1 Department of Microbiology and Immunology, 2 Structural and Computational Biology and Molecular Biophysics Program and 3 Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: ß-lactamase/compensating mutations/protein core/protein evolution/thermal value
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A second mechanism by which core substitutions are tolerated is by the accumulation of compensatory mutations, which relieve the stress imparted by the original mutation (Poteete et al., 1991; Serrano et al., 1993
; Baldwin et al., 1996
). To date, examples of compensatory mutations in protein cores are relatively rare. This is presumably due to the need for multiple, specific substitutions to occur at positions surrounding the original mutation in order to suppress its defect (Lesk and Chothia, 1980
; Baldwin et al., 1996
). By the time the necessary compensatory mutations have arisen, it is likely that the original mutant would have been eliminated from the population. However, why certain core positions are still able to be compensated by secondary mutations remains unclear. A ß-lactamase model has been used to analyze how core substitutions are accommodated in a protein (Huang et al., 1996
).
The most common mechanism of bacterial resistance to ß-lactam antibiotics, such as the penicillins and cephalosporins, is the production of ß-lactamases. These enzymes cleave the lactam ring of the antibiotic to render it inactive. Based on primary sequence homology, ß-lactamases have been grouped into four classes (Joris et al., 1988; Ambler et al., 1991
). Classes A, C and D utilize a catalytic serine residue in the active site, while class B enzymes require a zinc atom for catalysis (Joris et al., 1988
; Carfi et al., 1995
). The crystal structures of several class A enzymes have been solved (Moews et al., 1990
; Herzberg, 1991
; Jelsch et al., 1993
). The structures show that these enzymes share a similar three-dimensional structure and, along with primary sequence homology, suggest a common evolutionary origin for these proteins.
TEM-1 ß-lactamase, a class A enzyme, is the most prevalent plasmid encoded ß-lactamase found in Gram negative bacteria. Random replacement mutagenesis was previously used to completely randomize the 263 codons of the TEM-1 ß-lactamase gene (blaTEM-1) (Huang et al., 1996). This mutagenesis strategy targets three contiguous codons in blaTEM-1 and converts the wild-type sequence to all possible amino acid sequences within that three codon window (Huang et al., 1996
). The set of all possible mutants for a given three amino acid window constitutes a random library. Eighty-eight random libraries encompassing the entire TEM-1 ß-lactamase gene were selected for mutants that confer wild-type levels of ampicillin resistance to Escherichia coli. Sequence data of functional mutants revealed that 43 of the 263 residues tested do not tolerate amino acid substitutions and are thought to be essential for TEM-1 ß-lactamase structure and function (Huang et al., 1996
). Many of the conserved residues, such as F66 and L76, are core positions. This result is consistent with the general observation that core amino acids are less frequently substituted than surface residues (Bowie et al., 1990
; Huang et al., 1996
). However, while amino acid substitutions at residues F66 and L76 were conserved based on the in vitro mutagenesis results, the degree of sequence conservation at positions 66 and 76 in the class A ß-lactamase gene family is strikingly different (Figures 1 and 2
). Residue 76 is substituted rather freely in other gene family members, while residue 66 is conserved in nearly all class A ß-lactamases. In the wild-type TEM-1 ß-lactamase structure, F66 and L76 are nearly equidistant from the active site pocket, lying approximately 12 and 10 Å, respectively, from the catalytic serine at position 70 (Figure 3
) (from Brookhaven PDB entry 1BTL). F66 and L76 are both completely buried and interact with several surrounding amino acids (Jelsch et al., 1993
). Therefore, upon initial inspection, the environments surrounding both F66 and L76 are similar. In other members of the class A ß-lactamase gene family, it appears that a series of mutations have evolved at surrounding positions to compensate for the non-conservative substitutions at position 76. As a result, hydrophobic interactions are maintained in enzymes having a hydrophobic amino acid at position 76, while a buried hydrogen bond network is found in enzymes having a hydrogen bond donor at position 76 (Huang et al., 1996
).
|
|
|
In an attempt to determine why position 76 is more tolerant to substitutions than position 66, the thermal values for atoms surrounding and including both positions were examined in three class A ß-lactamases whose crystal structures have been solved: TEM-1 ß-lactamase, the PC1 ß-lactamase from Staphylococcus aureus and the ß-lactamase from Bacillus licheniformis 749/C (Moews et al., 1990; Herzberg, 1991
; Jelsch et al., 1993
). Both the PC1 and the 749/C ß-lactamases have hydrogen bond donors at position 76 (an asparagine and a threonine, respectively). Because thermal values reflect the range of movement a particular atom may have, they may explain why position 76 is more tolerant to substitution than position 66 (Petsko and Ringe, 1984
). In a study involving 25 temperature-sensitive (ts) mutants of T4 lysozyme, it was found that all of the mutations altered amino acid side chains having lower than average thermal factors (Alber et al., 1987
). It was concluded that amino acids with well-defined conformations form specific intramolecular interactions that make large contributions to the thermal stability of the protein (Alber et al., 1987
). It was further interpreted that residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, and that, in general, such amino acids make less of a contribution to protein stability (Alber et al., 1987
). The actual number of atoms in contact with position 66 and 76 further shows how much structural constraint exists in these regions. It is presumed that the greater the number of contacts, the greater the constraint. The hypothesis is that there are relatively few atoms in contact with position 76 and these atoms have a wider range of motion than atoms surrounding position 66. As a result, position 76 would be more tolerant of substitutions because there is less constraint in this region. Meanwhile, it is expected that a greater number of atoms are in contact with position 66, and these atoms have lower thermal values. This would result in greater constraints on position 66 due to its environment. It was found that there is little correlation between the thermal values of atoms in contact with positions 66 and 76 and the ability of both positions to be substituted. Therefore, unlike the T4 lysozyme model, the thermal values and number of contacts around positions 66 and 76 do not explain why substitutions at position 76 are tolerated more than substitutions at position 66. However, additional analyses suggest a volume requirement may be at least partially responsible for the small tolerance of substitution at position 66.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Escherichia coli BW313 [Hfr lysA(6162), dut1, ung1, thi1, recA1, spoT1] was used to propagate plasmid DNA prior to mutagenesis (Kunkel et al., 1987). DNA was introduced into E.coli ES1301 [lacZ53, mutS201::Tn5, thyA36, rha5, metB1, deoC, IN (rrnD-rrnE)] immediately following mutagenesis (Siegel et al., 1982
). Escherichia coli XL1-Blue [recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, [F':: Tn10 (Tetr)proAB,
lacIq (lacZ)M15]] was used in determining antibiotic susceptibility and specific activity levels (Stratagene, Inc., Calif., USA). Mutagenesis was performed on the plasmid pBG66, which contains the wild-type blaTEM-1 gene and a cat gene encoding chloramphenicol acetyltransferase. This 4.8 kb plasmid also contains the f1 and ColEI origins of DNA replication (Palzkill and Botstein, 1992
).
Random and site-directed mutagenesis
Oligonucleotide primers used for mutagenesis and DNA sequencing were synthesized by Genosys Biotechnologies, Inc., Texas, USA. The random library L76X was constructed (Huang and Palzkill, 1997). The F66X random library was constructed in the same manner using the following oligonucleotide: F66X, 5'-CTTTAAAAGTGCTCATCATTGGSNNACGTTCTTCGGGGCGAAAACTC-3', where N represents any base and S represents a C or a G. The mutagenesis template was pBG66 containing a SalI linker insertion at codon 65 of the blaTEM-1 gene (Huang et al., 1996
). This SalI insert mutant also contained a single base deletion, rendering this mutant non-functional. Single-stranded DNA of the SalI insert mutant was used in the mutagenesis reaction, which was performed as previously described (Huang et al., 1996
). Individual mutants from these libraries were obtained by streaking the libraries on LB agar plates containing 12.5 µg/ml chloramphenicol, and incubating the plates at 37°C for 18 h resulting in colonies containing the mutagenized pBG66 plasmid. Colonies from the chloramphenicol plates were replica plated onto duplicate 50, 100, 500 µg/ml and 1 mg/ml ampicillin LB agar plates. One set of plates was incubated 18 h at 37°C and the other at 30°C. This selection allowed various ß-lactamase-mediated phenotypes to be discerned by growth characteristics on the ampicillin plates at 30 and 37°C. The ß-lactamase genes from colonies growing under different ampicillin concentrations and temperatures were sequenced in attempt to isolate as many of the 19 different mutations possible for each residue. Fifteen of the 19 possible mutations at position 66 were isolated by sequencing 128 clones. Thirteen of the 19 possible mutations at position 76 were isolated by sequencing 48 clones. The F66D, F66E, F66G, F66P, L76A, L76D, L76E, L76F, L76K and L76Y mutants were constructed by oligonucleotide directed mutagenesis, as described by Kunkel et al. (1987). The sequence of these mutants was confirmed by the dideoxy chain termination method using PCR products as template (Hanke and Wink, 1994
).
Antibiotic susceptibility
Minimum inhibitory concentrations (MICs) were determined by broth microdilution. Escherichia coli XL1-Blue cells harboring the pBG66 plasmid, either containing wild-type or a selected mutant ß-lactamase, were grown overnight in 2 ml 2xYT media supplemented with 12.5 µg/ml chloramphenicol. Each culture was diluted so that 1x104 cells could be inoculated into microtiter wells containing 100 µl LB media having twofold increases of each antibiotic tested (ampicillin and cephaloridine). After an 18 h incubation at 37°C, the plates were examined, and the lowest concentration of antibiotic that inhibited visual growth was recorded as the MIC.
Specific activity of ß-lactamase mutants
Escherichia coli XL1-Blue cells harboring the pBG66 plasmid, either containing wild-type or a selected mutant ß-lactamase, were grown overnight at 37°C in 2 ml of 2xYT medium supplemented with 12.5 µg/ml chloramphenicol. One hundred microliters of the overnight culture were used to inoculate 10 ml 2xYT with 12.5 µg/ml chloramphenicol. The 10 ml culture was grown to log phase (A600 = 0.5). The periplasmic contents of each culture were extracted using an osmotic shock procedure (Neu and Heppel, 1965). The total protein concentration of the periplasmic extract was determined by the method of Bradford (1976). ß-Lactamase activity assays were performed in 0.5 ml phosphate buffer (pH 7.0). Ampicillin (300 µM) or cephaloridine (1.0 M) was present in each reaction. Ampicillin hydrolysis was monitored as the loss of absorbance at 235 nm in a Beckman DU-640 spectrophotometer. Cephaloridine hydrolysis was monitored as the loss of absorbance at 260 nm. Specific activity was calculated as µM of antibiotic hydrolyzed per minute divided by the concentration of total protein in the reaction. The extinction coefficient used for ampicillin was
= 900 M1cm1, and for cephaloridine was
= 10 020 M1cm1 (Minami et al., 1980
). All reactions were carried out at 30°C. Reaction mixtures were prewarmed at 30°C for 5 min before the addition of protein, and the reactions were monitored in the spectrophotometer using a water-jacketed, temperature controlled cuvette. Since all 40 mutants were not assayed at once, wild-type specific activity measurements were determined before each group of mutants to serve as an assay control. Because exact duplication of specific activity measurements is difficult, the percentage of wild-type activity for each given mutant was used for analysis.
Crystal contact and thermal value analysis
Potential hydrogen bond and van der Waals contact partners were identified by examining Brookhaven PDB data files 1BTL, 3BLM and 4BLM corresponding to TEM-1 ß-lactamase, the PC1 ß-lactamase from Staphylococcus aureus, and the ß-lactamase from Bacillus licheniformis 749/C, respectively (Moews et al., 1990; Herzberg, 1991
; Jelsch et al., 1993
). Atoms surrounding either position 66 or 76 were identified using the SYBYL software package (Tripos). Atoms within 4.0 Å of residue 66 and 76 were labeled as potential contact atoms, because they are within a reasonable van der Waals radius of the amino acid being examined. Atoms which may form hydrogen bonds with backbone nitrogen groups or the side chains of N76 in the PC-1 ß-lactamase and T76 in the 749/C enzyme were also included in the 4.0 Å radius.
The relative mobility of each atom in the X-ray crystal structures of TEM-1, PC1 and 749/C ß-lactamases is reflected by the B value of a particular atom. The B values for all of the atoms in contact with positions 66 and 76 in the ß-lactamase structures were obtained from the Brookhaven PDB files. The average thermal value for residues 66 and 76 in each model were determined by taking the sum of the thermal values for each atom in a particular amino acid and dividing by the number of atoms.
In order to compare packing density at F66 and L76, the three PDB files were modified so that they contained only the residues surrounding F66 and L76. The accessible surface in square Ångströms for all the residues within the structure subsets were then calculated (with respect to a probe sphere 1.40 Ångströms in diameter) using ACCESS (http://www.csb.yale.edu/download/download_descrip.html) (Lee and Richards, 1971). The volumes for these regions were then calculated from the output of ACCESS using VOLUME, under the default specifications (http://www.csb.yale.edu) (Richards, 1985
). VOLUME determines the volume of a polyhedron surrounding selected atoms in a protein when the polyhedral faces are determined by one of three procedures based on the Voronoi construction (Richards, 1985
).
To address the relative environment of F66 and L76 within the three structures, we analyzed the ability of positions 66 and 76 to accept other side chains using Rotamer Related, the option which counts and ranks position specific rotamers on the WHAT IF server (http://swift.embl-heidelberg.de/johnny/new/) (Vriend, 1990). The ranking system scores the most ideal amino acid a 1 and the amino acid furthest from ideal a 20.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
According to the hypotheses described above, it was expected that a greater number of ß-lactamase mutants with substitutions at position 76 would confer significant levels of resistance to both antibiotics compared with mutants with substitutions at position 66. This proved to be the case as several position 76 ß-lactamase mutants did confer resistance to both ß-lactams (Table I). Since lower amounts of cephaloridine are toxic to TEM-1 ß-lactamase mutant-containing bacteria, the range of observed MICs is not as broad for the cephaloridine data as for the ampicillin data. Therefore, the MIC data for cephaloridine are not as discriminating as the data from the ampicillin MICs. The results from the cephaloridine MIC data, however, correlate well with the ampicillin data in that mutations which were most effective in conferring cephaloridine resistance also conferred the greatest resistance to ampicillin.
|
|
A direct comparison of the constraint existing in each environment was done by calculating the average thermal displacements for positions 66 and 76 in each of the ß-lactamases studied. The unidirectional mean square thermal displacement (µ2) is related to the crystallographic thermal factor B by the equation B = 82(µ2). The B values are comprised of contributions made by thermal motion, segmental motion, lattice disorder and errors in the molecular model. However, thermal motion has been found to dominate the observed B values in several protein X-ray crystal structures (Petsko and Ringe, 1984
). The ability of position 76 to tolerate more substitutions than position 66 in the protein core leads to the prediction that there are fewer structural constraints around position 76. The sum of the thermal factors for atoms from each amino acid, at both positions, was divided by the number of atoms in the residue, and the resulting averages were compared. It was discovered that the average thermal factor of position 66 was slightly higher than the average thermal factor at position 76 in all three ß-lactamases (Table II
). Therefore, according to the average thermal factor parameter, there does not appear to be a significant difference in the constraint found at positions 66 and 76.
|
|
|
|
|
Structural constraint was further examined by comparing the position 66 and 76 packing densities, as measured by the Voronoi volumes at, and surrounding, both positions. If a correlation between the Voronoi volumes at positions 66 and 76 exists, it would suggest that there is a volume requirement for the amino acids that may occupy these positions. No correlation was observed regarding Voronoi volumes of the environments surrounding both positions; however, a strong correlation between the volumes of the amino acids occupying positions 66 and 76 was found (Table IV). In each of the three ß-lactamases, position 66 occupies a greater volume than position 76.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To test the relative tolerance of positions 66 and 76 to substitution in the TEM-1 enzyme, we constructed mutants containing every amino acid substitution at both sites and examined the ability of these mutants to confer bacterial resistance to two common ß-lactam antibiotics. The antibiotic susceptibility studies showed that mutations at position 76 do appear to be less deleterious than mutations at position 66 in TEM-1 ß-lactamase. Typical of core residues, position 76, and to a lesser extent, position 66 were tolerant of mutations in which the replacement amino acids were hydrophobic and were within one methyl group in volume change. However, several non-conservative hydrophilic amino acid substitutions and other small volume amino acid substitutions at position 76 resulted in mutant ß-lactamases with significant levels of activity. Of these non-conservative substitutions conferring partial activity, glutamate is the only one found at position 76 in a class A enzyme, the blaU gene from Streptomyces cacaoi. It was concluded that the ability of position 76 to tolerate several non-conservative substitutions and still retain partial activity is directly correlated to the ability of compensating mutations to arise in response to position 76 mutations. Many of the possible mutations at position 76 will not completely eliminate enzyme activity, and the chance of secondary mutations arising to restore the mutant to wild-type levels of activity is increased.
If a non-conservative mutation is to be maintained in the protein core, subsequent compensatory mutations are required. Because protein cores are believed to evolve through a constrained, step-by-step process that does not dramatically affect the main-chain fold, the probability that a compensating mutation will arise before the original mutant is eliminated from a population is low. However, if the original substitution does not significantly compromise the protein's structure or function, then the probability that a secondary mutation will arise to compensate for the original substitution before it is eliminated is increased (Bordo and Argos, 1989; Lim and Sauer, 1989
; Malcolm et al., 1990
; Baldwin et al., 1996
). An analysis of T4 lysozyme has shown that even when interacting buried residues are exchanged, complete compensation is not achieved (Baldwin et al., 1996
). Therefore, it appears that several mutations are needed to reconstitute the activity of the wild-type protein. One conclusion from the lysozyme study was that a reversion of a deleterious mutation back to the wild-type residue would be the easiest way for a protein to compensate for a non-conservative substitution rather than a series of secondary mutations. The results described here also suggest that a reversion mutation would be the easiest and possibly the only way to compensate for a non-conservative substitution at a typical core position, such as F66. However, if the core position is able to retain partial activity in response to a non-conservative substitution, then second-site compensating mutations would provide a feasible pathway to restore wild-type activity levels as well.
In order to determine why substitutions at position 76 are less detrimental to ß-lactamase structure, the environments around positions 66 and 76 were analyzed. By calculating the average thermal factors of positions 66 and 76 in the three ß-lactamases examined, it appears that the amount of structural constraint at both positions in each enzyme are similar. Furthermore, in the three ß-lactamases tested, the amino acid occupying position 76 makes a similar number of van der Waals or hydrogen bond contacts compared with F66. Those residues making contact with position 76 also have similar thermal values compared with the F66 contact residues. Both of these results suggest that there are equivalent structural constraints on positions 66 and 76. However, the Voronoi volumes occupied by positions 66 and 76 in the three enzymes does suggest that there is a requirement for larger amino acids at position 66. This agrees favorably with our mutant activity data which shows that larger amino acids are able to confer partial activity to position 66 mutants, while a variety of amino acids may confer partial activity to position 76 mutants. The rotamer re-packing data provides an estimate of how optimal the existing amino acids are at each of the positions tested. It appears that the core of the TEM-1 enzyme is the most evolved of the three enzymes examined because both F66 and L76 rank second in preferred amino acids at these positions. Meanwhile, the wild-type amino acids at positions 66 and 76 in the PC1 and 749/C enzymes rank no higher than 7th. Particularly interesting is the fact that the asparagine at position 76 in the PC1 enzyme is ranked 14th suggesting that substitutions for this residue would be tolerated, and in many cases, preferred. However, these results may also reflect a requirement for asparagine at position 76 for a folding pathway or other purpose.
The extent that hydrophobicity, side chain volume and steric hindrance contribute to protein core constraint has been well documented (Lesk and Chothia, 1980; Bordo and Argos, 1989
; Poteete et al., 1991
; Rennell et al., 1991
; Markiewicz et al., 1994
; Terwilliger et al., 1994
). Additional studies of the Cro protein of phage
, staphylococcal nuclease and kanamycin nucleotidyltransferase have shown that there is no simple pattern to which these properties contribute to protein stability (Shortle and Lin, 1985
; Matsumura et al., 1986
; Pakula et al., 1986
). A correlation between low thermal factors, fractional accessible surface area and detrimental amino acid substitutions has demonstrated the importance of these two parameters as well (Alber et al., 1987
). In the case of positions 66 and 76 in the three ß-lactamases examined, side chain volume requirements may explain why position 66 tolerates fewer substitutions than position 76.
It is not clear whether position 76 was the first amino acid to be substituted in the PC1 or 749/C ß-lactamases, or if a neighboring position mutated first and a secondary mutation occurred at position 76 to accommodate the adjacent hydrogen bonding amino acid. Positions 66 and 76 are both surrounded by amino acids that display various degrees of conservation in the class A ß-lactamase gene family as well as in the random library selection reported previously (Huang et al., 1996). Even though the environments surrounding positions 66 and 76 appear similar, there are explanations which could further account for the conservation of F66 in the class A ß-lactamase gene family. For example, F66 may play an important role in the folding of ß-lactamase while position 76 does not. It is also possible that among the atoms within contact distance of position 66, there are interactions which cannot be disrupted. Such important contact(s) may not exist at position 76.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ambler,R.P., Coulson,F.W., Frere,J.-M., Ghuysen,J.-M., Joris,B., Forsman,M., Levesque,R.C., Tiraby,G. and Waley,S.G. (1991) Biochem. J., 276, 269272.[ISI][Medline]
Baldwin,E., Xu,J., Hajiseyedjavadi,O., Baase,W.A. and Matthews,B.W. (1996) J. Mol. Biol., 259, 542559.[ISI][Medline]
Bordo,D. and Argos,P. (1989) J. Mol. Biol., 211, 975988.[ISI]
Bowie,J.U., Reidhaar-Olson,J.F., Lim,W.A. and Sauer,R.T. (1990) Science, 247, 13061310.[ISI][Medline]
Bradford,M.M. (1976) Anal. Biochem., 72, 248254.[ISI][Medline]
Carfi,A., Pares,S., Duee,E., Galleni,M., Duez,C., Frere,J.M. and Dideberg,O. (1995) EMBO J., 14, 49144921.[Abstract]
Chothia,C. and Gerstein,M. (1997) Nature, 385, 579581.[ISI][Medline]
Hanke,M. and Wink,M. (1994) Biotechniques, 17, 858860.[ISI][Medline]
Herzberg,O. (1991) J. Mol. Biol., 217, 701719.[ISI][Medline]
Huang,W. and Palzkill,T. (1997) Proc. Natl Acad. Sci. USA, 94, 88018806.
Huang,W., Petrosino,J., Hirsch,M., Shenkin,P.S. and Palzkill,T. (1996) J. Mol. Biol., 258, 688703.[ISI][Medline]
Jelsch,C., Mourey,L., Masson,J.-M. and Samama,J.-P. (1993) Proteins, 16, 364383.[ISI][Medline]
Joris,B., Ghuysen,J.-M., Dive,G., Renard,A., Dideberg,O., Charlier,P., Frere,J.-M., Kelly,J.A., Boyington,J.C., Moews,P.C. and Knox,J.R. (1988) Biochem. J., 250, 313324.[ISI][Medline]
Koolman,J. and Rohm,K.-H. (1996) Color Atlas of Biochemistry. Thieme, New York.
Kraulis,P.J. (1991) J. Appl. Crystallogr., 24, 946950.[ISI]
Kunkel,T.A., Roberts,J.D. and Zakour,R.A. (1987) Methods Enzymol., 154, 367382.[ISI][Medline]
Lee,B. and Richards,F.M. (1971) J. Mol. Biol., 55, 379400.[ISI][Medline]
Lesk,A.M. and Chothia,C. (1980) J. Mol. Biol., 136, 225270.[ISI][Medline]
Lim,W.A., Hodel,A., Sauer,R.T. and Richards,F.M. (1994) Proc. Natl Acad. Sci. USA, 91, 423427.[Abstract]
Lim,W.A. and Sauer,R.T. (1989) Nature, 339, 3136.[ISI][Medline]
Malcolm,B.A., Wilson,K.P., Matthews,B.W., Kirsch,J.F. and Wilson,A.C. (1990) Nature, 345, 8689.[ISI][Medline]
Markiewicz,P., Kleina,L.G., Cruz,G., Ehret,S. and Miller,J.H. (1994) J. Mol. Biol., 240, 421433.[ISI][Medline]
Matsumura,M., Kataoka,S. and Aiba,S. (1986) Mol. Gen. Genet., 204, 355358.[ISI][Medline]
Minami,S., Yotsuji,A., Inoue,M. and Misuhashi,S. (1980) Antimicrob. Agents Chemother., 18, 382385.[ISI][Medline]
Moews,P.C., Knox,J.R., Dideberg,O., Charlier,P. and Frere,J.M. (1990) Proteins, 7, 156171.[ISI][Medline]
Neu,H.C. and Heppel,L.A. (1965) J. Biol. Chem., 240, 36853692.
Pakula,A.A., Young,V.B. and Sauer,R.T. (1986) Proc. Natl Acad. Sci. USA, 83, 88298833.[Abstract]
Palzkill,T. and Botstein,D. (1992) Proteins, 14, 2944.[ISI][Medline]
Parker,A.C. and Smith,C.J. (1993) Antimicrob. Agents Chemother., 37, 10281036.[Abstract]
Petsko,G.A. and Ringe,D. (1984) Annu. Rev. Biophys. Bioengng, 13, 331371.[ISI][Medline]
Poteete,A.R., Dao-Pin,S., Nicholson,H. and Matthews,B.W. (1991) Biochemistry, 30, 14251432.[ISI][Medline]
Rennell,D., Bouvier,S.E., Hardy,L.W. and Poteete,A.R. (1991) J. Mol. Biol., 222, 6787.[ISI][Medline]
Richards,F.M. (1985) Method. Enzymol., 115, 440464.[ISI][Medline]
Rogers,M.B., Parker,A.C. and Smith,C.J. (1993) Antimicrob. Agents Chemother., 37, 23912400.[Abstract]
Serrano,L., Day,A.G. and Fersht,A.R. (1993) J. Mol. Biol., 233, 305312.[ISI][Medline]
Shortle,D. and Lin,B. (1985) Genetics, 110, 539555.
Siegel,E.C., Wain,S.L., Meltzer,S.F., Binion,M.L. and Steinberg,J.L. (1982) Mutat. Res., 93, 2533.[ISI][Medline]
Terwilliger,T.C., Zabin,H.B., Horvath,M.B., Sandberg,W.S. and Schlunk,P.M. (1994) J. Mol. Biol., 236, 556571.[ISI][Medline]
Vriend,G. (1990) Protein Engng, 4, 221223.[Abstract]
Received February 1, 1999; revised May 18, 1999; accepted June 4, 1999.