, and deviations from the expected phosphorescence intensities for Trp19 and Trp99 in the absence of energy transfer.

Apoproteins↗
PubMed1993

Mutagenic analysis of the interior packing of an alpha/beta barrel protein. Effects on the stabilities and rates of interconversion of the native and partially folded forms of the alpha subunit of tryptophan synthase.

A series of single and double amino acid replacements in four beta strands of the alpha subunit of tryptophan synthase from Salmonella typhimurium, and alpha/beta barrel protein, was made to study the interior packing of the barrel and to clarify its folding mechanism. The urea-induced unfolding of the alpha subunit is thought to involve a stable intermediate in which the amino folding unit (residues 1-188; helices 0-5, strands 1-6) remains folded while the carboxy folding unit (residues 189-268; helices 6-8, strands 7-8) becomes disordered [Beasty, A. M., & Matthews, C. R. (1985) Biochemistry 24, 3547; Miles, E. W., Yutani, K., & Ogasahara, K. (1982) Biochemistry 21, 2586]. Mutations in strands 1 (A18G and A18V), 6 (Y175Q), 7 (L209V), and 8 (G230A, G230V, and I232V) at the interface between these two folding units show that the effects on the stabilities of the native and intermediate conformations critically depend on the site of the replacement. Although all of these mutations decrease the stability of the native conformation, only the replacements in strand 6, Y175Q, and possibly strand 8, I232V, also perturb the intermediate. Comparisons of the effects of three pairs of double mutants with the effects of the constituent single mutants on stability show that strands 6 and 7 interact in both the intermediate and native conformations, while strands 1 and 8 interact only in the native conformation. Kinetic studies of unfolding indicate that the interactions which occur in the native conformation arise in the preceding transition state. These results demonstrate that the carboxy folding unit adopts an organized structure in the intermediate, contrary to our previous interpretation. The general implication is that the state of folding of one segment of a protein can depend on the presence of another, more stable element of structure.

Amino Acid Sequence↗
PubMed1993

Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding.

The refolding kinetics of Escherichia coli trp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroism and 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy. Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms). These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves. The transition curves were fit to a two-state model, and the resulting free energies of folding in the absence of denaturant were found to be similar (approximately 3.3 kcal/mol). Three subsequent slow refolding phases exhibited relaxation times and amplitudes similar to those previously observed for tryptophan fluorescence [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface. This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure.

Anilino Naphthalenesulfonates↗
PubMed1993

A reexamination of the folding mechanism of dihydrofolate reductase from Escherichia coli: verification and refinement of a four-channel model.

The mechanism of folding of dihydrofolate reductase from Escherichia coli was reinvestigated by studying the unfolding and refolding kinetics using absorbance and fluorescence spectroscopies. The original kinetic model proposed that folding involved a series of native, intermediate, and unfolded forms which interconverted through four independent channels linked by slow cis/trans isomerization reactions at Xaa-Pro peptide bonds [Touchette, N. A., Perry, K. M., & Matthews, C. R. (1986) Biochemistry 25, 5445]. Recently, alternative sequential models have been proposed [Frieden, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4413; Kuwajima et al. (1991) Biochemistry 30, 7693] which challenge the original proposal. Stopped-flow studies of the intrinsic tryptophan fluorescence demonstrated the presence of three (and tentatively four) kinetic phases in unfolding which correlated well with four phases previously observed in refolding experiments. By monitoring the binding of the inhibitor methotrexate during folding at varying relative concentrations of inhibitor to protein, it was found that the selective loss of the slow-folding phases at substoichiometric levels could only be explained by a four-channel folding model. Double-jump experiments (native-->unfolded-->native) showed that the four refolding channels are populated within 20 s at 15 degrees C and are not likely to be due to proline isomerization. Reverse double-jump experiments (unfolded-->native-->unfolded) demonstrated that interconversions between native conformers are more rapid than originally proposed. Interestingly, the majority of the protein folds through a channel to a native conformer that is minimally populated at equilibrium. This implies that although the folding of dihydrofolate reductase is ultimately under thermodynamic control, kinetic factors contribute to the transient populations of native species during folding.

Binding Sites↗
PubMed1993

Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants.

Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoproteins↗
PubMed1993

Tryptophan replacements in the trp aporepressor from Escherichia coli: probing the equilibrium and kinetic folding models.

Mutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction. The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wild-type aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes. Comparison of equilibrium fluorescence (Royer, C.A., Mann, C.J., & Matthews, C.R., 1993, Protein Sci. 2, 1844-1852) and circular dichroism transition curves induced by urea shows that replacement of either Trp 19 or Trp 99 results in noncoincident behavior. Unlike the wild-type protein (Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020), tertiary and/or quaternary structures are disrupted at lower denaturant concentration than is secondary structure. The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained. The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591-597) provides a simple explanation for this phenomenon.

Apoproteins↗
PubMed1993

Pathways of protein folding.

Advances in spectroscopy, protein engineering, and peptide synthesis have had a dramatic impact on the understanding of the structures and stabilities of transient folding intermediates. The data available from a variety of proteins point to the existence of three common stages of folding. 1. Initially, the unfolded protein collapses to a presumably more compact form containing substantial nonpolar surfaces and secondary structure. This species has little thermodynamic stability and encompasses an ensemble of conformations that are in dynamic equilibrium and may contain non-native elements of structure. This reaction occurs in less than 5 ms and, from a thermodynamic perspective, may be a noncooperative transition. The relatively high content of secondary structure implies that this manifold of states must be far smaller than the manifold for the unfolded protein. 2. The next phase involves the further development of secondary and the beginnings of specific tertiary structure throughout the protein as well as of measurable stability. Nativelike elements of structure appear, possibly in the form of subdomains that are yet to be properly docked. In many instances, the packing is not as tight as is ultimately found in the native conformation, suggesting that the side chains are in general more mobile. Some elements of surface structure, such as loops and the peripheries of sheets and helices, are not yet well defined. This stage, which may consist of more than a single kinetic step, occurs in the 5-1000 ms time range. The ensemble of conformations is much reduced from the first stage; however, it is far from a single, highly populated form. 3. The final stage in folding corresponds to the concerted formation of many noncovalent interactions throughout the protein. The solidlike interior packing is achieved; the final secondary structure forms and the surface structures settle into place. The breadth of these conformational changes reflects the global cooperativity characteristic of protein folding reactions. A pictorial representation of the kinetic and thermodynamic aspects of this process is shown in Figure 1. This folding scheme emphasizes the progressive development of structure and stability through an ever-slowing set of reactions. Because the product of each stage of folding, with the exception of the final step, is an ensemble of related but not identical species, it is an oversimplification to describe the process as a pathway. Perhaps it is better described as a series of transitions between manifolds of structures that are in dynamic equilibrium within any given set.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics↗
PubMed1992

Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.

The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.

Arginine↗
PubMed1992

Amino acid replacements can selectively affect the interaction energy of autonomous folding units in the alpha subunit of tryptophan synthase.

Amino acid replacements were made at the interface between two autonomous folding units in the alpha subunit of tryptophan synthase from Salmonella typhimurium to test their mutual interaction energy. The results of equilibrium studies of the urea-induced unfolding reaction of the wild-type and mutant proteins in which phenylalanine 22 is replaced by leucine, isoleucine, and valine can be understood in terms of a selective decrease in the interaction energy between the two folding units; the intrinsic stability of each folding unit is not significantly altered. Kinetic studies of the rate-limiting step in unfolding show that the interaction energy appears in the transition state preceding the native conformation. Comparisons of the individual effects of these nonpolar side chains show that both hydrophobic and steric effects play important roles in the interaction energy between the folding units. The implication of these results is that the high cooperativity observed in the folding of many globular proteins may be reduced by appropriate amino acid replacements.

Amino Acids↗
PubMed1992

Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains.

A single polypeptide chain containing two dihydrofolate reductase (DHFR) sequences from Escherichia coli was constructed to determine if a repeat sequence fusion protein could be expressed in an active form. The possibility that intersequence interactions could play a significant role for this enzyme is suggested by the results of Hall and Frieden (1989, Proc. Natl Acad. Sci. USA, 86, 3060-3064) who observed a substantial decrease in the yield of active enzyme when folded in the presence of a large C-terminal fragment. The fusion protein [DHFR(Cys152Glu)--Ile--DHFR (Met1Gln)] was efficiently expressed in E. coli cells and has an activity which is twice that of the wild-type enzyme in the standard assay. The Michaelis constants of the fusion protein for the substrate, dihydrofolate and the cofactor, NADPH, are essentially unchanged from those of the wild-type protein. The urea-induced in vitro unfolding reaction of the fusion protein at low concentrations was found to be fully reversible and follow a three state model, suggesting that the two domains unfold independently. At higher protein concentrations the unfolding transition broadened and shifted to a higher urea concentration. Size-exclusion chromatography results are consistent with the formation of aggregates at the higher protein concentration, even in the absence of denaturant.

Amino Acid Sequence↗
PubMed1991

Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy.

The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein. The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS)

Circular Dichroism↗
PubMed1990

Folding and stability of trp aporepressor from Escherichia coli.

Equilibrium and kinetic studies of the urea-induced unfolding of trp aporepressor from Escherichia coli were performed to probe the folding mechanism of this intertwined, dimeric protein. The equilibrium unfolding transitions at pH 7.6 and 25 degrees C monitored by difference absorbance, fluorescence, and circular dichroism spectroscopy are coincident within experimental error. All three transitions are well described by a two-state model involving the native dimer and the unfolded monomer; the free energy of folding in the absence of denaturant and under standard-state conditions is estimated to be 23.3 +/- 0.9 kcal/mol of dimer. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration in the manner expected from the law of mass action for the two-state model. We find no evidence for stable folding intermediates. Kinetic studies reveal that unfolding is governed by a single first-order reaction whose relaxation time decreases exponentially with increasing urea concentration and also decreases with increasing protein concentration in the transition zone. Refolding involves at least three phases that depend on both the protein concentration and the final urea concentration in a complex manner. The relaxation time of the slowest of these refolding phases is identical with that for the single phase in unfolding in the transition zone, consistent with the results expected for a reaction that is kinetically reversible. The two faster refolding phases are presumed to arise from slow isomerization reactions in the unfolded form and reflect parallel folding channels.

Apoproteins↗
PubMed1990

Role of diffusion in the folding of the alpha subunit of tryptophan synthase from Escherichia coli.

The rate-limiting step in the folding of the alpha subunit of tryptophan synthase has been proposed to be the association of two folding units. To probe the role of diffusion in this rate-limiting step, the urea-induced unfolding and refolding of the protein was examined in the presence of a number of viscosity-enhancing agents. The analysis was simplified by studying the effect of these agents on folding unit dissociation, the rate-limiting unfolding reaction, and the reverse of the rate-limiting step in refolding. In the presence of ethylene glycol, the relaxation times for unfolding to the same final conditions increased with increasing concentration of the cosolvent. When the effects of the cosolvent on protein stability were taken into account, the rates were found to show a unitary linear dependence on the viscosity of the solution. Similar results were obtained with glycerol and low concentrations of glucose, demonstrating that the effect is general and not specific to any viscogenic agent. These results clearly demonstrate that the rate-limiting folding unit association/dissociation reaction in the alpha subunit of tryptophan synthase involves a diffusional process.

Diffusion↗
PubMed1990

Multiple replacements at position 211 in the alpha subunit of tryptophan synthase as a probe of the folding unit association reaction.

Equilibrium and kinetic studies on the folding of a series of amino acid replacements at position 211 in the alpha subunit of tryptophan synthase from Escherichia coli were performed in order to determine the role of this position in the rate-limiting step in folding. Previous studies [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T., Onuffer, J. J., & Matthews, C. R. (1986) Biochemistry 25, 2965-2974] have shown that the rate-limiting step corresponds to the association/dissociation of the amino (residues 1-188) and carboxy (residues 189-268) folding units. In terms of the secondary structure, the amino folding unit consists of the first six strands and five alpha helices of this alpha/beta barrel protein. The carboxy folding unit comprises the remaining two strands and three alpha helices; position 211 is in strand 7. Replacement of the wild-type glycine at position 211 with serine, valine, and tryptophan at most alters the rate of dissociation of the folding units; association is not changed significantly. In contrast, glutamic acid and arginine dramatically decelerate and accelerate, respectively, both association and dissociation. The difference in effects is attributed to long-range electrostatic interactions for these charged side chains; steric effects and/or hydrogen bonding play lesser roles. When considered with previous data on replacements at other positions in the alpha subunit [Hurle, M. R., Tweedy, N. B., & Matthews, C. R. (1986) Biochemistry 25, 6356-6360], it is clear that beta strands 6 (in the amino folding unit) and 7 (in the carboxy folding unit and containing position 211) dock late in the folding process.

Amino Acids↗
PubMed1989

Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase.

To test the possibility that long-range interactions might influence the folding and stability of dihydrofolate reductase, a series of single and double mutations at positions 28 and 139 were constructed and their urea-induced unfolding reactions studied by absorbance and circular dichroism spectroscopy. The alpha carbons of the two side chains are separated by 15 A in the native conformation. The replacement of Leu 28 by Arg and of Glu 139 by Gln resulted in additive effects on both kinetic and equilibrium properties of the reversible unfolding transition; no evidence for interaction was obtained. In contrast, the Arg 28/Lys 139 double replacement changed the equilibrium folding model from two state to multistate and showed evidence for interaction in one of the two kinetic phases detected in both unfolding and refolding reactions. The results can be explained in terms of a long-range, repulsive electrostatic interaction between the cationic side chains at these two positions.

Amino Acids↗