In Vitro Mutagenesis and Overexpression of the Escherichia coli trpA Gene and the Partial Characterization of the Resultant Tryptophan Synthase Mutant a-Subunits”

A mutagenesis approach was initiated in order to examine further the folding behavior of the a-subunit of the Escherichia coli tryptophan synthase. A random single base pair saturation mutagenesis procedure (Myers, R. M., Lerman, L. S., and Maniatis, T. Science 229, 242-247) was applied in vitro to sub- cloned fragments of the trpA gene, which codes for this polypeptide. Mutagenesis plasmid vectors were con- structed containing three fragments of the trpA gene which together code for about half of the total amino acid residues of the a-subunit. The vectors were constructed such that each strand of each trpA fragment could be altered. These trpA fragments were mutagen-ked in vitro (using nitrous acid, formic acid, hydra- zine, and potassium permanganate), and several thousands of mutants have been isolated. Thirty-two mu- tants, contained within the first two trpA fragments (which encompass the first 206 base pairs of the trpA gene and encode the first 63 residues of the a-subunit) have been sequenced. Of these, 20 (63%) contained single base pair alterations, 12 (37%) contained multiple alterations, and 17 (53%) of these base pair alter- ations resulted in single amino acid substitutions. Selected necessary for the transformation steps, the bacteriophage M13 origin of replication necessary for single-stranded DNA production, and a GC-rich region (the GC clamp) which was critical for mutant trpA fragment isolation and which also served as a priming site for DNA sequence analysis. In addition, the availability of the pGC plasmids, which contain polylinkers adjacent to the GC clamp, allows each fragment to be inserted for mutagenesis in the opposite orientations with respect to the GC clamp. During the construction of the mutagenesis vectors, emphasis was placed on maintaining or creating unique restriction sites for the mobilization of each trpA fragment into and out of the mutagenesis vectors during the mutagenesis protocol and for the replacement of the wild-type trpA fragment with the mutant trpA fragment in the overexpression vector, ptactrpA, while still maintaining a proper reading frame, Details of these constructions are given in the Mini- print section.

** To whom correspondence should be addressed. may be particularly critical for the normal folding of the polypeptide.
Early studies (1) suggesting that the three-dimensional structure of a protein is determined by its amino acid sequence have been borne out by extensive studies on the unfolding and refolding behavior of a variety of proteins. These studies have been exhaustively reviewed over the years (see Refs. 2-4 for reviews). While it is clear that proteins may tolerate a great number of amino acid substitutions without significantly affecting the native conformation and function of the protein, it is also evident (5-11) that a more limited number of residue types may be allowable at particularly critical sites in yielding a normal stable tertiary structure.
In principle, the isolation of a large number of mutations in a gene should aid in the identification of critical residues or sequences that determine the rate of folding or the stability of the final product encoded by that gene, and an increasing number of investigators are addressing the protein folding problem in this way (12). Such studies on several proteins appear to have progressed to a point where particular critical residues can be identified. A random mutagenic approach with the staphylococcal nuclease gene has been used to identify specific nonactive site residues that altered activity. Presumably, such alterations (potentially, folding mutations) led to abnormal conformations with decreased stability and activity of the enzyme (6). Temperature-sensitive mutations in the phage P22 tail spike endorhamnosidase have been useful in obtaining early intermediates in the folding of the protrimer form of this enzyme (7-10). More recently, two temperaturesensitive mutant forms of /3-lactamase (11) have been shown to be blocked near the end of the proposed folding pathway for this enzyme.
Several missense mutations of the Escherichia coli trpA gene, which codes for the a-subunit of tryptophan synthase, have been employed in studies on the stability and folding properties of this polypeptide (13)(14)(15)(16)(17)(18)(19)(20)(21). This gene-enzyme system is particularly useful for applying such a mutagenesis approach. First of all, the trpA gene can be readily cloned and easily manipulated. Second, the enzyme consists of a single polypeptide chain of molecular weight, 28,700, containing no prosthetic groups or disulfide bonds, and its folding properties have been described in some detail by Matthews and coworkers (13,15,16). It is composed of two domains, an Nterminal domain consisting of residues 1-188 and a smaller C-terminal domain which includes residues 189-268 (17). Studies of refolding after guanidine hydrochloride (18,19) and urea (13,15) treatments suggest that one or more stable intermediates appear during the folding process. The principal that the N-terminal domain is more stable than the C-terminal domain when the two fragments were unfolded separately with guanidine hydrochloride; hydrogen exchange studies by Beasty and Matthews (16) confirmed the independent folding of these two domains and indicated that the Nterminal domain folds to a large extent as a single entity prior to the folding of the C-terminal domain.
In this study a random single base pair saturation chemical mutagenesis scheme designed by Myers et al. (22) was applied to subcloned DNA fragments of the trpA gene. This method introduces all types of single-base substitutions into the subcloned fragments and utilizes a selection procedure based only on single base pair differences from the wild-type trpA fragments. Subsequently, the mutant trpA fragments can be substituted for the wild-type fragment in the trpA gene, contained in an overexpression vector. This approach was desirable for several reasons. First of all, the mutant selection step makes no presumptions regarding a folding mutant phenotype which would be difficult to predict uniquely. All types of altered proteins, containing both acceptable and unacceptable amino acid changes, become available for study instead of only those that lead, for example, to a n enzymatically inactive protein.
Second, since the crystal structure of this protein has not yet been solved, it is difficult to predict, with any precision, those particular residues or sequences of residues that might be expected to be uniquely critical to the stability of the folded product or to the kinetics of folding. In this respect, both the randomness of the alterations and the potential variability of amino acid substitutions at any residue site are particularly useful. Third, the capability of replacing the wild-type trpA fragments with mutated trpA fragments in the intact trpA gene that is contained within an overexpression vector allows us to obtain substantial amounts of the altered proteins with relative ease. Last, this method has the added advantage of being relatively rapid in generating a very large number of different mutations.
This reports describes ( a ) the details of the mutagenesis procedure as applied to the trpA gene; ( b ) the construction of a n overexpression vector containing the trpA gene, the kinetics of wild-type a-subunit production and its purification from cells containing this vector; and ( c ) the expression and preliminary characterization of the resulting mutant a-subunits. Preliminary reports of this work have been presented (23,24).

EXPERIMENTAL PROCEDURES'
The details concerning all the materials, strains, plasmids, and methods employed are given in the Miniprint section. An outline of the plasmid constructions and the mutagenesis procedure is given below.
Constructwn of ptactrpA, the Overexpression Vector Containing the trpA Gene-The trpA gene was isolated as a 2246-bp2 fragment from q580pt190 DNA which contains the entire trp operon. Also located on this fragment were the trp operon terminator genes, trpt and trpt' (25,26), and 1103 bp at the 3'-end of the trpB gene. The details of * The abbreviations used are: bp, base pair; SDS, sodium dodecyl sulfate.
this initial subcloning to produce pSP6trpA are shown in Fig. 1. In order to overproduce the a-subunit, it was necessary to insert a strong promoter 5"distaI to the trpA gene. The tac promoter (27,28) was utilized for this purpose. In addition, in order to make this vector useful in the mutagenesis experiments, it was desirable to eliminate most of the trpB gene that was subcloned into pSP6trpA. Accordingly, pSP6trpA was cleaved at the single HpaI site located 72 bp 5"distal from the trpA initiation codon and treated with Ba131 to remove much of the residual portion of the trpB gene. Sequence analysis indicated that the trpB DNA was trimmed to within 5 bp of the trpA initiation codon. The tac promotor derived from ptac12H3 was then inserted 5"distal to the trpA gene; a BglII linker was inserted between the tuc promoter and the trpA gene to facilitate the removal of the trpA gene from the plasmid. The steps in this construction are given in Fig. 1.
The Mutagenesis Procedure- Fig. 2 presents the general scheme of the procedure. The experimental details for each step are given in the Miniprint section. The essential features include chemical treatment, selection of mutant trpA fragments, and subcloning of mutant fragments into the overexpression vector.
Chemical mutagens were chosen (22) that cause damage to the bases but do not cause strand scission. Moreover, it was desirable to choose chemical mutagens that yield a variety of base changes to increase the randomness and sites of the mutations. The chemical mutagens used and their potential base changes are as follows: nitrous acid, C to T and A to G; formic acid, A and G to all bases; hydrazine, C and T to all bases; and potassium permanganate, T to all bases. Each chemical mutagenesis was done separately in vitro on singlestranded forms of the mutagenesis vectors containing the trpA fragments. Conditions of each chemical treatment were determined so that 10-20% of the trpA fragments contained in the mutagenesis vector will contain a single lesion and, in addition, were designed

In Vitro
Mutagenesis of the E. coli trpA Gene such that approximately 80-90% of the mutagenized fragments would contain single base changes. Subsequently, the mutational alterations were fixed by converting the single-stranded trpA fragments into double-stranded form using the adjacent GC-rich region (the GC clamp) as the priming site and subcloning them into unmutated backbone.
Since only 10% of the transformants obtained contains mutant trpA fragments, it was necessary to apply a selection step. This selection utilizes a urea-formamide gradient (DNA-melting) polyacrylamide gel system and recovers trpA fragments containing bp differences from the wild-type fragments. This procedure requires the presence of the GC clamp (a 300-bp GC-rich sequence), which remains helical over the entire range of formamide denaturant used in the gradient, and permits the recovery of all sequence-altered trpA fragments of variable melting properties (29). These altered trpA fragments are reinserted into the same double-stranded backbone as above, individual transformants are screened for trpA fragments melting differences, and, after conversion of the vectors to singlestranded form, the fragments are sequenced by the dideoxy method using the adjacent GC clamp again as the priming site.
Selected mutant trpA fragments are then substituted for the corresponding wild-type fragments in the overexpression vector.
Construction of the Mutagenesis Vectors Containing Fragments of the trpA Gene-Three fragments of the trpA gene were inserted separately between sites B and C during the construction of the mutagenesis vectors (for the first step: Fig. 2). Plasmids ptactrpA or a derivative of it, ptactrpAXb, served as a source of the trpA fragments.
Four plasmids (pMHP8, pMHP9, pGC1, and pGC2) were utilized as the plasmid backbones into which the three trpA fragments were subcloned for mutagenesis, mutant trpA fragment isolation, and DNA sequence analysis of mutant trpA fragments. Each of these plasmids contained a pBR322 origin of replication which was necessary for the transformation steps, the bacteriophage M13 origin of replication necessary for single-stranded DNA production, and a GC-rich region (the GC clamp) which was critical for mutant trpA fragment isolation and which also served as a priming site for DNA sequence analysis. In addition, the availability of the pGC plasmids, which contain polylinkers adjacent to the GC clamp, allows each fragment to be inserted for mutagenesis in the opposite orientations with respect to the GC clamp.
During the construction of the mutagenesis vectors, emphasis was placed on maintaining or creating unique restriction sites for the mobilization of each trpA fragment into and out of the mutagenesis vectors during the mutagenesis protocol and for the replacement of the wild-type trpA fragment with the mutant trpA fragment in the overexpression vector, ptactrpA, while still maintaining a proper reading frame, Details of these constructions are given in the Miniprint section.

RESULTS AND DISCUSSION
Three fragments of the trpA gene have been subjected to the mutagenesis procedure as outlined in Fig. 2. Fragment I of the trpA gene consists of the first 109 bp of the gene and encodes the first 36 residues of the &-subunit; fragment 11 contains the next 97 bp and codes for residues 36-69; and fragment 111 contains the next 161 bp and codes for residues 69-121. Thus, with mutagenesis vectors containing these fragments, 372 of the total 804-bp trpA sequence can be mutagenized, and the first 121 amino acid residues of the a-subunit may be altered. This portion of the polypeptide chain constitutes approximately 65% of the N-terminal domain. Although a systematic treatment of the entire gene is planned, our initial efforts are directed toward the examination of this region of the polypeptide chain because of its importance in the initial step(s) of the proposed folding pathway.
Six mutagenesis vectors have been constructed which contain the three trpA fragments each in the forward and reverse orientation with respect to the GC damp. The forward orientation corresponds to the 5' > 3' orientation within the trpA gene. Thus, when these vectors are converted to singlestranded forms for the first step in mutagenesis, each strand of each fragment can be mutagenized. Such constructions permit a more random and complete mutagenesis of each fragment. Of these, three (pMHtrpATR, pMHtrpAIIF, and pGCtrpAIIIR) have been mutagenized. Thus, the noncoding strands of trpA fragments I and 111 and the coding strand of trpA fragment I1 served as the substrates for mutagenesis and as the template strands for sequence analysis. Fig. 3 and Table I  and sequence analyses of mutant trpA fragments I and 11. Included are the results of all (32) trpA fragments that have been sequenced so far. Although too few mutants have been examined to warrant a rigorous statistical analysis, an examination of the results suggests that the method does appear to produce a random distribution of alterations. Of the 52 base pair sites that have been altered, 9 represent multiple hits at a single base pair site. The cluster of altered sites between base pair positions 170 and 180 probably reflects the fact that most of these are nitrous acid mutants and this Crich region is the most likely target for this mutagen. For the most part, the mutagen specificity is similar to that obtained with the @-globin promoter ( 2 2 ) . As expected, nitrous acid deamination resulted in C to T and A to G transitions with the C to T transitions predominating. Formic acid depurination resulted in A to T and G to T transversions about equally.
Hydrazine, which breaks pyrimidine rings, resulted mainly in C to T and T to C transitions. Finally, potassium permanganate resulted in T to C transitions in the two fragments that were sequenced.
The chemical treatment used here was slightly harsher than that used for the @-globin promoter (22) and resulted in a lower fraction of single-site mutations (63 verses 90%). This treatment was deliberate since we anticipated that because of codon degeneracy, a substantial fraction of single sites might not result in an amino acid alteration. Indeed, we did find that 25% of the singly altered trpA fragments resulted in no alteration in the amino acid sequence. Similarly, four of the 17 mutants that resulted in single amino acid alterations arose from multiple base pair alterations in the DNA.
It seems clear that this procedure does generate a vast number of mutants representing, primarily, single site DNA alterations. The mutation sites appear random and there is every reason to think that, with the number of mutants already in our collection and the ability to mutagenize either strand of each trpA fragment, the entire sequence can be saturated with mutations. An examination of the codons for randomly chosen amino acid residues in several regions of the a-subunit indicates that with such a saturation mutagenesis, an average of six different amino acid substitutions is possible for any residue in the sequence. These results can be achieved in the absence of a selection for any functional phenotype that the polypeptide might or might not exhibit.
Expression of the trpA Gene inptactrpA and the Purification of the a-Subunit-In order to examine the properties of asubunits that contain these mutational alterations, it was necessary to introduce the mutant trpA fragments into a trpA gene-containing plasmid (last step, Fig. 2). To facilitate the isolation of mutant a-subunits for study, it was also desirable that the trpA gene in this plasmid is overexpressed. Plasmid ptactrpA (or derivatives of it) satisfies these requirements. In this plasmid, the trpA gene is under the control of the tac promoter which is one of the strongest promoters characterized in vivo and in vitro and has been used to efficiently express a variety of gene products (28,(30)(31)(32)(33)(34)(35)(36). This promoter is a fusion of the -35 sequence of the trp promoter and the -10 sequence of the lacUV5 promoter (27). The construction of the tac promoter places the -35 and the -10 promoter regions 16 bp apart, similar to the strong ribosomal RNA promoters (28). Since the tac promoter contains the lac operator region, it is controlled by the lac repressor and can be induced by lactose or by isopropylthio-P-D-galactoside. Moreover, the tac promoter carries the Shine-Dalgarno sequence for the &galactosidase gene. Hence, a fused ribosomal binding site is possible using the Shine-Dalgarno sequence of the tac promoter and the initiation codon of the gene fused to the tac promoter. In addition, ptactrpA contains the efficient trp terminators, trpt and trpt ' (25, 26). This circumvents the possible problem of plasmid instability due to a high level of transcription beyond the trpA gene which may interfere with plasmid replication (37, 38).
The use of ptactrpA for a-subunit production and purification required an examination of the kinetics and the extent of induction and the stability of the plasmid. Fig. 4 shows the results of the induction kinetics of a-subunit activity. It is seen that a-subunit specific activity increased to nearly 900 units/mg in crude extracts from 22-h induced cultures. It can also be noted that cell growth continues apparently normally after induction. Similar growth rates occur in the absence of induction or with cells containing no plasmid. Correlated with the specific activity increases, the relative amounts of asubunit (i.e. the 30-kDa polypeptide) increased to a maximum of 33% of the total cellular protein (Fig. 5).
A potentially serious problem was the level of a-subunit produced in the absence of inducer. Under these conditions, approximately 5-7s of the total cellular protein (55 units/ mg, specific activity) was detectable in strain RB797 after 24 h of growth (Figs. 4 and 5). Possible origins of the noninduced synthesis of the a-subunit were the bacterial chromosome or the incomplete repression of the tac promoter. Since in subsequent studies mutant a-subunit protein synthesis is to be controlled by the tac promoter, it was important to determine how much of this uninduced level was due to the wild-type chromosomal a-subunit. Chromosomal levels of a-subunit production were determined in strain RB797 in the absence of ptactrpA. Strain RB797, containing no plasmid, was grown for 2 h and then induced for 6 h. Under these conditions, the 30-kDa polypeptide was expressed at 0.6% of total cellular protein (Fig. 5). This polypeptide appears to be of chromosomal origin, rather than plasmid encoded, since a similar value is obtained with cells that contain the plasmid from which the tac-trpA DNA insert is removed. This suggests that the level of this polypeptide is less than 2% chromosomally derived. This estimate may be somewhat exaggerated by the presence of another host polypeptide since specific activities of the a-subunit (4-6 units/mg) from extracts of induced cells containing no plasmid or insertless plasmid are less than 1% that of fully induced ptactrpA-containing cells. These results  indicate that the uninduced levels appear to be due to the incomplete repression of the tac promoter and suggest that, given the degree of precision of the methodology for later studies with purified proteins, this potential wild-type contamination level should not be a significant problem. If necessary, however, trpA-deleted strains can be employed.
As mentioned above, strain RB797 which carries h i q and recA mutations is employed as the host strain for ptactrpA. The laciq mutation, which results in the overproduction of the lac repressor (28), was thought necessary (38) because of the potential problem that cell growth may be inhibited by the high levels of a particular protein that is continuously overproduced. Therefore, sufficient cell growth can occur before induction is initiated and high levels of a protein become potentially toxic. Although we routinely employ a laciq host strain, overproduction of the a-subunit does not appear to be toxic to growth of this strain (see above, Fig. 4) nor is the initial repression of the tuc promoter absolutely required (see results below with the non-laciq strain, HB101).
It is also worth noting that problems of functional stability of this vector were encountered. Initially, ptactrpA was maintained in E. coli RB986, a recA+ strain. While maintained in this strain, the plasmid tended to form predominantly dimers. Interestingly, the level of induction of the a-subunit from the dimerized plasmid was observed to be even less than the uninduced level for RB797. To pursue this observation, a monomeric and dimeric form of ptactrpA were each transformed into several E. coli strains carrying a recA mutation, HBlOl and RB797. After 4 h of induction with lactose, strain HBlOl which contained the monomeric form showed induced levels of a-subunit activity, 1200 units/mg, whereas strain HBlOl which contained the dimeric form showed uninduced levels of a-subunit activity (Fig. 6). Hence, it appears that overexpression of the a-subunit occurred only when ptactrpA was in the monomeric form. The same behavior was noted with RB797. The wide variability in expression or induction efficiency (as low as 5%) reported by others (28,(30)(31)(32)(33)(34)(35)(36) using tac promoter-containing vectors may be related to this phenomenon. The only correlation seen here is that loss of expression appears to parallel plasmid dimerization during culture maintenance. Whether or not there is a causal relationship is uncertain. It may be related to the fact that the multicopy ptactrpA plasmid contains another lac operator region in the backbone (pSP64). Another overexpression multicopy vector we have constructed carries the tac-trpA region plus only the pBR322 origin and the chloramphenicol acetyltransferase gene from pBR329 and does not exhibit these properties.
Because of the overproduction of the cu-subunit, it was also possible to develop a simplified purification scheme for this protein. This was necessary for its anticipated use in isolating the relatively large number of mutant a-subunits. Previously reported purification methods by 40) and by Kirschner and co-workers (41), each of which required multiple column steps, seemed to be far too cumbersome for our needs. Accordingly, a one-step ion-exchange column chromatography procedure using DEAE-cellulose was developed (see Miniprint section). Typically, 15 mg of wildtype cu-subunit could be obtained in 75% yield from extracts of fully induced 200-ml cultures. SDS-polyacrylamide gel electrophoresis of purified preparation indicated the presence of slight (less than 0.5%) contaminating polypeptide. Preliminary experiments indicate that most of the mutant @-subunits can also be obtained in homogeneous form using this procedure. respectively. Cell-free extracts were prepared, and the indole to tryptophan specific activity measurements were made. Cell-free extracts (50 pg) and the purified a-subunit (3 pg) were electrophoresed and stained with Coomassie Blue.

In Vitro
Mutagenesis of the E. coli trpA Gene Expression and Stability of Mutant a-Subunits-Selected mutants of trpA fragments I and I1 were subcloned into the respective modified overexpression vectors, ptactrpAITc and ptactrpAIIP (see Miniprint section). The presence of a trpA fragment in the mutant ptactrpA plasmids was confirmed for each mutant by restriction endonuclease mapping.
Each strain carrying a plasmid with a mutant trpA gene was then induced. Induction of each mutant protein and the preparation of crude extracts were performed as described previously for the wild-type a-subunit. Initially, an induction time of 22 h was chosen since that time was optimum for the wild-type protein production. Each cell-free extract containing mutant a-subunit protein was electrophoresed through an SDS-polyacrylamide gel (Fig. 7) and then scanned to determine the relative amount of the mutant a-subunit. As shown in Fig. 8A (open bars), the level of expression of several of the mutant a-subunits was comparable to that of the wild type, that is about 30% of the total protein. However, several of the mutant a-subunits were detected in much smaller quantities. The decreased levels varv from about 2% for YC4. SL33, GS44, AT47/AT59, and F154 through about 12% for GD51 to 17% for PS28 and 21% for PH53 and the double mutant PH53/LQ58. One possible explanation is that the conformational changes due to the mutations may be severe enough so that they are recognized as denatured polypeptides and degraded. Thus, the turnover rate of these mutant proteins may be much faster than that of wild type, and a 22-h induction period was too long to detect the presence of these mutant a-subunits. To determine if these putatively unstable proteins could be detected following a shorter induction time, cell-free extracts containing each mutant a-subunit were prepared after an induction period of 6 h. Again each cell-free extract was electrophoresed through an SDS-polyacrylamide gel (Fig. 7), and the gel was scanned to determine the relative amount of a-subunit protein (Fig. 8A, solid bars). Except for FI54, all that had lower than wild-type levels after 22 h show substantial increases in a-subunit levels.
&Subunit Actiuating Activity of the Mutants-Most, if not all, trpA missense mutant proteins (isolated by conventional methods) have been shown to combine with the &subunit and activate it for catalysis of the indole to tryptophan reaction. This property has been used to determine whether or not the conformational states of the different mutant asubunits that we have generated are altered to a point where they cannot activate the &subunit. Extracts from 6-h induced cultures were chosen since, as mentioned above, most   Fig. 7 and calculating the relative amount of a-subunit

(solid bars, 6-h induction; open bars, 22-h induction). In B, the relative specific activities for &-combining activity (solid bars) and intrinsic activity (open bars) of a-subunits in extracts from 6-h induced cultures.
contain substantial levels of the a-subunit. The specific activity of each is shown in Fig. 8B (solid bars). This specific activity has been corrected for the relative amount of asubunit detected in each extract.
Intrinsic Enzymatic Actiuity of Mutants-The a-subunit has enzymatic activity by itself in one of the partial reactions (indoleglycerol phosphate to indole) catalyzed by tryptophan synthase. Each of the extracts obtained after 6-h induction was examined for this activity, and the results (Fig. 823, open bars) have been corrected for the relative amount of a-subunit present. Table I1 presents a comparison of the properties of the mutant a-subunits that we have examined so far in crude extracts. They possess properties of interest both for structure-function considerations and for potential defects in folding.
Seven have either greatly reduced (YC4) or no intrinsic activity (FS22, AT471 AT59, PH53/LQ58, PH53, FI54, and DN60). Moreover, there are two subclasses within this group: those which are typical of many a-subunits from missense mutants in that they can activate the &subunit (FS22, AT47/AT59, and DN60) and those which cannot (YC4, PH53/LQ58, PH53, and FI54). As noted above, this latter group is unusual for missense types. Another novel type of functional alteration detected is exemplified by mutants PL28, SL33, GS44, and GD51. These have mutant a-subunits with normal intrinsic activity but are unable to activate the &-subunit in the indole to tryptophan reaction. This pattern of functional defects observed with this

In Vitro
Mutagenesis of the E. coli trpA Gene activity activity "The stability property refers to the relative amount of mutant a-subunit detected after the 22-h induction * A "+" indicates that the DroDertv is 275% of the wild-type value; a "0" indicates that the property is ~5 0 % of period.

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the wild-type value.
" " small set of our mutants illustrates the utility of the selection procedure employed here. The conventional procedure for identifying most trpA missense mutant strains demanded that they accumulate indoleglycerol in the medium ( i e . they have limited intrinsic a-subunit activity) and they utilize indole for growth (i.e. they can activate the &subunit in the indole to tryptophan reaction). From the large collection of such E.
coli trpA missense mutants with amino acid alterations in this region of the a-subunit, only two sites, at residue positions 22 and 49, have been found to be essential for function and/or structure (42, 43). Thus, many of the unusual functionally defective a-subunits would not have been detected. It should also be noted that our protocol yields information, as expected, about what amino acid substitutions have no effect on function, and studies may be done to determine if they can display conformational effects without functional changes.
Two groups of mutants are obvious candidates for more detailed and immediate study of their folding properties. The implication of prolyl isomerization event(s) postulated (16) for the folding of the N-terminal domain makes the five prolyl mutants potentially interesting. Three of the five prolyl residues encoded by trpA fragments I and I1 have been altered (PS21, PS28, PL28, and PH53); and in SP6, a prolyl residue has been introduced. Perhaps as attractive are the stability mutants (YC4, Ps28, SL33, GS44, AT47/AT59, GD51, and FI54), many of which are also defective in &activating activity. Both of these defects appear in a-subunits with alterations in the region between residues 28 and 54. Although it is hazardous to speculate because of the relatively few mutant a-subunits examined, the localization of these defects may suggest that this is a region particularly critical for folding (and/or perhaps for &-subunit association) of the polypeptide chain. Comparative sequence analyses of a-subunits from yeast (44) and a variety of bacterial sources3 (45-48) indicate that for this portion of the polypeptide chain, residues 8, 26, 28, 44, 49-51, 56, 57, 59, 61, 62, 64, and 65 are invariant. The clustering of invariant residues in the 44-65 region lends support to this suggestion. The mutagenesis protocol described here allows us to address this question directly by looking at a much larger population of mutants already in our collection for alterations in this region as well as in the following region encoded by trpA fragment 111.
The availability of the overexpression vector for a-subunit production should allow us to obtain substantial quantities of mutant proteins for further study.