Biomedical subjects

C R Matthews

Publications and source records attributed to C R Matthews.

At least 37 records · Page 2Linked to original sources

PubMed1998

Single-tryptophan mutants of monomeric tryptophan repressor: optical spectroscopy reveals nonnative structure in a model for an early folding intermediate.

A monomeric version of the dimeric tryptophan repressor from Escherichia coli, L39E TR, has previously been shown to resemble a transient intermediate that appears in the first few milliseconds of folding [Shao, X., Hensley, P., and Matthews, C. R. (1997) Biochemistry 36, 9941-9949]. In the present study, the optical properties of the two intrinsic tryptophans were used to compare the structure and dynamics of the monomeric form with those of the native, dimeric form. The urea-induced unfolding equilibria of Trp19/L39E TR (Trp99 replaced with Phe) and Trp99/L39E TR (Trp19 replaced with Phe) mutants were monitored by circular dichroism and fluorescence spectroscopies at pH 7.6 and 25 degrees C. Coincident normalized transitions show that the urea denaturation process for each single-tryptophan mutant follows a two-state model involving monomeric native and unfolded forms. The free energies at standard state in the absence of denaturant for Trp19/L39E TR and Trp99/L39E TR are less than that for L39E TR, indicating that both tryptophans are involved in stabilizing the monomer. Fluorescence and near-UV circular dichroism spectroscopies indicate that the tryptophan side chains in monomeric Trp19/L39E TR and Trp99/L39E TR occupy hydrophobic, well-structured environments that are distinctively different from those found in their dimeric counterparts. Acrylamide quenching experiments show that both Trp19 and Trp99 are partially exposed to solvent in the native state, with Trp99 having a slightly greater degree of exposure. Measurements of the steady-state anisotropies of Trp19/L39E and Trp99/L39E TR demonstrate that the motions of both tryptophan side chains are restricted in the folded conformation. On the basis of these data, it can be concluded that this monomeric form of the tryptophan repressor adopts a well-folded, stable conformation with nonnative tertiary structure. When combined with previous results, the current findings demonstrate that the development of higher order structure during the folding of this intertwined dimer does not follow a simple hierarchical model.

Bacterial Proteins↗

PubMed1997

Construction and characterization of monomeric tryptophan repressor: a model for an early intermediate in the folding of a dimeric protein.

Tryptophan repressor (TR) from Escherichia coli is a homodimer whose highly helical subunits intertwine in a complex fashion. A monomeric version of Trp repressor has been constructed by introducing a pair of polar amino acids at the hydrophobic dimer interface. Analytical ultracentrifugation was used to show that the replacement of leucine at position 39 with glutamic acid results in a monomer/dimer equilibrium whose dissociation constant is 1.11 x 10(-)4 M at 25 degrees C and pH 7.6. Tryptophan fluorescence, both near- and far-UV circular dichroism, and NMR spectroscopies demonstrated that, at the micromolar concentrations where the monomer predominates, secondary and tertiary structure are present. Hydrophobic dye-binding experiments showed that nonpolar surface is accessible in the monomeric form. The urea-induced equilibrium unfolding of monomeric L39E TR was monitored by circular dichroism, fluorescence, and absorbance spectroscopies. Coincident transitions show that the urea denaturation process follows a simple two-state model involving monomeric native and unfolded forms. The free energy at standard state in the absence of denaturant was estimated to be 2.37 +/- 0.15 kcal mol-1, and the sensitivity of the unfolding transition to denaturant, the m value, was 0.86 +/- 0.04 kcal mol-1 M(urea)-1 at pH 7.6 and 25 degrees C. The thermal denaturation transition occurred over a broad temperature range, suggesting either that the enthalpy change is small or that intermediates may exist. Kinetic studies showed that both the refolding and unfolding of the monomer were complete in the mixing dead time of stopped-flow CD and fluorescence spectroscopy, 5 ms. These structural, thermodynamic, and kinetic results are very similar to those previously reported for an early, monomeric intermediate in the folding of the wild-type TR dimer [Mann, C. J., & Matthews, C. R. (1993) Biochemistry 32, 5282-5290]. The construction of a stable, monomeric form of TR that strongly resembles a transient folding intermediate should provide useful insights into the nature of the early events in the folding of TR.

Bacterial Proteins↗

PubMed1997

Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor.

The urea-induced equilibrium unfolding of the Escherichia coli Trp repressor (TR) is a two-state process, involving the native dimeric and unfolded monomeric species. Kinetic studies, however, reveal the presence of transient intermediates that appear only during the folding of the 107-residue protein [Gittelman, M. G., & Matthews, C. R. (1990) Biochemistry 29, 7011-7020]. In order to gain insight into the complex kinetic folding mechanism, the sequence of TR was reduced to the amino-terminal 66 residues, corresponding to the dimerization domain. Two polypeptides, 2-66 and NHis-7-66, were shown to be dimeric at 25 degrees C by size exclusion chromatography and to retain native-like spectroscopic features as evidenced by near- and far-UV circular dichroism and fluorescence spectroscopy. The equilibrium properties of the urea-induced folding of these core fragments were examined by intrinsic tryptophan fluorescence and circular dichroism and found to be well described by a two-state model. At 25 degrees C, the stabilities of both fragments are 14 kcal mol(-1), as compared to the 24 kcal mol(-1) observed for full-length TR. In contrast, the thermal denaturation of [2-66]2 and full-length TR are three-state processes; the midpoint of the transition monitored by absorbance at 292 nm precedes that monitored by circular dichroism at 222 nm. Global analysis of the thermal data as a function of monomer concentration suggests that both the full-length and [2-66]2 TR variants unfold via a dimeric intermediate. Taken together, these results demonstrate that the [2-66]2 fragment constitutes a well-structured, independently folding subdomain of TR that may be useful in elucidating the properties of the transient intermediates observed in the folding of the full-length protein. The dimeric intermediate observed in the thermal denaturation of [2-66]2 suggests that it may be possible to further reduce the core sequence while maintaining the ability to dimerize.

Bacterial Proteins↗

PubMed1997

Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL.

The chaperonin GroEL binds nonnative proteins in its central channel through hydrophobic interactions and initiates productive folding in this space underneath bound co-chaperone, GroES, in the presence of ATP. The questions of where along the folding pathway a protein is recognized by GroEL, and how much structure is present in a bound substrate have remained subjects of discussion, with some experiments suggesting that bound forms are fully unfolded and others suggesting that bound species are partially structured. Here we have studied a substrate protein, human dihydrofolate reductase (DHFR), observing in stopped-flow fluorescence experiments that it can rapidly bind to GroEL at various stages of folding. We have also analyzed the structure of the GroEL-bound protein using hydrogen-deuterium exchange and NMR spectroscopy. The pattern and magnitude of amide proton protection indicate that the central parallel beta-sheet found in native DHFR is present in a moderately stable state in GroEL-bound DHFR. Considering that the strands are derived from distant parts of the primary structure, this suggests that a native-like global topology is also present. We conclude that significant native-like structure is present in protein-folding intermediates bound to GroEL.

Amides↗

PubMed1997

Probing minimal independent folding units in dihydrofolate reductase by molecular dissection.

Molecular dissection was employed to identify minimal independent folding units in dihydrofolate reductase (DHFR) from Escherichia coli. Eight overlapping fragments of DHFR, spanning the entire sequence and ranging in size from 36 to 123 amino acids, were constructed by chemical cleavage. These fragments were designed to examine the effect of tethering multiple elements of secondary structure on folding and to test if the secondary structural domains represent autonomous folding units. CD and fluorescence spectroscopy demonstrated that six fragments containing up to a total of seven alpha-helices or beta-strands and, in three cases, the adenine binding domain (residues 37-86), are largely disordered. A stoichiometric mixture of the two fragments comprising the large discontinuous domain, 1-36 and 87-159, also showed no evidence for folding beyond that observed for the isolated fragments. A fragment containing residues 1-107 appears to have secondary and tertiary structure; however, spontaneous self-association made it impossible to determine if this structure solely reflects the behavior of the monomeric form. In contrast, a monomeric fragment spanning residues 37-159 possesses significant secondary and tertiary structure. The urea-induced unfolding of fragment 37-159 in the presence of 0.5 M ammonium sulfate was found to be a well-defined, two-state process. The observation that fragment 37-159 can adopt a stable native fold with unique, aromatic side-chain packing is quite striking because residues 1-36 form an integral part of the structural core of the full-length protein.

Binding Sites↗

PubMed1996

Mutagenic and thermodynamic analyses of residual structure in the alpha subunit of tryptophan synthase.

The alpha subunit of tryptophan synthase from Escherichia coli has been previously shown to contain residual structure at 5 M urea, conditions where the secondary structure is entirely disrupted and the tyrosine residues are exposed to solvent [Saab-Rincón, G., Froebe, C. L., & Matthews, C. R. (1993) Biochemistry 32, 13981-13990]. The residual structure can be monitored by one-dimensional NMR spectroscopy studies of histidine 92 whose C epsilon proton is sensitive to the slow exchange between this form and the unfolded protein. The temperature dependence of the cooperative urea-induced unfolding transition between intermediate and unfolded forms demonstrates that this process involves negative values for both the enthalpy and entropy changes at 25 degrees C. The effects of replacements of several nonpolar side chains adjacent to histidine 92 on the slopes and midpoints of the unfolding transition curve show that these side chains participate in the residual structure. A 15-residue peptide spanning histidine 92 and the mutated residues, however, is not sufficient to define this structure. These results demonstrate that the residual structure in the alpha subunit is stabilized by the hydrophobic effect and may involve side chains which are distant in sequence to histidine 92.

Amino Acid Sequence↗

PubMed1995

Characterization of the slow folding reactions of trp aporepressor from Escherichia coli by mutational analysis of prolines and catalysis by a peptidyl-prolyl isomerase.

Escherichia coli trp aporepressor (TR) is a highly helical, dimeric protein whose folding has been shown to involve three phases whose relaxation times range from 200 ms to 50 s at 25 degrees C and pH 7.6 [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. All three phases are urea and protein concentration independent below 3 M urea, suggesting that cis/trans proline isomerization might limit the folding of TR under these conditions. This hypothesis was tested by measuring the sensitivity of the folding reaction to site-directed mutagenesis and to cyclophilin, a peptidyl-prolyl isomerase. Each of the four proline residues in TR was replaced singly as well as simultaneously, and the effects on the folding mechanism were assessed. All of these mutants, including the version lacking prolines (des-Pro TR), retain three slow, denaturant-independent folding phases similar to those observed for wild-type TR. However, the pattern of catalysis of the two slower folding phases in wild-type and mutant TRs by cyclophilin shows that cis/trans isomerization of the Thr44/Pro45 peptide bond can limit folding in proteins containing Pro45. The observation of three urea-independent, slow folding phases in des-Pro TR demonstrates that proline isomerization is not solely responsible for this complex folding behavior. Other types of isomerization or conformational rearrangement reactions appear to limit the folding of this dimeric protein under strongly folding conditions.

Amino Acid Isomerases↗

PubMed1995

Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy.

Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding. The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable biomolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidine-HCl induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mol dimer)-1 when fit to a two-state model involving the native dimer and the unfolded monomer. The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration. The unfolding kinetics displayed single-exponential behavior, consistent with a unimolecular reaction. The refolding kinetics, which are dependent on both peptide and guanidine concentration, are well described by a simple bimolecular association reaction. A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N2[symbol: see text]2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M-1 S-1 and 3.3 x 10(-3) S-1, respectively. The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model.

Amino Acid Sequence↗

PubMed1995

Characterization of the tryptophan binding site of Escherichia coli tryptophan holorepressor by phosphorescence and optical detection of magnetic resonance of a tryptophan-free mutant.

The L-tryptophan binding site of the Escherichia coli tryptophan holorepressor (trpR) is characterized by low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Measurements are made on a tryptophan-free mutant of trpR, W19/99F, in which both intrinsic tryptophan residues of apo-trpR have been replaced with phenylalanine. Thus, essentially all of the phosphorescence that is observed from trpR originates from the bound L-tryptophan corepressor. The phosphorescence and ODMR results for the bound corepressor agree quite well with those obtained previously for the corepressor site in both single tryptophan-containing mutants, W19F and W99F [Burns, L.E., & Maki, A.H. (1994) J. Fluorescence 4, 217-226]. A red shift of the L-tryptophan phosphorescence origin as well as a decrease in the D-E ODMR frequency result from an increase in the local polarizability upon binding at the corepressor binding site. A large decrease in the ODMR line widths signals a reduction of local heterogeneity upon binding. Subsequent binding of trpR to a self-complementary DNA sequence that mimics the trp operator, 5'-CGTACTAGTTAACTAGTACG-3', produces a further decrease in line widths and additional changes in the ODMR frequencies, attributable to an increase in both the D and E parameters. This result demonstrates that binding of holo-trpR to the operator affects the local environment of the bound corepressor.

Bacterial Proteins↗

PubMed1995

Detection of a stable intermediate in the thermal unfolding of a cysteine-free form of dihydrofolate reductase from Escherichia coli.

The reversible temperature-induced unfolding of a cysteine-free mutant (C85S/C152E, des-Cys) of dihydrofolate reductase from Escherichia coli has been studied by absorbance and by both far- and near-ultraviolet circular dichroism spectroscopies. The non-coincidence of all three transition curves demonstrated the existence of a highly populated partially-folded form near 39 degrees C at pH 7.8. This intermediate retains substantial secondary structure and partially excludes one or more of the five tryptophans from solvent; however, the intermediate has lost specific tertiary packing around its aromatic residues. Increases in enthalpy, entropy, and heat capacity are observed for both the native/intermediate and intermediate/unfolded transitions; the majority of the changes in these parameters occurs in the first transition. These results suggest that the thermal unfolding reaction of des-Cys dihydrofolate reductase involves a stable intermediate whose properties resemble those of a molten globule.

Circular Dichroism↗

PubMed1995

Local and global dynamics during the folding of Escherichia coli dihydrofolate reductase by time-resolved fluorescence spectroscopy.

Time-resolved fluorescence techniques were utilized to monitor the kinetic refolding reaction of Escherichia coli dihydrofolate reductase (DHFR). Measurements of emission and anisotropy decay lifetimes of both the five intrinsic tryptophan residues and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) during the folding reaction were used to characterize the compactness and development of tertiary structure in transient intermediates formed during the folding of DHFR. Experiments monitoring bound ANS show that a rapidly-formed intermediate (< 20 ms) has a rotational time of approximately 10 ns and, therefore, a compactness similar to that for the native conformation. All of the tryptophan residues in this burst phase species rotate as freely as in the unfolded state. In the set of four intermediates which then appear over the next few hundred milliseconds, the apparent rotational time measured by ANS fluorescence increases to a maximum rotational time of approximately 20 ns. An increase in the average tryptophan lifetime for these intermediates suggests these side chains become excluded from solvent and associated dynamic quenching mechanisms. As the folding reaction proceeds to a set of four native conformers the bound ANS rotational time then decreases to approach that for the native protein, 10.5 ns, and the average tryptophan rotational time increases to the same value. During these rate-limiting, final steps in folding, the static quenching effects which reflect the formation of specific tertiary contacts involving tryptophans also appear.

Adenine↗

PubMed1995

Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR.

The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates. The first forms within 5 ms and has substantial secondary structure but little stability. The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789). Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period. A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms). The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet. The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices. Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms. Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms. Only approximately half of the population of molecules form this set of stable hydrogen bonds. Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction.

Amino Acid Sequence↗

PubMed1995

A strategy for testing the suitability of cysteine replacements in dihydrofolate reductase from Escherichia coli.

Amino acid sequences in proteins can contain residues which complicate biochemical, biophysical, or protein engineering studies but which are not essential for folding or activity. Their replacement with other naturally-occurring amino acids which are not subject to such complications but which maintain essential properties of the protein is a desirable goal. A simple strategy for testing various mutants for their suitability is described for a pair of cysteine residues in dihydrofolate reductase (DHFR) from Escherichia coli. Using a reconstructed gene which preserves the amino acid sequence and introduces a variety of unique restriction sites, the cysteines at positions 85 and 152 were replaced by site-directed and cassette mutagenesis. The enzymatic activity, stability, and folding mechanism of six double mutant DHFR proteins were examined with the purpose of identifying a suitable alternative to wild type DHFR. The Cys85-->Ala and Cys152-->Ser double mutant DHFR was found to retain the four channel folding mechanism and have activity and stability which are comparable to the wild type enzyme. The replacement of the cysteines improved the resistance of DHFR to the irreversible loss of activity at high temperature.

Amino Acid Sequence↗

PubMed1994

Development of nonpolar surfaces in the folding of Escherichia coli dihydrofolate reductase detected by 1-anilinonaphthalene-8-sulfonate binding.

The development of nonpolar surfaces during the folding of Escherichia coli dihydrofolate reductase (DHFR) was studied by monitoring the time-dependent fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) included in the refolding solution. Stopped-flow refolding experiments demonstrated a rapid increase in fluorescence intensity within the dead time of mixing (5 ms), indicating that the earliest detectable folding intermediate contains hydrophobic surfaces which are capable of binding ANS. A further increase in fluorescence intensity over the next 300 ms coincides with the formation of a set of four intermediates which are known to contain a specific tertiary contact [Kuwajima, K., Garvey, E. P., Finn, B. E., Matthews, C. R., & Sugai, S. (1991) Biochemistry 30, 7693-7703]. Experiments performed in the presence of polar fluorescence quenching agents indicate that the binding sites for ANS in the burst phase species are more exposed to solvent than those in the subsequent set of intermediates. When considered along with the above study of the formation of secondary structure by stopped-flow circular dichroism, these results imply that DHFR initially forms a molten globule intermediate. Subdomains containing specific tertiary structure and more solvent-excluded ANS binding sites then form before ultimately being converted to native or native-like conformations during the rate-limiting steps in the folding of DHFR. The occurrence of similar kinetic phases observed by ANS binding during the folding of a number of other proteins suggests that this may be a common scheme for protein folding reactions.

Acrylamide↗

PubMed1994

Thermodynamic properties of the transition state for the rate-limiting step in the folding of the alpha subunit of tryptophan synthase.

To gain insight into the physical properties of the transition state for the rate-limiting step in the folding of the alpha subunit of tryptophan synthase from Escherichia coli, the urea dependence of the unfolding reaction was examined as a function of temperature. Consistent with a previous, more limited study [Hurle, M.R., Michelotti, G.A., Crisanti, M.M., & Matthews, C.R. (1987) Proteins 2, 54], the activation entropy for unfolding was found to be negative above 4 M urea. The present study extends this finding to show that both the activation entropy and enthalpy decrease with increasing urea concentrations between 4 and 7.5 M. The change in the heat capacity from the native to the transition state is positive and appears to increase with the denaturant concentration. The urea and temperature dependences of the unfolding rates were analyzed in terms of the denaturant-binding model of Tanford [Tanford, C. (1970) Adv. Protein Chem. 24, 1]. The values for the activation enthalpy and activation entropy of binding are in good agreement with those obtained from a calorimetric study of urea binding to unfolded proteins [Makhatadze, G.I., & Privalov, P.L. (1992) J. Mol. Biol. 226, 491]. These results show that (1) the binding of urea to the transition state of the alpha subunit has thermodynamic properties which are similar to those for urea binding to unfolded proteins, (2) the transition state is distinct from the unfolded conformation and exposes only a fraction of its urea-binding sites to solvent, and (3) the negative value for the activation entropy for unfolding reflects, in part, the ordering of urea on newly exposed surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1994

1H, 15N and 13C resonance assignments, secondary structure, and the conformation of substrate in the binary folate complex of Escherichia coli dihydrofolate reductase.

By using fully 15N- and 15N/13C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific 1H and 15N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the 13C alpha resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected 15N and 13C experiments. A smaller but significant subset of side-chain 1H and 13C assignments were also determined. In this complex, only one 15N or 13C resonance was detected per 15N or 13C protein nucleus, which indicated a single conformation. Proton-detected 13C experiments were also performed with unlabeled DHFR, complexed with 13C-7/13C-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on 1H NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667-9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.

Amino Acid Sequence↗

PubMed1993

Urea-induced unfolding of the alpha subunit of tryptophan synthase: one-dimensional proton NMR evidence for residual structure near histidine-92 at high denaturant concentration.

The urea-induced unfolding reaction of the alpha subunit of tryptophan synthase was monitored by examining the chemical shifts and peak areas of the C epsilon protons of the four histidine residues with 1D NMR spectroscopy. In a native base-line region defined by tyrosine absorbance and far-UV circular dichroism spectroscopy, histidine-146 appears to undergo a rapid, local unfolding reaction at increasing denaturant concentrations. As the native form is converted to a previously detected stable intermediate between 2 and 3 M urea [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784], histidines-92 and -146 in the amino folding unit (residues 1-188) and histidines-195 and -244 in the carboxy folding unit (residues 189-268) all experience a change in their environments which is slow on the NMR time scale. The subsequent conversion of this intermediate to a newly detected, stable, partially folded form populated at 5 M urea appears to have no effect on any of the histidines at 25 degrees C when an intermolecular association process involving His-244 is taken into account. Strikingly, a slow exchange process involving only His-92 is observed to begin at 5 M urea where the unfolding transitions monitored by absorbance or far-UV circular dichroism spectroscopy are essentially complete. This residual tertiary structure unfolds in a cooperative fashion as the urea concentration is increased to 8 M.

Amino Acid Sequence↗

PubMed1993

Collapse of parallel folding channels in dihydrofolate reductase from Escherichia coli by site-directed mutagenesis.

The rate-limiting steps in the folding of dihydrofolate reductase from Escherichia coli have been shown to involve the conversion of a set of four intermediates to a corresponding set of native conformers via four parallel channels [Jennings et al. (1993) Biochemistry 32, 3783-3789]. Fluorescence and absorbance studies of the unfolding and refolding of the C85S/C152E double mutant at various final urea concentrations reveal two slow folding reactions, two fewer than observed in the wild-type protein. Refolding in the presence of substoichiometric levels of the inhibitor methotrexate shows that the two remaining slow reactions correspond to two parallel channels which lead to a pair of native conformers capable of binding the inhibitor. A combination of stopped-flow circular dichroism and cofactor binding studies confirms that the four parallel channels observed in the wild-type protein have collapsed into two channels in the mutant. Kinetic and equilibrium studies of the single cysteine mutants suggest that replacements of Cysteine-85 which perturb the hydrophobic core containing this side chain are responsible for the simplification of the kinetic mechanism. These results demonstrate that at least two of the parallel folding channels in dihydrofolate reductase arise when tertiary structure develops and are not dependent upon cis/trans isomerization at prolyl peptide bonds.

Circular Dichroism↗