Circular Permutation Analysis as a Method for Distinction of Functional Elements in the M20 Loop of Escherichia coliDihydrofolate Reductase
1999; Elsevier BV; Volume: 274; Issue: 27 Linguagem: Inglês
10.1074/jbc.274.27.19041
ISSN1083-351X
AutoresT. Nakamura, Masahiro Iwakura,
Tópico(s)Protein Structure and Dynamics
ResumoA functional element of an enzyme can be defined as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. In order to elucidate the distribution of functional elements in an active site flexible loop (the M20 loop) of Escherichia colidihydrofolate reductase, systematic cleavage of main chain connectivity was performed using circular permutation. Our analysis is based on the assumption that a permutation within a functional element would significantly reduce enzyme function, whereas ones outside or at the boundaries of the elements would affect the function only slightly. Thus, a functional element would be assigned as the minimum peptide chain between the identified boundaries. Comparison of the activities of the circularly permuted variants revealed that the peptide chain around the M20 loop could be divided into four regions (regions 1–4). Region 1 was found to play an important role in overall tertiary fold because most variants permuted at region 1 did not accumulate inE. coli cells stably. A distinction between region 2 and region 3 was in agreement with the extent of movements calculated from the coordinates of α carbons, supporting the idea that the movement of peptide backbone is a key feature of enzyme function. The boundary between region 3 and region 4 coincided with that between the M20 loop and the following α helix. From equilibrium binding studies, region 2 was found to be involved in the binding of nicotinamide substrates, whereas region 4 appeared to be very important for the binding of pterin substrates. A functional element of an enzyme can be defined as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. In order to elucidate the distribution of functional elements in an active site flexible loop (the M20 loop) of Escherichia colidihydrofolate reductase, systematic cleavage of main chain connectivity was performed using circular permutation. Our analysis is based on the assumption that a permutation within a functional element would significantly reduce enzyme function, whereas ones outside or at the boundaries of the elements would affect the function only slightly. Thus, a functional element would be assigned as the minimum peptide chain between the identified boundaries. Comparison of the activities of the circularly permuted variants revealed that the peptide chain around the M20 loop could be divided into four regions (regions 1–4). Region 1 was found to play an important role in overall tertiary fold because most variants permuted at region 1 did not accumulate inE. coli cells stably. A distinction between region 2 and region 3 was in agreement with the extent of movements calculated from the coordinates of α carbons, supporting the idea that the movement of peptide backbone is a key feature of enzyme function. The boundary between region 3 and region 4 coincided with that between the M20 loop and the following α helix. From equilibrium binding studies, region 2 was found to be involved in the binding of nicotinamide substrates, whereas region 4 appeared to be very important for the binding of pterin substrates. circularly permuted dihydrofolate DHF reductase tetrahydrofolate trimethoprim The active site of an enzyme contains amino acid residues involved in enzyme function. Some residues in the active site bind to the substrate or cofactor, and others are involved in the catalysis itself. In addition, residues away from the active site sometimes promote the catalytic reaction through intramolecular interactions (1Miller G.P. Benkovic S.J. Biochemistry. 1998; 37: 6327-6335Crossref PubMed Scopus (65) Google Scholar, 2Miller G.P. Benkovic S.J. Biochemistry. 1998; 37: 6336-6342Crossref PubMed Scopus (60) Google Scholar). Enzyme-catalyzed reactions proceed, in general, through multiple steps, including binding of the substrate, catalysis, and release of product. At each step, certain amino acid residues play critical roles. However, an isolated collection of these directly functioning residues is not sufficient to obtain catalytic activity. Amino acid residues should be connected covalently to make up a polypeptide chain with a specific amino acid sequence that determines a proper tertiary structure. The functioning residues on the peptide backbone are then arranged to give the proper configuration for effective catalytic activity. Because enzymes cleaved at certain sites have been demonstrated to show catalytic activity as high as that of uncleaved enzyme (3Luger K. Hommel U. Herold M. Hofsteenge J. Kirschner K. Science. 1989; 243: 206-210Crossref PubMed Scopus (193) Google Scholar, 4Buchwalder A. Szadkowski H. Kirschner K. Biochemistry. 1992; 31: 1621-1630Crossref PubMed Scopus (75) Google Scholar, 5Protasova N.Y. Kireeva M.L. Murzina N.V. Murzin A.G. Uverski V.N. Gryaznova O.I. Gudkov A.T. Protein Eng. 1994; 7: 1373-1377Crossref PubMed Scopus (44) Google Scholar, 6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar, 7Yang Y.R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11980-11984Crossref PubMed Scopus (48) Google Scholar, 8Graf R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11591-11596Crossref PubMed Scopus (95) Google Scholar, 9Horlick R.A. George H.J. Cooke G.M. Tritch R.J. Newton R.C. Dwivedi A. Lischwe M. Salemme F.R. Weber P.C. Horuk R. Protein Eng. 1992; 5: 427-431Crossref PubMed Scopus (32) Google Scholar, 10Zhang T. Bertelsen E. Benvegnu D. Alber T. Biochemistry. 1993; 32: 12311-12318Crossref PubMed Scopus (83) Google Scholar, 11Hahn M. Piotukh K. Borriss P. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10417Crossref PubMed Scopus (78) Google Scholar, 12Mullins L.S. Wesseling K. Kuo J.M. Garrett J.B. Raushel F.M. J. Amer. Chem. Soc. 1994; 116: 5529-5533Crossref Scopus (19) Google Scholar, 13Viguera A.R. Blanco F.J. Serrano L. J. Mol. Biol. 1995; 247: 670-681Crossref PubMed Scopus (128) Google Scholar, 14Kreitman R.J. Puri R.K. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6889-6893Crossref PubMed Scopus (108) Google Scholar, 15Vignais M.L. Corbier C. Mulliert G. Branlant C. Branlant G. Protein Sci. 1995; 4: 994-1000Crossref PubMed Scopus (18) Google Scholar, 16Kommar A.A. Jaenicke R. FEBS Lett. 1995; 376: 195-198Crossref PubMed Scopus (41) Google Scholar), chain connectivity must not be absolutely required for catalytic activity. This means that chain connectivity is crucial for catalytic activity in some regions, but in others, it is not. The detailed configuration of the functioning residues seems to depend on local peptide backbone rather than the overall polypeptide chain. In this paper, we define a "functional element" as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. Such functional elements would be distributed across the primary structure. When the enzyme is properly folded, these elements would associate to make a functional active site (Fig. 1). Identifying such functional elements would be very informative in revealing the architecture of enzyme function. Here, we propose a novel approach for identifying the functional elements of an enzyme. Based on our definition of a functional element, alteration of main chain connectivity (peptide bond cleavage) seems to be a useful approach for locating functional elements. Possible consequences of peptide bond cleavage are illustrated in Fig. 1. If the main chain is cleaved within a region that is indispensable for protein folding (designated as the folding element), the resulting protein will not be able to fold to a stable conformation (case 1). In the other cases, the properties of the cleaved protein would vary depending on the location of the cleavage site. Cleavage within a functional element (case 2) will break the local configuration, leading to loss of the function contributed by the element, whereas those outside (case 3) or at the boundary of (case 4) the functional elements will not, resulting in a minor effect on the enzyme function. Even if functional elements are located side by side, distribution of the functional elements can be recognized by the presence of the boundaries (compare case 2 and case 4). Based on this idea, systematic peptide bond cleavage and characterization of the products are crucial to locating functional elements. The simplest way to cleave a peptide bond is to break the polypeptide chain into two fragments by manipulation of the coding gene. However, this dissection method is poorly suited for the purpose of obtaining a variety of cleaved proteins without disrupting the overall tertiary fold, because production of properly folded protein requires the association of the fragmentary chains by long-range interaction. Circular permutation analysis, in which the original N and C termini of a protein are connected by an appropriate linker and new termini are created at a position of interest (Fig.1), can overcome such entropic problems because it allows a peptide bond to be broken without fragmenting the protein into two pieces. To date, a number of circularly permuted proteins with various folded structures have been reported (3Luger K. Hommel U. Herold M. Hofsteenge J. Kirschner K. Science. 1989; 243: 206-210Crossref PubMed Scopus (193) Google Scholar, 4Buchwalder A. Szadkowski H. Kirschner K. Biochemistry. 1992; 31: 1621-1630Crossref PubMed Scopus (75) Google Scholar, 5Protasova N.Y. Kireeva M.L. Murzina N.V. Murzin A.G. Uverski V.N. Gryaznova O.I. Gudkov A.T. Protein Eng. 1994; 7: 1373-1377Crossref PubMed Scopus (44) Google Scholar, 6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar, 7Yang Y.R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11980-11984Crossref PubMed Scopus (48) Google Scholar, 8Graf R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11591-11596Crossref PubMed Scopus (95) Google Scholar, 9Horlick R.A. George H.J. Cooke G.M. Tritch R.J. Newton R.C. Dwivedi A. Lischwe M. Salemme F.R. Weber P.C. Horuk R. Protein Eng. 1992; 5: 427-431Crossref PubMed Scopus (32) Google Scholar, 10Zhang T. Bertelsen E. Benvegnu D. Alber T. Biochemistry. 1993; 32: 12311-12318Crossref PubMed Scopus (83) Google Scholar, 11Hahn M. Piotukh K. Borriss P. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10417Crossref PubMed Scopus (78) Google Scholar, 12Mullins L.S. Wesseling K. Kuo J.M. Garrett J.B. Raushel F.M. J. Amer. Chem. Soc. 1994; 116: 5529-5533Crossref Scopus (19) Google Scholar, 13Viguera A.R. Blanco F.J. Serrano L. J. Mol. Biol. 1995; 247: 670-681Crossref PubMed Scopus (128) Google Scholar, 14Kreitman R.J. Puri R.K. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6889-6893Crossref PubMed Scopus (108) Google Scholar, 15Vignais M.L. Corbier C. Mulliert G. Branlant C. Branlant G. Protein Sci. 1995; 4: 994-1000Crossref PubMed Scopus (18) Google Scholar, 16Kommar A.A. Jaenicke R. FEBS Lett. 1995; 376: 195-198Crossref PubMed Scopus (41) Google Scholar). Construction of circularly permuted (CP)1 variants has revealed that the N and C termini of proteins can be moved to alternative positions without lethal damage and that the order of peptide synthesis is not critical for the final tertiary structure of a protein. In most cases, newly created termini were directed to sites such as interdomain hinges or surface loops. On the other hand, random circular permutation was applied to the catalytic chains of aspartate transcarbamoylase to investigate possible rules for inserting termini in various regions of the three-dimensional structures of protein (8Graf R. Schachman H.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11591-11596Crossref PubMed Scopus (95) Google Scholar). In the same way that alanine scanning has been used to elucidate the role of side chains (17Cunningham B.C. Wells J.A. Science. 1989; 244: 1081-1085Crossref PubMed Scopus (1114) Google Scholar), systematic circular permutation analysis will be useful for estimating the role of main chain connectivity. We chose an active site flexible loop of Escherichia colidihydrofolate reductase (DHFR) (EC 1.5.1.3) as the target of an investigation of functional elements. DHFR catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) and plays an important role in supplying the cofactor for one-carbon transfer reactions, e.g. the reaction catalyzed by thymidylate synthase (18Blakley R.L. Blakley R.L. Benkovic S.J. Dihydrofolate Reductase in Folates and Pteridines. 1. Wiley, New York1984: 191-253Google Scholar). The flexible loop connecting β strand A and α helix B of E. coli DHFR (Ala-9∼Leu-24) has been called loop I (19Volz K.W. Matthews D.A. Alden R.A. Freer S.T. Hansch C. Kaufman B.T. Kraut J. J. Biol. Chem. 1982; 257: 2528-2536Abstract Full Text PDF PubMed Google Scholar) or the M20 loop (20Bystroff C. Oatley S.J. Kraut J. Biochemistry. 1990; 29: 3263-3277Crossref PubMed Scopus (269) Google Scholar) (Fig.2). This loop has attracted much attention because of its variable conformations in crystal structures (20Bystroff C. Oatley S.J. Kraut J. Biochemistry. 1990; 29: 3263-3277Crossref PubMed Scopus (269) Google Scholar, 21Bystroff C. Kraut J. Biochemistry. 1991; 30: 2227-2239Crossref PubMed Scopus (205) Google Scholar). Sawaya and Kraut (22Sawaya M.R. Kraut J. Biochemistry. 1997; 36: 586-603Crossref PubMed Scopus (615) Google Scholar) summarized the tertiary structures of the enzymes in various ligation states and provided snapshots of the enzymes at each step of the catalytic cycle. They observed that a conformational oscillation of the M20 loop occurred as part of the catalytic cycle. The catalytic role played by the M20 loop was examined by the use of a deletion mutant in which the hairpin-forming residues (Met-16∼Ala-19) had been replaced by a single glycine (23Li L. Falzone C.J. Write P.E. Benkovic S.J. Biochemistry. 1992; 31: 7826-7833Crossref PubMed Scopus (101) Google Scholar). The results suggested that those residues contributed to the acceleration of hydride transfer reaction without significantly affecting the binding of DHF and NADPH and release of THF. NMR studies indicate that the M20 loop oscillates at a frequency similar tok cat, which is limited by the release of the product, THF (24Falzone C.J. Wright P.E. Benkovic S.J. Biochemistry. 1994; 33: 439-442Crossref PubMed Scopus (147) Google Scholar, 25Fierke C.A. Johnson K.A. Benkovic S.J. Biochemistry. 1987; 26: 4085-4092Crossref PubMed Scopus (476) Google Scholar). The reaction catalyzed by E. coliDHFR proceeds through binding of DHF to the holoenzyme followed by proton and hydride transfer reaction, release of NADP+, binding of NADPH, and release of THF. Thus, the M20 loop is likely to play multiple roles. This conclusion is also supported by evidence from the deletion mutant indicating that the M20 loop seems to contribute to the hydride transfer reaction (23Li L. Falzone C.J. Write P.E. Benkovic S.J. Biochemistry. 1992; 31: 7826-7833Crossref PubMed Scopus (101) Google Scholar) and that the movement of the loop may be a limiting factor in substrate turnover (24Falzone C.J. Wright P.E. Benkovic S.J. Biochemistry. 1994; 33: 439-442Crossref PubMed Scopus (147) Google Scholar). In order to elucidate the role of the M20 loop in detail, it would be necessary to understand the flexible loop as an assembly of independent regions. Therefore, the idea of functional elements will be greatly helpful. To this end, systematic circular permutation analysis was performed to investigate whether the M20 loop could be further divided into multiple functional elements. Circularly permuted forms of DHFR have been reported for the enzymes of mouse (4Buchwalder A. Szadkowski H. Kirschner K. Biochemistry. 1992; 31: 1621-1630Crossref PubMed Scopus (75) Google Scholar) and E. coli (5Protasova N.Y. Kireeva M.L. Murzina N.V. Murzin A.G. Uverski V.N. Gryaznova O.I. Gudkov A.T. Protein Eng. 1994; 7: 1373-1377Crossref PubMed Scopus (44) Google Scholar). In designing the peptide linker connecting the original N and C termini ofE. coli DHFR, we have shown that the five-glycine linker is the most favorable for maintaining the overall structure and function (6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar). The results obtained in this study suggested the presence of three boundaries, indicating that the M20 loop could be divided into multiple functional elements. E. coli JM109 was used as cloning and expression host. Methotrexate affinity resin was obtained from Sigma. DEAE-Toyopearl 650 m was purchased from Tosoh Co. (Tokyo, Japan). Restriction enzymes, T4 DNA ligase, and Taqpolymerase were obtained from Takara Shuzo Co. (Kyoto, Japan). All primer DNAs for mutagenesis were synthesized by JbioS Ltd. (Saitama, Japan). All other chemicals were of reagent grade. Construction of the circularly permuted genes was carried out as described previously (6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar). Coding sequences of circularly permuted proteins were amplified with polymerase chain reaction on a tethered dimer gene. Then, an overexpression promoter, a ribosome-binding site, and BamHI sites were attached to the coding sequence. Resulting genes were inserted into BamHI site of a high copy vector, pUC19, and sequenced with the dideoxy method to confirm the construction. E. coli JM109 cells were transformed with the expression plasmid and trimethoprim (TMP) resistance was tested on the agar plate as described previously (6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar). CP variants giving TMP resistance were further purified. Purification of the CP variants was carried out mainly by methotrexate affinity chromatography, taking advantage of adequate prepurification steps from cell-free extracts (26Iwakura M. Furusawa K. Kokubu T. Ohashi S. Shimura Y. Tsuda K. J. Biochem. 1992; 111: 37-45Crossref PubMed Scopus (36) Google Scholar). Protein concentration was determined by the absorbance at 280 nm using the extinction coefficient (ε280 = 31,100m−1·cm−1 (27Touchette N.A. Perry K.P. Matthews C.R. Biochemistry. 1986; 25: 5445-5452Crossref PubMed Scopus (128) Google Scholar)) for wild-type and circularly permuted DHFR. The activity of DHFR was determined spectrophotometrically at 15 °C by following the disappearance of NADPH and DHF at 340 nm (ε340 = 11,800m−1·cm−1 (28Stone S.R. Morrison J.F. Biochemistry. 1982; 21: 3757-3765Crossref PubMed Scopus (91) Google Scholar)). The standard assay mixture contained 50 μm DHF, 100 μmNADPH, 14 mm β-mercaptoethanol, MTEN buffer (50 mm 2-morpholinoethanesulfonic acid, 25 mmtris(hydroxymethyl)aminomethane, 25 mm ethanolamine, and 100 mm NaCl, pH 7.0 (29Morrison J.F. Stone S.R. Biochemistry. 1988; 27: 5499-5506Crossref PubMed Scopus (86) Google Scholar)), and the enzyme in a final volume of 2.0 ml. The reaction was started by the addition of DHF. Circular dichroism spectra were obtained by scanning from 250 to 190 nm with 15 or 20 s averaging times on an Aviv 62DS spectrometer using either a 1- or a 2-mm cuvette. Protein concentrations were 0.05–0.2 mg/ml. The raw data were converted to mean residue ellipticity (MRE) by the equation MRE = (Θ × 100)/(C × D × NA), where Θ is the ellipticity in degrees, C is the molar protein concentration, D is the path length in cm, and NAis the number of residues in the protein. Urea-induced unfolding of proteins was studied by monitoring ellipticity at 222 nm. All samples were dialyzed against 10 mm potassium phosphate, pH 7.8, 0.2 mm EDTA, and 1 mm β-mercaptoethanol. The proteins were diluted to varying final urea concentrations in the above buffer and incubated at least 12 h prior to the data collection. Final protein concentrations ranged from 0.1 to 0.5 mg/ml. Equilibrium unfolding data obtained from the ellipticity at 222 nm were analyzed based on a two-state model as described previously (30Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Crossref PubMed Scopus (1622) Google Scholar, 31Iwakura M. Jones B.E. Luo J. Matthews C.R. J. Biochem. 1995; 117: 480-488Crossref PubMed Scopus (66) Google Scholar). Coordinates of α carbons were obtained from Protein Data Base codes 1rx1, 1rx4, and1rb3. Conformations of the M20 loop in these structures are closed, occluded, and open, respectively. Calculations were carried out using the AMBER computer program (Oxford Molecular). Mutant genes with single alanine substitutions in the region from Leu-8 to Asp-27 (excluding Ala-9, Ala-19, and Ala-26), were constructed and expressed in E. coli cells (alanine scanning) according to a similar method described previously (32Ohmae E. Ishimura K. Iwakura M. Gekko K. J. Biochem. 1998; 123: 839-846Crossref PubMed Scopus (23) Google Scholar). For Ala-9, Ala-19, and Ala-26 sites, glycine scanning was performed. All the mutant proteins were overexpressed and were found to accumulate stably in E. coli cells. The relative activity of the mutant proteins were estimated as follows: each E. coli transformant was grown in 2 ml of 2× YT culture medium (10 g/l NaCl, 16 g/l tryptone, 10 g/l yeast extracts) containing 200 mg/l of ampicillin, at 37 °C, until the absorbance at 660 nm reached approximately 1.5. One ml of cell culture was harvested by centrifugation, and the cells were suspended in 0.2 ml of 10 mm potassium phosphate, pH 7.8, 0.2 mm EDTA containing 1 mg/ml lysozyme. After gently mixing for 30 min, cells were disrupted by sonication for 10 s. Cell debris was removed by centrifugation, and 10 μl of the resultant supernatant was used for the DHFR assay. To normalize the activity to the number of cells used, the measured activity (ΔA 340/min) was divided by the absorbance at 660 nm ((ΔA 340/min)/A 660). Enzyme activity relative to wild-type DHFR is defined as the ratio of (ΔA 340/min)/A 660 for the mutant protein to (ΔA 340/min)/A 660 for the wild-type DHFR. Equilibrium dissociation constants (K d) were determined from fluorescence emission spectra of enzyme-ligand complexes using an Aviv ATF105 spectrofluorometer. The solution contained 1 μm enzyme and various concentrations of ligand (DHF, THF, NADPH, or NADP+) in MTEN buffer, pH 7.0. The emission spectra were scanned from 500 to 300 nm at 15 °C, with an excitation wavelength of 290 nm. DNA sequencing was carried out using an ABI PRIZM 310 genetic analyzer. N-terminal amino acid sequence determination (Edman degradation) and mass spectrometric measurements were carried out as described previously (6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar). NADPD was prepared using alcohol dehydrogenase from Thermoanaerobium brockii (Sigma) and purified on a Mono-Q column (Amersham Pharmacia Biotech). All circularly permuted genes were created by polymerase chain reaction on a tethered dimer gene. Because a five-glycine peptide had been shown to be the most favorable linker in the circularly permuted DHFR with N terminus at Met-16 (cpM16; similar abbreviations are used for all CP variants unless noted) (6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar), this peptide linker was employed in all variants (from cpL8 to cpD27). For the construction of the circularly permuted genes (except that of cpM16), the codon for methionine (ATG) was introduced at the 5′-ends of the coding sequences for translation initiation. For cpM16, because the codon for Met-16 was also used as the initiation codon; an extra ATG initiation codon was not added. However, an extra ATG codon was needed for the gene of cpM20, because the N terminus of the purified cpM20 was Pro-21 that was resulted from the removal of Met-20 by the methionyl-aminopeptidase of E. coli (33Hirel P.H. Schmitter J.M. Dessen P. Fayat G. Blanquet S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8247-8251Crossref PubMed Scopus (676) Google Scholar). The constructed genes were cloned into a high copy plasmid, pUC19, sequenced, and used to transform E. coliJM109. The E. coli transformants harboring DHFR activity were selected for on agar plates containing TMP, a competitive inhibitor of DHFR. The transformants corresponding to from cpL8 to cpI14 were TMP-sensitive, whereas those corresponding to from cpG15 to cpD27 were TMP-resistant. The permutant proteins at the positions from Leu-8 to Ile-14 could not be obtained because of poor accumulation in the E. coli cells as observed by SDS-polyacrylamide gel electrophoresis of the crude extracts. This suggests that peptide connectivity of the region is crucial for protein folding, namely, it is a folding element. Based on these results, the peptide chain around the M20 loop could be classified into two regions separated by a boundary at Ile-14 or Gly-15. Then, permutant proteins from cpG15 to cpD27 were purified and further analyzed. N-terminal amino acid sequences and molecular masses were in agreement with the sequences of the constructed genes (Table I). According to reaction of methionyl-aminopeptidase (33Hirel P.H. Schmitter J.M. Dessen P. Fayat G. Blanquet S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8247-8251Crossref PubMed Scopus (676) Google Scholar), the N-terminal methionines of the translated polypeptides may or may not have been processed. Specifically, N-terminal methionines were observed in cpM16, cpE17, cpN18, cpM20, cpW22, cpN23, cpL24, and cpD27 but not in cpG15, cpA19, cpP21, cpP25, or cpA26.Table ISummary of the properties of the circularly permuted variantsProteinN-terminal sequenceMolecular massΔGH2OaF app (fraction unfolded), data were fitted to the following equation: Fapp = Kapp/(1 + Kapp), where Kapp = exp{−(ΔGH2O − A[urea])/RT. R, gas constant, T, temperature (K).AaF app (fraction unfolded), data were fitted to the following equation: Fapp = Kapp/(1 + Kapp), where Kapp = exp{−(ΔGH2O − A[urea])/RT. R, gas constant, T, temperature (K).Isotope effectbRatio of the activity with NADPH to that with NADPD. Activities were assayed in the standard assay mixture.CalculatedMeasuredDakcal · mol −1kcal · mol −1 · m −1Wild-typeMISLI17,99917,9976.7 ± 0.2cThe errors are 95% confidence limits.2.2 ± 0.11.2cpG15GMENA18,28418,2845.2 ± 0.31.9 ± 0.1NDdNot determined.cpM16MENAM18,28418,2835.2 ± 0.31.7 ± 0.22.8cpE17MENAM18,41618,4196.2 ± 0.32.0 ± 0.12.8cpN18MNAMP18,41618,4197.1 ± 0.32.2 ± 0.12.9cpA19AMPWN18,28418,2905.3 ± 0.41.7 ± 0.12.0cpM20MMPWN18,41618,4215.5 ± 0.41.9 ± 0.11.9cpP21PWNLP18,28418,2864.7 ± 0.21.7 ± 0.12.4cpW22MWNLP18,41618,4195.3 ± 0.21.8 ± 0.12.6cpN23MNLPA18,41618,4215.0 ± 0.21.8 ± 0.12.8cpL24MLPAD18,41618,4154.9 ± 0.31.4 ± 0.11.4cpP25PADLA18,28418,2864.4 ± 0.61.7 ± 0.23.2eAssayed with 100 μm DHF.cpA26ADLAW18,28418,2893.9 ± 0.31.5 ± 0.1NDcpD27MDLAW18,41618,4092.3 ± 1.20.2 ± 0.4NDa F app (fraction unfolded), data were fitted to the following equation: Fapp = Kapp/(1 + Kapp), where Kapp = exp{−(ΔGH2O − A[urea])/RT. R, gas constant, T, temperature (K).b Ratio of the activity with NADPH to that with NADPD. Activities were assayed in the standard assay mixture.c The errors are 95% confidence limits.d Not determined.e Assayed with 100 μm DHF. Open table in a new tab In our study, the purpose of the circular permutation is to investigate whether or not the cleavage of chain connectivity at each position breaks a functional element. Therefore, the position of the cleavage is an essential consideration in the construction of variants, but the identity of the N-terminal residue is not. Nevertheless, in order to assess the effect of the extra N-terminal residue, we constructed and characterized variants in which Met and Ala were added to the N terminus of cpM16 and cpP21, respectively. Thus, these new variants were designated as cpMM16 and cpMAP21, respectively. Measurements of the activities of cpMM16 and cpMAP21 showed that the N-terminal structure was not critical for the properties of CP variants (see below). The activity profiles of CP variants are shown in Fig.3. When the k catvalues were plotted against the position of the new termini in the original loop, a significant landscape with three peaks was observed. Because peaks were observed at cpM16, cpM20, and cpL24, cleavages of peptide bonds at Met-16, Met-20, and Leu-24 did not have a significant effect on the catalytic function. If we let these three points define the boundaries (boundaries I, II, and III; see Fig. 3), then the peptide chain around the M20 loop could be divided into four regions. Boundary I, which was recognized from the minor peak at cpM16, is close to the boundary assigned by the TMP sensitivity of the transformants. Thus, the region N-terminal to boundary I seems to involve several residues. Boundaries II and III, which were assigned based on the major peaks at cpM20 and cpL24, respectively, clearly divide the peptide chain into multiple functional elements. Thek cat value for cpMM16 and cpMAP21 were 0.4-fold and 3.0-fold of cpM16 and cpP21, respectively (data not shown). Replacement of cpM16 and cpP21 by cpMM16 and cpMAP21, respectively, did not change the characteristics of the plot significantly. Therefore, the boundaries determined from the plot of Fig. 3 are valid even though the terminal structures are not identical among the CP variants. As a result, we assigned the four regions as follows: region 1, ∼Met-16; region 2, Met-16∼Met-20; region 3, Met-20∼Leu-24; and region 4, Leu-24∼. Far UV CD spectra of CP variants are shown in Fig.4. A mutant DHFR with a five-glycine peptide at its C-terminal end, cpM1, was used as a control (6Iwakura M. Nakamura T. Protein Eng. 1998; 11: 707-713Crossref PubMed Google Scholar). The spectra of the variants from cpM16 to cpN23 were quite similar to that of cpM1. This result supports the notion that peptide bond cleavage at the positions Met-16 through As
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