©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tryptophan-containing -Subunits of the Escherichia coli Tryptophan Synthase
ENZYMATIC AND UREA STABILITY PROPERTIES (*)

(Received for publication, March 2, 1995; and in revised form, April 7, 1995)

Shin-Geon Choi (§) Stephen E. O'Donnell (1) Krishna D. Sarken John K. Hardman (¶)

From the Cell and Developmental Section, Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama 35487 and the Department of Biology and Chemistry, University of Montevallo, Montevallo, Alabama 35115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Early studies suggested that the Escherichia coli tryptophan synthase -subunit unfolded in a two-step process in which there was a stable intermediate composed of a native -1 folding unit (residues 1-188) and a completely unfolded -2 folding unit (residues 189-268). More recent evidence has indicated that such a structure for the intermediate seems unlikely. In this report, single Trp residues (absent in the wild-type -subunit) are substituted separately for Phe residues at positions 139 (in -1) and 258 (in -2) to produce the F139W, F258W, and F139W/F258W mutant -subunits. The UV absorbance and fluorescence properties of the F139W/F258W double mutant are identical with those of equimolar mixtures of the single mutants, suggesting that the Trp residue at each position can independently report the behavior of its respective folding unit. Each mutant -subunit is wild-type enzymatically, and when UV absorbance is monitored, the urea-induced unfolding of the three tryptophan-containing -subunits is virtually identical to the wild-type protein. These wild-type properties make these proteins attractive candidates for a fluorescence examination of the behavior of the individual folding units and the structure of potential intermediate(s) and as host proteins for the insertion of our existing destabilizing and/or stabilizing mutational alterations.


INTRODUCTION

The -subunit of the Escherichia coli and/or Salmonella typhimurium tryptophan synthase has long been used as a model to explore the stability and unfolding and folding properties of proteins and polypeptides. The -subunit consists of a single polypeptide chain of 268 amino acid residues and contains no prosthetic groups. Structurally(1) , it is a member of the / barrel family, belonging to a subgroup that includes phosphoribosylanthranilate isomerase, indoleglycerolphosphate synthase, and triosephosphate isomerase (2) and is composed of 11 -helices and 8 -strands (). Studies on the unfolding properties of the E. coli protein by urea and/or guanidine were initiated more than a decade ago by the Yutani(3, 4) , Matthews(5) , and Miles (6, 7) groups. The unfolding behavior indicated the presence of a partially unfolded, stable intermediate during the unfolding process. This conclusion was supported by the finding that there was a hypersensitive trypsin site at Arg(6) , subsequently determined to be located within a region (residues 178-191) that is highly mobile in the crystal structure(1) . Cleavage at this site yields two fragments, an N-terminal fragment (-1) composed of -strands 1-6 of the barrel structure and a C-terminal fragment (-2) containing the remaining barrel -strands 7 and 8. In guanidine, each of these can unfold and refold independently, and when combined, the -2 folding unit unfolds prior to -1. These results led initially to the notion that a stable unfolding intermediate existed containing a native -1 unit and an essentially completely unfolded -2 unit. In a subsequent extensive analysis by Matthews' groups(8, 9, 10, 11, 12) , a refolding pathway was elaborated, which was generally consistent with such a structural intermediate. Although the existence of separate folding units appears valid, more recent studies by the Matthews (13, 14, 15) and Yutani groups (16, 17) have substantially modified this picture of the structure of the unfolding intermediate.

The Matthews group has employed several singly and doubly altered -subunits in examining temperature effects on urea denaturation by UV and proton NMR methods. It was concluded that the -2 unit can assume an organized structure in the intermediate state. The total structure is proposed to be a loosened form of the barrel structure in which -strand 6 (in -1) and -strand 7 (in -2) have altered packing, although they continue to interact. Not inconsistent with these results, the Yutani group, utilizing calorimetry, CD, and tyrosine fluorescence, has described the equilibrium intermediate structure as a molten globule form.

To utilize our collection of 70-80 trpA mutants(18, 19) , which exhibit a broad spectrum of thermal instability, we have constructed several wild-type-like host proteins into which we could insert these mutational changes. The goal in preparing such wild-type-like host -subunits was to be able to monitor separately the behavior of the putative individual -1 and -2 folding units while still utilizing the intact protein. To this end, we have substituted a single tryptophan residue within the -1 folding region (at position 139) in one such host protein and one tryptophan residue within the -2 folding region (at position 258) in another. The normal -subunit contains no tryptophan. We report here the suitability (i.e. the spectral properties and wild-type behavior) of these tryptophan-containing -subunits to serve as host proteins and to provide additional information regarding both the unfolding and refolding pathways for each folding unit and the structure of potential intermediate(s).


MATERIALS AND METHODS

Chemicals and Enzymes

All chemicals (reagent or ultrapure grade) and enzymes for mutagenesis and sequencing were obtained from Sigma or New England Biolabs. Ultrapure urea was from Life Technologies, Inc. and was used without further purification. Urea concentrations were determined by refractive index measurements.

Mutagenesis

Oligonucleotides were synthesized on a Milligen Biosearch model 8700 DNA synthesizer and purified by reverse phase chromatography on Waters oligo-pak columns. For the Phe Trp change, the oligonucleotide 5`-GAGTCCGCGCCCTGGCGGCAGGCCGCG-3` was used. The sequence alterations from the wild-type sequence (TTCCGG) are underlined and represent those that encode Phe-Arg in the -subunit. There is a 3-base pair change, which results in the Phe Trp substitution only but introduces an additional BglI site into the trpA gene that can be used for the initial mutant selection. For the Phe Trp change, the oligonucleotide 5`-GGCGCGACTGAAAGTTTGGGTCCAACCGATGAAAGCGGCG-3` was used. The changes from the wild-type sequence (TTTGTA), again underlined, result in a Phe Trp substitution only, but in this case there is the loss of an RsaI site in trpA, and this is used for initial mutant selection. The sequence alterations were confirmed by DNA sequencing.

The mutagenesis/sequencing vector for Phe mutagenesis was similar to those described before (18, 20) except that a trpABsaHIXhoI sequence (encoding residues 124-177 in the -subunit) from ptactrpAMK (20) was inserted immediately 3`-distal to the GC clamp. The mutagenesis/sequencing vector for Phe mutagenesis was pGCtrpAVI as described before(20) . The procedures for oligonucleotide-directed mutagenesis and confirmation of the sequence change by restriction nuclease/DNA sequencing are described elsewhere (20) . The mutagenized fragments containing the altered sequences at positions 139 and 258 were subcloned as described before (20) into modified expression vectors of pCATtrpA and ptactrpAMK, respectively. These subclonings yielded expression vectors pCATtrpF139W and ptactrpFW258, which encode the F139W and F258W mutant -subunits. The double mutant, F139W/F258W, was constructed by substituting the trpABglII-XhoI fragment (encoding residues 1-177) from pCAT trpF139W for the corresponding wild-type fragment in ptactrpF258W to yield ptactrpF139W/F258W.

- and -Subunit Purifications

The -subunits (wild type, F139W, F258W, and F139W/F258W) were purified from E. coli strain RB797 containing, respectively, ptactrpAMK, pCATtrpF139W, ptactrpF258W, and ptactrpF139W/F258W. Growth conditions and -subunit purification protocols were identical for all and have been described before(20) . The -subunit was obtained also as previously detailed(20) .

Protein and Activity Assays

Wild-type -subunit concentrations were determined using the molar extinction coefficients at 278 nm of 12,700 cm. Concentrations of the tryptophan-containing -subunits were determined initially by the microbiuret assay (21) and by comparison with the microbiuret results with the wild-type protein; the molar extinction coefficients at 278 nm were estimated for the F139W, F258W, and F139W/F258W -subunits. The concentration of the -subunit was estimated with the E of 6.5(22) . The enzymatic activities of the -subunits in the tryptophan synthase - and -reactions were performed according to Lim et al.(23) ; the -reaction assays were according to Sarkar et al.(20) . Values of k for the wild-type -subunit (in the presence of the -subunit) in the -, -, and -reaction assays were 0.18 s, 11.9 s, and 6.21 s, respectively.

Spectrophotometric Measurements

All measurements were recorded at ambient temperatures, invariably 23-24 °C. The buffer in all experiments is 10 mM potassium phosphate (pH 7.8), 0.2 mM disodium EDTA, and 1 mM -mercaptoethanol. Corrections were made for buffer and denaturant. UV absorbance measurements for equilibrium studies were made with a Perkin-Elmer dual-beam 3A spectrophotometer. A Perkin-Elmer luminescence/fluorescence spectrometer model LS50B was used for the fluorescence spectral analysis. The experimental data for the unfolding experiments presented here represent the average of three to six experiments. The standard deviation for each measurement is ±5-10% or less. The data analysis program is a standard non-linear regression utilizing the Levenberg-Marquardt method as described in Press et al.(24) .


RESULTS AND DISCUSSION

Enzymatic and Spectroscopic Properties of the Tryptophan-containing -Subunits

The goal in selecting residue sites within the -subunit at which to insert tryptophan residues to produce suitable wild-type host proteins was that the substitution would not itself lead to major changes in function and/or stability to denaturants. The selection of Phe (the second residue in helix 4) in the -1 folding unit and Phe (a middle residue in helix 9) in the -2 folding unit was based on a variety of considerations such as lack of conservation at the site, relative residue size, hydrophobicity, secondary structure-forming tendencies, and solvent accessibility. Despite such considerations, it was difficult to predict accurately the effect of tryptophan substitutions at these sites. It was important, therefore, at the outset to characterize some of their enzymatic spectroscopic and urea stability properties.

The tryptophan-containing -subunits were examined for their enzymatic activity in the three tryptophan synthase reactions: the -, -, and -reactions. The -reaction measures the functional integrity of the -active site with respect to the catalysis of the aldolysis of indole-3-glycerol phosphate to indole and D-glyceraldehyde-3-phosphate. The -reaction measures the structural capacity of an -subunit to (a) allow access of indole from the solvent to the -active site; (b) provide the proper alignment of the intramolecular indole channel between the subunits; and (c) bring about proper conformational changes in the -subunit to activate its catalysis of indole plus L-serine to L-tryptophan. The -reaction measures the ability of an -subunit to (a) catalyze an accelerated rate of aldolysis of indole-3-glycerol phosphate at the -active site; (b) transfer the enzymatically formed indole intramolecularly to the -active site; and (c) undergo conformational changes induced by the -subunit (following binding of L-serine to the -active site) that lead to the increased rate of indole-3-glycerol phosphate utilization. -Subunits F139W, F258W, and F139W/F258W had relative specific activities, respectively, of 96, 89, and 105% that of the wild-type -subunit in the -reaction; 98, 92, and 106% that of the wild-type -subunit in the -reaction; and 102, 104, and 100% that of the wild-type -subunit in the -reaction.

All have expected increases in UV absorbance. Mutant -subunits F139W, F258W, and F139W/F258W have estimated molar extinction coefficients at 278 nm of 16,700 cm, 16,690 cm, and 20,800 cm, respectively, compared to that of 12,700 cm of the wild-type -subunit. These increases in molar extinction values (above that of the wild-type -subunit) of 4000 cm, 3990 cm, and 8100 cm, respectively, are consistent with the introduction of either one or two tryptophan residues. A molar extinction value of 4400 cm for L-tryptophan is obtained under similar conditions. The fluorescence behavior of the tryptophan-containing mutants is shown in Fig. 1. All exhibit an emission shift to lower wavelengths and fluorescence intensity increases of varying magnitude (relative to free tryptophan), suggesting that the tryptophan residues are buried in less polar environments within the respective proteins. The spectrum and relative fluorescence intensities of a equimolar mixture of F139W and F258W are virtually identical to the double mutant.


Figure 1: Fluorescence spectra, emission , and the relative fluorescence intensities for L-tryptophan and the F139W, F258W, and F139W/F258W -subunits (solid lines) and a mixture of F139W plus F258W -subunits (dashed lines). All concentrations were 5 µM, the excitation wavelength was 295 nm, and the scan rate was 120 nm/min with slit widths of 5 nm.



Urea Stabilities of the Tryptophan-containing -Subunits

For comparative purposes, the urea stability behavior of the wild-type and mutant -subunits was monitored by UV absorbance changes according to the methods of Matthews and co-workers (5, 25) . Fig. 2shows the absorbance changes upon urea addition to the wild-type and mutant -subunits; the differences reflect the differences in their respective extinction coefficients. Applying corrections for these differences and assuming that the absorbance changes at 6 M urea represent essentially complete unfolding(5, 25) , the fractional unfolding versus urea concentrations is given in Fig. 3, A-D, where f = ( - )/( - ), is the observed extinction coefficient, and and are the extinction coefficients of the native and unfolded forms. All commence unfolding at 2.2-2.5 M urea and are essentially completely unfolded at 5-5.5 M urea. -Subunit F139W appears virtually identical to the wild-type -subunit; -subunits F258W and F139W/F258W appear to be only slightly more stable. In addition, the unfolding transitions for all appear to be completely reversible (Fig. 3, A-D, opencircles).


Figure 2: Urea-induced unfolding of the wild-type (), F139W (), F258W (), and F139W/F258W () -subunits. The protein concentrations were 35 µM.




Figure 3: The fractional unfolding, F, versus urea concentration for wild-type (A), F139W (B), F258W (C), and F139W/F258W (D) -subunits (solid circles). The solidline is that for a two-step, three-state unfolding transition. The opencircles represent the calculated F observed upon dilution from 6 M urea to the indicated urea concentration. PanelsA`-D` present the fractions of native (f), intermediate (f), and unfolded (f) forms during unfolding of wild-type (A`), F139W (B`), F258W (C`), and F139W/F258W (D`) -subunits.



A quantitative comparison of these equilibrium data was made by statistically modeling each data set for a two-step, three-state unfolding model, following the classical treatment for multistep unfolding processes in which there are populated intermediates:

where K is the apparent equilibrium constant, K is the product of the K values for the individual steps, and Z is the fractional change in the measured parameter for the product of each step. For the case of a three-state, two-step model as suggested for the -subunit, namely

this relationship can be reformulated as:

where K and K are the equilibrium constants for the N I, I U transitions, respectively, and Z = ( - )/( - ) where is the extinction coefficient of the intermediate form. The results of these statistical analyses for the wild-type and tryptophan-containing -subunits are shown by the solidlines on Fig. 3, A-D. For all the -subunits (wild-type, F139W, F258W, and F139W/F258W), the fit to a two-step model appears to fit the data well. Assuming a linear dependence of free energy on urea concentration, the free energy of unfolding in the absence of denaturant, G2, was estimated for each step according to Pace(26) :

and

where m, m (the slopes) are the cooperativity factors for each plot, and C is the urea concentration. The G2, m, and Z values for the four -subunits are given in Table 1. The values for the wild-type -subunit compare favorably to those obtained by Beasty et al.(25) and are shown in parentheses. Mutant -subunit differences from the wild-type (at zero urea concentrations) in G2 and G2 vary between -0.6 and -1.4 kcal/mol.



As pointed out by Beasty et al.(25) and Cupo and Pace(27) , because of potential errors due to the large extrapolation required to determine G2 and the legitimacy of a linear relationship in this extrapolated range, a more appropriate comparison of stability would be at a urea concentration other than zero. A suitable reference point for comparison would be the urea concentration at the midpoints of the two transitions (N I, I U) for the reference wild-type -subunit, at which G and G are zero. Utilizing the Z values obtained, the relative proportions of native (f), intermediate (f), and unfolded (f) species for wild-type and tryptophan-containing mutants can be determined; these are shown in Fig. 3, A`-D`, together with the urea concentration (C), at which the intermediate is maximal. Only relatively minor differences from the wild-type -subunit are apparent. The midpoints, C and C for each transition for each protein are given also in Table 1. Values of G (Table 1) obtained for the mutant proteins at 2.51 and 3.73 M (the midpoints of the N I and I U transitions for wild-type -subunit) vary between -0.26 and 0.33 Kcal/mol. Thus, it appears that the replacement of a phenylalanine by a tryptophan residue at either or both the 139- or 258-residue sites does not appear to significantly alter the stability to urea.

The apparent wild-type enzymatic and urea stability (as monitored by UV absorbance) properties of these tryptophan-containing -subunits would seem to make them ideal host proteins for the introduction of our collection of potentially stabilizing/destabilizing mutational alterations. Moreover, a comparison of both the UV and fluorescence properties of the native forms of F139W and F258W and that of the double mutant, F139W/F258W, indicates that the tryptophan residue in each of these regions of the protein is displayed independently spectroscopically. Therefore, the tryptophan residues substituted at positions 139 and 258 can function as separate reporter groups for monitoring the behavior of the putative -1 and -2 folding units in the intact -subunit. The equilibrium and kinetic unfolding and refolding properties of these mutant -subunits monitored by fluorescence changes, currently underway, should provide interesting information regarding the intermediate(s) in these pathways and also serve as the wild-type reference behavior for a study of the effects of our other -subunit mutants.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
This work is in partial fulfillment of the Ph.D. requirements for the Dept. of Biological Sciences, University of Alabama.

To whom correspondence should be addressed.


REFERENCES

  1. Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988)J. Biol. Chem. 263, 17857-17871 [Abstract/Free Full Text]
  2. Faber, G. K., and Petsko, G. A.(1980)Trends Biochem. Sci. 15, 228-234
  3. Yutani, K., Ogasahara, K., Suzuki, M., and Sugino, Y.(1979)J. Biochem. (Tokyo) 85,915-921 [Abstract]
  4. Iwahashi, H., Yutani, K., Ogasahara, K., Tsunasawa, S., Kyogoko, Y., and Sugino, Y.(1983) Biochim. Biophys. Acta 744, 189-192
  5. Matthews, C. R., and Crisanti, M. M.(1981)Biochemistry 20, 784-792 [Medline] [Order article via Infotrieve]
  6. Miles, E. W., and Higgins, W.(1978)J. Biol. Chem. 253, 6266-6269 [Abstract]
  7. Miles, E. W., Yutani, K., and Ogasahara, K.(1982)Biochemistry 21, 2586-2592 [Medline] [Order article via Infotrieve]
  8. Crisanti, M. M., and Matthews, C. R.(1981)Biochemistry 20, 2700-2706 [Medline] [Order article via Infotrieve]
  9. Beasty, A. M., and Matthews, C. R.(1985)Biochemistry 24, 3547-3553 [Medline] [Order article via Infotrieve]
  10. Hurle, M. R., and Matthews, C. R.(1987)Biochim. Biophys. Acta 913, 179-184 [Medline] [Order article via Infotrieve]
  11. Hurle, M. R., Micheloti, G. A., Crisanti, M. M., and Matthews, C. R.(1987) Proteins 2, 54-63 [Medline] [Order article via Infotrieve]
  12. Chrunyk, B. A., and Matthews, C. R.(1990)Biochemistry 29, 2149-2154 [Medline] [Order article via Infotrieve]
  13. Tsuji, T., Chrunyk, B. A., Chen, X., and Matthews, C. R.(1993)Biochemistry 32, 5566-5575 [Medline] [Order article via Infotrieve]
  14. Saab-Rincon, G., Froebe, C. L., and Matthews, C. R.(1993)Biochemistry 32, 13981-13990 [Medline] [Order article via Infotrieve]
  15. Chen, X., and Matthews, C. R.(1994)Biochemistry 33, 6356-6362 [Medline] [Order article via Infotrieve]
  16. Ogasahara, K., Matsushita, E., and Yutani, K.(1993)J. Mol. Biol. 234, 1197-1206 [CrossRef][Medline] [Order article via Infotrieve]
  17. Ogasahara, K., and Yutani, K.(1994)J. Mol. Biol. 236, 1227-1240 [Medline] [Order article via Infotrieve]
  18. Milton, D. L., Napier, M. L., Myers, R. M., and Hardman, J. K.(1986)J. Biol. Chem. 261, 16604-16615 [Abstract/Free Full Text]
  19. Lim, W. K., Brouillette, C., and Hardman, J. K.(1992)Arch. Biochem. Biophys. 292, 34-41 [Medline] [Order article via Infotrieve]
  20. Sarken, K. D., and Hardman, J. K.(1995)Proteins Struct. Funct. Genet. 21,130-139 [Medline] [Order article via Infotrieve]
  21. Leggett-Bailey, J. (1962) in Techniques in Protein Chemistry, p. 249, Elsevier Scientific Publishing Co., Inc., New York
  22. Faeder, E. J., and Hammes, G. G.(1970)Biochemistry 9, 4043-4049 [Medline] [Order article via Infotrieve]
  23. Lim, W. K., Sarkar, S. A., and Hardman, J. K.(1991)J. Biol. Chem. 266,20205-20212 [Abstract/Free Full Text]
  24. Press, W. H., Flannery, B. P., Teukolsky, S. A., and Vetterling, W. T. (1986) Numerical Recipes: The Art of Scientific Computing, Cambridge University Press, Cambridge
  25. Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T., Onuffet, J. J., and Matthews, C. R. (1986)Biochemistry 25, 2965-2974 [Medline] [Order article via Infotrieve]
  26. Pace, C. N.(1986) Methods Enzymol.131,266-280 [Medline] [Order article via Infotrieve]
  27. Cupo, F. J., and Pace, C. N.(1983)Biochemistry22,2654-2658 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.