Genetically Engineered Zinc-chelating Adenylate Kinase from Escherichia coli with Enhanced Thermal Stability*

Véronique PerrierDagger , Simona Burlacu-Miron§, Serge Bourgeois, Witold K. Surewiczparallel , and Anne-Marie GillesDagger **

From the Dagger  Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, France, § INSERM U 350, Institut Curie-Recherche, 91405 Orsay Cedex, France,  Laboratoire de Sciences des Sols et Hydrologie, Institut National d'Agronomie, Centre de Grignon, 78850 Thiverval, France, and parallel  Department of Ophthalmology, University of Missouri, Columbia, Missouri 65212

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In contrast with adenylate kinase from Gram-negative bacteria, the enzyme from Gram-positive organisms harbors a structural Zn2+ bound to 3 or 4 Cys residues in the structural motif Cys-X2-Cys-X16-Cys-X2-Cys/Asp. Site-directed mutagenesis of His126, Ser129, Asp146, and Thr149 (corresponding to Cys130, Cys133, Cys150, and Cys153 in adenylate kinase from Bacillus stearothermophilus) in Escherichia coli adenylate kinase was undertaken for determining whether the presence of Cys residues is the only prerequisite to bind zinc or (possible) other cations. A number of variants of adenylate kinase from E. coli, containing 1-4 Cys residues were obtained, purified, and analyzed for metal content, structural integrity, activity, and thermodynamic stability. All mutants bearing 3 or 4 cysteine residues acquired zinc binding properties. Moreover, the quadruple mutant exhibited a remarkably high thermal stability as compared with the wild-type form with preservation of the kinetic parameters of the parent enzyme.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Adenylate kinase (AK,1 ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme which contributes to homeostasis of adenine nucleotides in living cells (1). Three classes of AKs, differing in size, subcellular localization, and substrate specificity were identified in vertebrates, AK1 in the cytosol, AK2 in the mitochondrial intermembrane space, and AK3 (called also GTP:AMP phosphotransferase) in the mitochondrial matrix. Only one form of AK was identified in bacteria. Mitochondrial adenylate kinases (AK2, AK3) and the vast majority of bacterial adenylate kinases belong to the class of long forms. They differ from AK1 and some bacterial AKs, the short variants, by a 28-residue long insertion organized into a small domain (2) called LID well exposed to the solvent (Fig. 1A) and undergoing a large movement during catalysis (3, 4).


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 1.   Pictorial representation of the three-dimensional structure of AKe and of the LID domain after substitution by 4 cysteines (A) and alignment of partial amino acid sequences of the LID domain in long isoforms of adenylate kinase (B). The model was obtained using the atomic coordinates of AKe (32). The 4 Cys side chains were substituted in the corresponding positions His126, Ser129, Asp146, and Thr149 without searching for side chain optimization after substitution. The figure was drawn using the MOLSCRIPT software (33). The residues critical for coordination of zinc atom in Gram-positive bacteria and their counterparts in Gram-negative organisms are boxed.

AKs from Gram-positive bacteria contain a structural zinc atom (5-7), a property which is due to the presence of 3 or 4 cysteine residues in the LID domain. Sequence alignment of AKs from Gram-positive and Gram-negative organisms, devoid of metal, showed that in the latter species the Cys residues are substituted with four other highly conserved amino acids, His, Ser, Asp, and Thr (Fig. 1B). This conservation suggests that these particular residues have some essential function, but different in the enzyme from the Gram-negative bacteria and eukaryotes. A noticeable exception is AK from the Gram-negative bacterium Paracoccus denitrificans. This enzyme not only conserves the Cys-containing sequence found in AK from Gram-positive species but binds zinc or iron (8).

In this study, we substituted His126, Ser129, Asp146, and Thr149 in Escherichia coli adenylate kinase with cysteine residues. Our aim was to know whether a motif composed of 3 or 4 Cys residues generates a metal-binding site in AK or whether other structural factors contribute to the specificity (zinc versus iron or any other metal) or to the strength of the protein/metal interaction. On the other hand, we wanted to know the relevance of the metal binding for catalysis or stability of AK. A number of variants of AKe containing one to four cysteine residues were thus obtained. In agreement with previous studies on zinc-binding AKs, we found that the 3 and 4 cysteine modified forms of AKe acquired zinc binding properties. Moreover, the 4 cysteine-containing AKe exhibited an increased stability against thermal denaturation as compared with the wild-type form, with full conservation of its catalytic properties.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Adenine nucleotides, coupling enzymes, T4 DNA ligase, T4 polynucleotide kinase, and restriction enzymes were from Boehringer Mannheim. T4 DNA polymerase was from Biolabs. T7 DNA polymerase and Deaza sequencing mixes kit were from Amersham Pharmacia Biotech. Blue Sepharose (Cibacron Blue 3 G-1 Sepharose CL-6B) was from Pharmacia LKB Biotechnologies Inc. TPCK-treated trypsin, soybean trypsin inhibitor, PMPS, 4-(2-pyridylazo)resorcinol (PAR), and DTNB were purchased from Sigma.

Bacterial Strains, Plasmids, and DNA Manipulations-- The E. coli NM554, CJ236, and BL21(DE3) strains were used for cloning experiments, site-directed mutagenesis, and recombinant protein overproduction, respectively (9, 10). Plasmid pDIA17 harboring the lacI gene provides additional transcriptional control, under nonpermissive conditions. Plasmid pEAK91 carries the E. coli adk gene (11) and was kindly provided by A. Wittinghofer (Max Planck Institut für Molekulare Physiologie, Dortmund). Plasmid pPV1003 is a pET22b derivative carrying the adk gene subcloned from pEAK91.

Site-directed Mutagenesis, DNA Sequence Analysis, and Growth Conditions-- Site-directed mutagenesis was carried out according to Kunkel et al. (12). A 91 bases long primer (see Table I) containing 7 mismatched bases allowed several simultaneous substitutions in the adk gene. Mutant plamids from 48 randomly selected clones were further analyzed. A panel of single, double, triple, and quadruple mutants was obtained. Some additional variants, not resulting from this procedure, were created individually with appropriate primers (see Table I). Absence of any other mutation in the adk gene was checked on all plasmids. Overproduction of various AK forms was performed by growing strain BL21(DE3)/pDIA17 containing pVP1003 derivatives in LB medium (13) supplemented with 100 mg/liter ampicillin and 30 mg/liter chloramphenicol. Overproduction was carried out by adding 1 mM isopropyl-beta -D-thiogalactoside when the culture reached an absorbance at 600 nm of 1.0. Bacteria were harvested by centrifugation 3 h after induction.

Purification of AKe and Activity Assays-- The adenylate kinase overproduced in E. coli was purified as described previously (14). When required, purified proteins were dialyzed against 50 mM ammonium bicarbonate, then lyophilized. Enzyme activity was determined at 30 °C using the spectrophotometric assay (15). Measurements were made at 334 nm (0.5 ml final volume) using an Eppendorf ECOM 6122 photometer. One unit of enzyme activity corresponds to 1 µmol of the product formed in 1 min at 30 °C and pH 7.4 (in the direction of ATP formation). Protein concentration was determined according to Bradford (16), using a Bio-Rad kit. SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (17).

Zinc Content-- The metal in various forms of AK was quantitated colorimetrically, using the metal-binding dye PAR as described previously (5, 7) and by atomic absorption spectrophotometry, using a graphite furnace instrument. The protein samples and the zinc standard solutions were diluted with water purified to 18.2 megohms/cm resistivity. In all cases, the background levels of zinc were insignificant.

Differential Scanning Calorimetry-- The thermal stability of different proteins was studied by differential scanning calorimetry using an ultrasensitive Microcal MC-2D instrument at a scanning rate of approximately 50 °C/h. Proteins in 50 mM Tris-HCl buffer (pH 7.4) were in the range of 1-1.5 mg/ml. Differential scanning calorimetry data were analyzed by the software provided by Microcal Inc., Northampton, MA.

Nomenclature-- The mutants were named according to the position of key residues in the motif 126His-X2-Ser-X16-Asp-X2-Thr149. Thus, the HSDT variant is the wild-type AKe. AKeC4 corresponds to the 4 Cys-substituted enzyme, AKeHC3 to HCCC, AKeC3T to CCCT, AKeC2DT to CCDT, AKeHSC2 to HSCC, and AKeHC2T to HCCT.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Overproduction and Purification of Cysteine-substituted Variants of AKe-- To create a 4-Cys-substituted mutant of AKe, a single nondegenerate 91-base oligonucleotide, was designed spanning the adk gene region corresponding to the LID domain in the protein (Table I). The 7 mismatched bases in the oligonucleotide allow simultaneous substitutions of His126, Ser129, Asp146, and Thr149 codons with cysteines. Out of 48 randomly selected clones, two-thirds carried one or several substitutions with cysteine residue(s) and one-third harbored the expected quadruple modification. Considering the length of the oligonucleotide and the relatively low yield of its synthesis, the mutagenesis reaction was fairly effective and produced in one step a panel of single, double, and triple mutants displaying different positions of substitution. Missing species were constructed with appropriate primers (Table I).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Oligonucleotides used for the construction of AKe cysteine-substituted mutants
The asterisk (*) indicates the position of the mismatches.

The overproduced variants of AKe (about 20% of the soluble E. coli proteins) exhibited high specific activity in crude extracts (90-120 units/mg of protein), close to that of the wild-type AKe, indicating that substitution with cysteine of any of the four targeted residues has no direct consequences on enzyme activity. Chromatography on blue Sepharose and Ultrogel AcA54 yielded homogeneous preparations of enzymes. The double Cys-substituted AKe species, HC2T, and CSDC were inactivated during purification in the absence of reducing agents. They conserved only 5% (23 units/mg of protein) and 37% (175 units/mg of protein) of wild-type activity.

Metal Binding-- The zinc content of different variants of AKe, was quantified either by atomic absorption spectrophotometry or with the metal-binding dye PAR. The enzymes were first reacted with PMPS (18, 19), the formation of the PMPS-sulfhydryl chromophore being followed at 250 nm. Linear incorporation of PMPS into the proteins was observed up to 3.4 ± 0.3 equiv./mole of C4 mutant, 2.3 ± 0.1 equiv./mole of HC3 and C3T mutants (Fig. 2A). The released Zn2+ (0.73-0.82 mol of zinc/mol of protein) was determined spectrophotometrically with PAR (Fig. 2B). Atomic absorption spectrophotometry confirmed that the quadruple and the triple Cys mutations conferred to the protein the ability to bind the metal (0.8 ± 0.1 mol of zinc/mol of protein). Less than 0.03 mol of zinc/mol of protein was found in the wild-type AKe. No iron was observed in AKeC4 when E. coli was cultivated in minimal medium supplemented with this metal (data not shown).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Titration of the mutated forms of AKe with PMPS. A, AKe (25 nmol in 0.6 ml of 10 mM Tris-HCl, pH 8.0) was treated with PMPS (1 mM solution in the same buffer) to give the indicated molar ratios of PMPS to enzyme. The absorbance at 250 nm was measured relative to the control value at the beginning of the titration. B, titration of each variant (3.5 µM in the same buffer) was also performed in the presence of PAR, where the absorbance developed at 500 nm reflects formation of a zinc-dye complex (epsilon m = 6.6 × 104). bullet , AKeC4; black-triangle, AKeHC3; and open circle , AKeC3T variants.

Reaction of Wild-type AKe and of Cysteine-substituted Enzymes with DTNB-- Wild-type AKe contains a buried cysteine residue in position 77. It reacted with DTNB only in the presence of urea over 2 M (15). The same was true for AK from Bacillus subtilis and AK from Bacillus stearothermophilus, although they contain, besides the conserved Cys77, 3 and, respectively, 4 other Cys residues in the LID domain (5, 7). It was, therefore, surprising to find that Zn2+-chelating AKe variants reacted with DTNB under native conditions (Fig. 3). The kinetics of the reaction with DTNB of these mutants was fitted to a single exponential equation. Over 0.5 mM DTNB, the values of kobs (5.10-3 s-1 for AKeC4, 7.10-3 s-1 for AKeHC3 and 14.10-3 s-1 for AKeC3T) were practically independent on the concentration of thiol reagent. Thus, the first order process might reflect the dissociation rate of the AKe-Zn2+ complex.
<UP>AK-Zn<SUP>2+</SUP> ⇋ Zn<SUP>2+</SUP></UP>+<UP>AK</UP><SUB>e</SUB> <LIM><OP><ARROW>→</ARROW></OP><UL><UP>DTNB</UP></UL></LIM><UP> AK*</UP> (Eq. 1)
As preincubation of AKeC4 with ZnCl2 did not affect the kobs (data not shown), one might assume that difference in affinity for metal is primarily due to the dissociation rate constant for protein-metal complex.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Reaction of AKe with DTNB under native conditions. AKe (in 10 mM Tris-HCl, pH 8.0) was treated with 20 µl of 10 mM DTNB, then the absorbance increase was read at 412 nm. The ratio of thiols reacted to moles of AKe was calculated using a molecular mass of 23.5 kDa. Symbols are the same as those used in Fig. 2, to which wild-type AKe (diamond ) and AKsub (down-triangle) were added.

The reactivity toward DTNB of the double substituted species of AKe was relevant for the structural changes into the LID domain of the bacterial enzyme. Thus, the HSC2 variant reacted with DTNB under denaturing conditions. This means that Cys146 and Cys149 in this mutant are not exposed to the solvent and do not form a disulfide bridge as might be expected. On the other hand, the C2DT variant easily forms an intramolecular disulfide bridge, as suggested by its lack of reactivity toward DTNB both under nondenaturing and denaturing conditions. An even more complicated behavior was found with CSDC and HC2T forms of AKe, both of which were inactivated during the purification. Under nondenaturing conditions, 1.3-1.6 mol of SH/mol of enzymes were titrated with DTNB, indicating that the extra thiol groups were free and accessible. The observed inactivation of these species of AKe is likely due to structural deformation of the LID domain, which propagates to the CORE of the molecule.

Thermal Stability and Proteolysis by Trypsin of Cys-modified Mutants of AKe-- In preliminary experiments, different proteins were heated for 10 min at various temperatures between 40 and 80 °C, after which the residual enzyme activity was determined. The wild-type AKe and the C3T, CSDT, HCDT, and HSDC mutants were half-inactivated at temperature between 51 and 54 °C; the C4 and HC3 variants exhibited a higher thermal stability (half-inactivation at 65 and 58 °C, respectively) than the wild-type AKe, whereas the C2DT mutant was less resistant (half-inactivation at 46 °C).

The thermal stability of the C4 and HC3 variants was further examined by microcalorimetry. The excess heat capacity curve for the wild-type AKe, C4, and HC3 mutants is shown in Fig. 4. The Tm values (63 and 55.7 °C, respectively, instead of 51.8 °C for the wild-type enzyme) were reproducible within 0.1 °C. Inspection of Fig. 4 suggests that, at least under the conditions of the calorimetric experiments, the cooperativity of the denaturation process decreases significantly in the case of C4 mutant. A detailed analysis of structural and energetic properties of this variant is described in a companion study (20).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Differential scanning calorimetric traces for wild-type AKe (A), HC3 (B) and C4 (C) mutants. The curves were obtained by smoothing raw calorimetric data and substracting from them the base lines using a cubic interpolation procedure.

Limited proteolysis was used as a test of conformational changes in AKe induced by various amino acid substitutions. Inactivation of the bacterial enzyme by TPCK-trypsin followed first order kinetics (7). The triple and quadruple Cys mutants of AKe showed similar or slightly higher resistance against trypsin digestion (t1/2 between 26 and 40 min) as compared with the wild-type enzyme. The other modified variants of AKe, except the HCDT mutant, exhibited a much lower resistance to proteolysis (t1/2 < 3 min). Sequence analysis of the proteolytic fragments indicated that 131R---V132 and 141K---F142 bonds located into the LID domain became sensible to the attack by trypsin; the 14-kDa fragment accumulated upon proteolysis corresponds to the segment 1-131 of the molecule (Fig. 5).


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 5.   Proteolysis by TPCK-trypsin of the wild-type and of the mutated forms of AKe. AKe at 1 mg/mL in 50 mM Tris-HCl, pH 7.4, was incubated at 30 °C with TPCK-trypsin (2 µg/mL). At different time intervals, 10-µl aliquots were withdrawn, boiled with electrophoresis buffer, and analyzed by SDS-polyacrylamide gel electrophoresis (12.5% gel) and Coomassie Blue staining. Lanes 1 and 2, wild-type AKe (0 and 25 min); lanes 3 and 4, HCDT mutant (0 and 25 min); lanes 5 and 6, C4 mutant (0 and 25 min); and lanes 7 and 8, CSDT mutant (0 and 3 min). Lane 9, standard proteins, from top to bottom, phosphorylase a (94,000), bovine serum albumin (66,200), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (21,100), and lysozyme (14,400).

Catalytic Properties of Cys-substituted AKe-- Table II shows the kinetic parameters of wild-type AKe, compared with two zinc-containing variants. The Km for nucleotide substrates was similar for the three variants of bacterial enzyme, and excess of AMP (above 0.3 mM) inhibited the activity of all forms at a similar extent. It should be mentioned that removal of metal ion did not affect the phosphorylating activity of apoAKeC4 or apoAKeHC3, confirming that zinc does not participate in the kinase activity.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Comparative kinetic parameters of wild-type (WT) AKe, AKeC4, and AKeHC3
Km (ADP) and Vmax (ADP) were determined from plots of 1/v versus 1/ADP2, which assumes that the two molecules of ADP bind to the enzyme with the same affinity. The apparent Km for AMP and for ATP was determined at a single fixed concentration of cosubstrates (1 mM ATP and 0.2 mM AMP). The Vmax (ATP, AMP) was obtained by extrapolating the reaction rates for infinite concentrations of ATP and AMP and assuming that the concentration of one nucleotide substrate does not affect the apparent Km for the second nucleotide substrate.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Zinc in proteins is responsible for a wide range of functions (21-24). The design of zinc binding sites generates proteins with new interesting properties (25-28). The fact that zinc is a structural component of AKs from Gram-positive bacteria (6) prompted us to create a similar metal site in the enzyme from Gram-negative species. As shown here, the presence of three or four cysteine residues in the consensus sequence 126Cys-X2-Cys-X16-Cys-X2-Cys149 led to a zinc binding site in E. coli AK. Moreover, a significant increase in thermostability of the C4 variant as compared with the wild-type AKe was observed.

The crystal structure of AKe shows that the LID domain forms a single distorted antiparallel beta -sheet, two turns and one loop structure (29). The beta -sheet is stabilized by hydrogen backbone interactions and attractive forces between few side chains inside the beta -sheet. The four amino acids (His126, Ser129, Asp146, and Thr149) replaced by cysteine residues in the mutagenized protein belong to this hydrogen binding network (Fig. 6). Three additional amino acid side chains (Arg131, Glu151, and Tyr133) stabilize the network of hydrogen bonds by connecting the beta -sheet segments in a sandwich-like structure. In the LID domain of adenylate kinase from Gram-positive bacteria, Zn2+ which is held by cysteine residues seems to substitute efficiently the hydrogen bond network (30). The crystal structure of adenylate kinase from B. stearothermophilus (entry 1.Zip in the Protein Data Bank) confirms this observation. Metal chelation not only preserves the above mentioned network but also enhanced the thermal stability of the protein. As the catalytic properties of the C4 and C3 containing variants of AKe are conserved, the zinc-chelating LID domain of the protein conserves also intact the ability to rotate and to move like a solid block on the ATP-binding pocket. In other words, the overall conformation of this domain remains intact, in agreement with circular dichroism and NMR structural analysis (20).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6.   Amino acid side chains forming the hydrogen bonding network into the LID domain of AKe. Gray ovals are the positions for the cysteine replacement.

The biochemical characteristics of the others variants of AKe might be also viewed in the light of hydrogen bond network located into the LID domain. The double mutants with vicinal thiols (C2DT and HSC2) conserve over 65% of the activity of the wild-type enzyme. On the contrary the two mutants, where each Cys residue is located on one side of the sandwich-like structure (HC2T and CSDC), are greatly affected in their activity. The loss of activity was independent on disulfide bridge formation as in the latter cases the SH groups are free.

Among the Cys-monosubstituted variants, the most conservative substitution concerns Ser129. The S129C mutant exhibited similar structural and catalytic properties with the wild-type enzyme and with another AKe mutant (S129F) previously described by Haase et al. (31). This last mutation, however, is conditionally lethal, and the bacteria do not survive at 42 °C. It was concluded that AKe might be involved in other essential cellular functions, independent of phosphotranferase activity, such as phospholipid synthesis. This attractive hypothesis still awaits for experimental proofs. All other single Cys variants (except HCDT form) of the AKe, although active and with similar thermal stability as the wild-type enzyme, exhibited a considerably lower resistance against trypsin digestion. In other words, despite the fact that the single amino acid substitutions were "conservative" in terms of hydrogen bond formation, some subtle conformational changes into the LID domain occur, yielding proteins with higher susceptibility to proteolytic digestion.

In conclusion, this study highlighted the importance of some key residues into the LID domain of the AKe. Quadruple and triple Cys mutations stabilized the protein by chelation with zinc. Double mutants are the most exposed to conformational changes leading to inactivation, irrespective of the presence or absence of disulfide bridges. All single Cys mutants are active but only one (HCDT) conserves the stability of the wild-type protein.

    ACKNOWLEDGEMENTS

We thank O. Bârzu, C. T. Craescu, H. Sakamoto, and C. Schulz for fruitful suggestions and comments, L. Serina for help in single strand DNA preparation, and M. Ferrand for excellent secretarial assistance.

    FOOTNOTES

* This work was supported by grants from the Centre National de la Recherche Scientifique (URA D1129), Institut Pasteur, and Institut National de la Santé et de la Recherche Médicale (U 350).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel.: 33 1 45 68 89 68; Fax: 33 1 45 68 84 05; E-mail: amgilles{at}pasteur.fr.

1 The abbreviations used are: AK, adenylate kinase; AK1, muscle cytosolic adenylate kinase; AK2 and AK3, mitochondrial adenylate kinases; AKe, E. coli adenylate kinase; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); PAR, 4-(2-pyridylazo)resorcinol; PMPS, p-(hydroxymercuri)phenylsulfonate; TPCK, L-1-tosylamino-2-phenylethyl chloromethyl ketone; LID, domain closing over ATP of AKe (residues 122-159).

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Noda, L. H. (1973) in The Enzymes (Boyer, P. D., ed), 3rd Ed., Vol. 8, pp. 279-305, Academic Press, New York
  2. Schulz, G. E., Schiltz, E., Tomasselli, A. G., Franck, R., Brune, M., Wittinghofer, A., and Schirmer, R. H. (1986) Eur. J. Biochem. 161, 127-132[Abstract]
  3. Schulz, G. E., Müller, C. W., and Diederichs, K. (1990) J. Mol. Biol. 213, 627-630[Medline] [Order article via Infotrieve]
  4. Vonrhein, C., Schlauderer, G. J., and Schulz, G. E. (1995) Structure 3, 483-490[Medline] [Order article via Infotrieve]
  5. Glaser, P., Presecan, E., Delepierre, M., Surewicz, W. K., Mantsch, H. H., Bârzu, O., and Gilles, A.-M. (1992) Biochemistry 31, 3038-3043[Medline] [Order article via Infotrieve]
  6. Gilles, A.-M., Glaser, P., Perrier, V., Meier, A., Longin, R., Sebald, M., Maignan, L., Pistotnik, E., and Bârzu, O. (1994) J. Bacteriol. 176, 520-523[Abstract]
  7. Perrier, V., Surewicz, W. K., Glaser, P., Martineau, L., Craescu, C. T., Fabian, H., Mantsch, H. H., Bârzu, O., and Gilles, A.-M. (1994) Biochemistry 33, 9960-9967[Medline] [Order article via Infotrieve]
  8. Deligiannakis, Y., Boussac, A., Bottin, H., Perrier, V., Bârzu, O., and Gilles, A.-M. (1997) Biochemistry 36, 9446-9452[CrossRef][Medline] [Order article via Infotrieve]
  9. Raleigh, E. A., Lech, K., and Brent, R. (1989) Current Protocols in Molecular Biology, Wiley Interscience, New York, Unit 1.4
  10. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89[Medline] [Order article via Infotrieve]
  11. Reinstein, J., Brune, M., and Wittinghofer, A. (1988) Biochemistry 27, 4712-4720[Medline] [Order article via Infotrieve]
  12. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382[Medline] [Order article via Infotrieve]
  13. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  14. Bârzu, O., and Michelson, S. (1983) FEBS Lett. 153, 280-284[CrossRef][Medline] [Order article via Infotrieve]
  15. Saint Girons, I., Gilles, A.-M., Margarita, D., Michelson, S., Monnot, M., Fermandjian, S., Danchin, A., and Bârzu, O. (1987) J. Biol. Chem. 262, 622-629[Abstract/Free Full Text]
  16. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  17. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  18. Hunt, J. B., Neece, S. H., Schachman, H. K., and Ginsberg, A. (1984) J. Biol. Chem. 259, 14793-14803[Abstract/Free Full Text]
  19. Giedroc, D. P., Keating, K. M., Williams, K. R., Konigsberg, W. H., and Coleman, J. E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8452-8456[Abstract]
  20. Burlacu-Miron, S., Perrier, V., Gilles, A.-M., Pistotnik, E., and Craescu, C. T. (1998) J. Biol. Chem. 273, 19102-19107[Abstract/Free Full Text]
  21. Vallee, B. L., and Auld, D. S. (1990) Biochemistry 29, 5647-5659[Medline] [Order article via Infotrieve]
  22. Vallee, B. L., and Auld, D. S. (1993) Biochemistry 32, 6493-6500[Medline] [Order article via Infotrieve]
  23. Vallee, B. L., and Auld, D. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2715-2718[Abstract]
  24. Berg, J. M., and Shi, Y. (1996) Science 271, 1081-1085[Abstract]
  25. Regan, L., and Clarke, N. D. (1990) Biochemistry 29, 10878-10883[Medline] [Order article via Infotrieve]
  26. Regan, L. (1993) Annu. Rev. Biophys. Biomol. Struct. 1, 257-287
  27. Schwabe, J. W., and Klug, A. (1994) Nat. Struct. Biol. 1, 345-349[Medline] [Order article via Infotrieve]
  28. Klemba, M., Gardner, K. H., Marino, S., Clarke, N. D., and Regan, L. (1995) Nat. Struct. Biol. 2, 368-373[Medline] [Order article via Infotrieve]
  29. Müller, C. W., Schlauderer, G. J., Reinstein, J., and Schulz, G. E. (1996) Structure 4, 147-156[Medline] [Order article via Infotrieve]
  30. Schlauderer, G. J., and Schulz, G. E. (1996) Protein Sci. 5, 434-441[Abstract/Free Full Text]
  31. Haase, G. H. W., Brune, M., Reinstein, J., Pai, E. F., Pingoud, A., and Wittinghofer, A. (1989) J. Mol. Biol. 207, 151-162[Medline] [Order article via Infotrieve]
  32. Müller, C. W., and Schulz, G. E. (1992) J. Mol. Biol. 224, 159-177[Medline] [Order article via Infotrieve]
  33. Kraulis, P. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
  34. Koivula, T., and Hemilä, H. (1991) J. Gen. Microbiol. 137, 2595-2600[Medline] [Order article via Infotrieve]
  35. Nakamura, K., Nakamura, A., Takamatsu, H., Yoshikawa, H., and Yamane, K. (1990) J. Biochem. (Tokyo) 107, 603-607[Abstract]
  36. Himmelreich, R., Hilbert, H., Plagens, H., Pirkl, E., Li, B.-C., and Herrman, R. (1996) Nucleic Acids Res. 24, 4420-4449[Abstract/Free Full Text]
  37. Brune, M., Schumann, R., and Wittinghofer, A. (1885) Nucleic Acids Res. 13, 7139-7151[Abstract]
  38. Gilles, A.-M., Sismeiro, O., Munier, H., Fabian, H., Mantsch, H. H., Surewicz, W. K., Craescu, C. T., Bârzu, O., and Danchin, A. (1993) Eur. J. Biochem. 218, 921-927[Abstract]
  39. Maskell, D. J., Szabo, M. J., Butler, P. D., Williams, A. E., and Moxom, E. R. (1991) Mol. Microbiol. 5, 1013-1022[Medline] [Order article via Infotrieve]
  40. Tomasselli, A. G., Frank, R., and Schultz, E. (1986) FEBS Lett. 202, 303-308[CrossRef][Medline] [Order article via Infotrieve]
  41. Konrad, M. (1993) J. Biol. Chem. 268, 11326-11334[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.