©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reconstitution of Escherichia coli RNase HI from the N-fragment with High Helicity and the C-fragment with a Disordered Structure (*)

(Received for publication, April 18, 1995; and in revised form, June 21, 1995)

Eiko Kanaya Shigenori Kanaya (§)

From the Protein Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Escherichia coli RNase HI variant with the Lys Ala mutation is purified in two forms, as nicked and intact proteins. The nicked K86A protein, in which the N-fragment (Met^1-Lys) and the C-fragment (Arg-Val) remain associated, is enzymatically active. These N- and C-fragments were isolated and examined for reassociation. These peptides did not associate to form the nicked K86A protein at pH 3.0 in the absence of salt, but were associated, with a yield of 30-80%, when the pH was raised to 5.5 or when salt was added. Measurements of the CD spectra show that the alpha-helices are partially formed in the N-fragment at pH 3.0 in the absence of salt and are almost fully formed either at pH 5.5 or at pH 3.0 in the presence of 0.15 M NaCl. In contrast, the C-fragment remains almost fully disordered under these conditions. The N-fragment with this high (native-like) helicity shows the characteristics of a molten globule with respect to the content of the secondary and tertiary structures, the ability to bind a fluorescent probe (1-anilinonaphthalene-8-sulfonic acid), and the behavior on the thermal transition. These results suggest that the N-fragment contains an initial folding site, probably the alphaI-helix, and the completion of the folding in this site provides a surface that facilitates the folding of the C-fragment. This folding process may represent that of the intact RNase HI molecule.


INTRODUCTION

Escherichia coli ribonuclease HI, which endonucleolytically hydrolyzes only the RNA strand of a DNA/RNA hybrid, is composed of a single polypeptide chain with 155 amino acid residues (Kanaya and Crouch, 1983). Crystallographic studies revealed that this protein is of the alpha+beta structure, and is composed of a five-stranded beta-sheet and five alpha-helices (Katayanagi et al., 1990, 1992; Yang et al., 1990). A similar folding topology has been weakly observed in other functionally unrelated proteins, such as hexokinase and glycerol kinase, and it is therefore defined as an RNase H-like fold by Holm and Sander(1994). Recently, the crystal structures of the catalytic domain of human immunodeficiency virus type-1 integrase (Dyda et al., 1994) and the Holliday junction resolving enzyme RuvC from E. coli (Ariyoshi et al., 1994) were shown to have this RNase H-like fold. These proteins are functionally unrelated to E. coli RNase HI (EC 3.1.26.4) and have little sequence similarity. Dyda et al.(1994) and Yang and Steitz(1995) have suggested that this folding topology represents the structures of similar types of enzymes that catalyze polynucleotidyl transfers.

We have previously introduced Ala-scanning mutagenesis into the basic protrusion of E. coli RNase HI to analyze the role of this region in substrate binding (Kanaya et al., 1991). Of the 16 mutant proteins in which the amino acid residues from His to Asn, except for Ala, were individually replaced by Ala, only the K86A protein, with the Lys Ala mutation, was purified in two forms. One is the intact protein, and the other is the nicked protein. In the nicked protein, the peptide bond between Lys and Arg, which is located nearly in the middle of the protein molecule, is cleaved, but the resultant N-fragment (Met^1-Lys) and the C-fragment (Arg-Val) remain associated (Kanaya et al., 1991). It remains to be determined which protease specifically cleaves this peptide bond, and why the LysAla mutation makes this peptide bond susceptible to proteolysis. The crystal structure of E. coli RNase HI, in which the N-terminal segment (1-87) is distinguished from the C-terminal segment (88-155), is schematically shown in Fig. 1. The N-terminal segment forms the initial four beta-strands (betaA-betaD) and three alpha-helices (alphaI-alphaIII), and the C-terminal segment forms the remaining beta-strand (betaE) and two alpha-helices (alphaIV, alphaV). Studies of the dissociation and association (complementation) of the N- and C-fragments, as well as their structural analyses, will be of special interest, because these studies should provide valuable information on the folding process of E. coli RNase HI, which may represent those of proteins with the RNase H-like fold.


Figure 1: Main chain folding of E. coli RNase HI. The crystal structure of E. coli RNase HI, determined by Katayanagi et al.(1990, 1992), was drawn as a ribbon model with the program MOLSCRIPT (Kraulis, 1991). The position of the peptide bond between Lys and Arg, which is cleaved by a protease when Lys is replaced by Ala, and the side chain of Lys are indicated. The solid and shaded regions correspond to the N- and C-fragments. The alphaIII-helix and the long loop between the alphaIII- and alphaIV-helices form the basic protrusion. This crystal structure of E. coli RNase HI is deposited in the Protein Data Bank, Brookhaven National Laboratory, with the accession number 2RN2.



Generally, a folding intermediate of a protein is very unstable, because of the very rapid completion of the following folding process to the native state. Folding intermediates have been analyzed by NMR measurements of hydrogen exchange (Matouschek et al., 1990; Miranker et al., 1991, 1993; Neri et al., 1992; Jennings and Wright, 1993) or by CD (Shortle and Meeker, 1989; Griko et al., 1994). Fragmentation and complementation experiments are expected to be an alternative strategy to analyze folding intermediates of proteins. If a protein molecule were cut into a few pieces that could be reassembled to form the nicked protein, structural analyses of the individual peptide fragments would provide information on the structure of the folding intermediates of the intact protein molecules. Since it was first reported by Richards and Vithayathil (1959) for RNase A, the complementation of peptide fragments has been reported for various proteins, such as staphylococcal nuclease (Taniuchi and Anfinsen, 1971), barnase (Sancho and Fersht, 1992; Kippen et al., 1994), adenylate kinase (Girons et al., 1987), chymotrypsin inhibitor-2 (Gay and Fersht, 1994; Gay et al., 1994a, 1994b), bovine pancreatic trypsin inhibitor (Oas and Kim, 1988; Staley and Kim, 1990), E. colitrp repressor (Tasayco and Carey, 1992), and isoleucine-tRNA synthetase (Shiba and Schimmel, 1992). Such peptide fragments were created by chemical synthesis, proteolysis and chemical cleavage of the proteins, or genetic methods. The structures of various peptide fragments (Bierzynski et al., 1982; Osterhout et al., 1989; Fontana, 1990; Beals et al., 1991; Chaffotte et al., 1991; Dyson et al., 1992; Sancho et al., 1992; Waltho et al., 1993;), including a dissected protein molecule (Peng and Kim, 1994), have also been analyzed, assuming that a peptide containing an initiation site for protein folding spontaneously folds into a conformation that is similar to that of a folding intermediate of the intact protein molecule.

In this study, we present thermal, chemical, and acid denaturations of the nicked K86A protein, structural analyses of the N- and C-fragments, and reconstitution of the nicked protein from the N- and C-fragments. We propose that the initial folding site in E. coli RNase HI resides in its N-terminal half region and that the formation of an alpha-helix in this region facilitates the following folding process of the C-terminal half region.


EXPERIMENTAL PROCEDURES

Cells and Plasmids

Plasmids pJAL600 (Kanaya et al., 1993) and pK86A (Kanaya et al., 1991) for the overproduction of the wild-type E. coli RNase HI and the K86A protein, respectively, were previously constructed. Competent cells of E. coli HB101(F, hsd S20, recA13, ara-14, proA2, lac Y1, gal K2, rps L20(str), xyl-5, mtl-1, sup E44, leuB6, thi-1) were from Takara Shuzo Co., Ltd. Cells were grown in Luria-Bertani (LB) medium (Miller, 1972) containing 100 mg/liter ampicillin.

Materials

Restriction and modifying enzymes were from Takara Shuzo Co., Ltd. Ampholines (pH 8-9.5) and Sephadex G-75 were from Pharmacia Biotech Inc. 1-Anilino-8-naphthalenesulfonic acid (ANS) (^1)was from Tokyo Kasei Kogyo Co., Ltd. Guanidine hydrochloride (GdnHCl) was from Wako Pure Chemical Industries, Ltd. Other chemicals were of reagent grade.

Overproduction and Purification

Plasmid pJAL86A, in which the transcription of the mutated rnhA gene encoding the K86A protein is controlled by the bacteriophage P(L) and P(R) promoters, was constructed to improve the production level of this protein, by replacing the rnhA gene in the plasmid pJAL600 with the mutated rnhA gene from the plasmid pK86A. The K86A protein was overproduced in E. coli HB101 cells harboring plasmid pJAL86A by raising the cultivation temperature from 30 to 42 °C, as described previously (Kanaya et al., 1989).

The K86A protein was purified as a mixture of the intact and nicked forms by the standard purification procedure (Kanaya et al., 1989). The intact and nicked K86A proteins were separated by cation-exchange HPLC on an Asahipak ES502C column (7.5 100 mm) from Asahi Chemical Industries Co., Ltd. Elution was performed by a linear gradient of 0.015-0.10 M Na(2)SO(4) in 20 mM sodium phosphate buffer (pH 6.5) over 30 min with a flow rate of 1.0 ml/min. Proteins were detected by a variable wavelength UV detector at 280 nm. The retention times of the intact and nicked K86A proteins were 23 and 26 min, respectively. The cellular production level of the intact K86A protein was 30-50 mg/liter, as judged by the intensity of the band visualized by Coomassie Brilliant Blue staining of the SDS protein gel. SDS-polyacrylamide gel electrophoresis was carried out on a 15% polyacrylamide gel, as described by Laemmli(1970). The intact and nicked K86A proteins were individually purified with yields of 20-40%.

The N-fragment (Met^1-Lys) and the C-fragment (Arg-Val), which remain associated in the nicked K86A protein, were separated by reverse-phase HPLC on an Aquapore RP-300 column (4.6 250 mm) from Brownlee Laboratories. Elution was performed by a linear gradient of 10-60% (v/v) acetonitrile with 0.1-0.05% trifluoroacetic acid, over 25 min with a flow rate of 1.0 ml/min. The retention times of the N-fragment, the intact K86A protein, and the C-fragment were 18.1, 23.4, and 24.7 min, respectively.

Protein Concentration

The concentrations of the intact and nicked proteins were determined from UV absorption, with an absorption coefficient (A) of 2.02 (Kanaya et al., 1990b), assuming that the A values of these proteins are the same as that of the wild-type protein. The concentrations of the N-and C-fragments were determined from UV absorption with the A values of 1.54 and 2.58, respectively. These values are 90% of the values that are calculated using 1576 M cm for tyrosine and 5225 M cm for tryptophan at 280 nm (Goodwin and Morton, 1946), because the A value determined for the wild-type protein is 90% of the calculated value. The molecular weights of the N- and C-fragments are 9,764 and 7,794, respectively. The N-fragment has four Tyr and two Trp residues, and the C-fragment has one Tyr and four Trp residues.

Kinetic Analysis of Enzymatic Activity

The RNase H activity was determined at 30 °C in the presence of 10 mM MgCl(2) by measuring the radioactivity of the acid-soluble digestion product from the ^3H-labeled M13 DNA/RNA hybrid, as described previously (Kanaya et al., 1991). In this condition, the Mg binding sites of the intact and nicked proteins are fully occupied by the catalytically essential Mg ion. For the kinetic analyses, the substrate concentration was varied from 0.1 to 3.0 µM (nucleotide phosphate concentration). The hydrolysis of the substrate with the enzyme follows Michaelis-Menten kinetics, and the kinetic parameters, K and V(max), were determined from the Lineweaver-Burk plot.

Isoelectric Focusing

Isoelectric focusing was carried out using a 110-ml column (Pharmacia) according to the method of Vesterberg (1971) by adding the sample (1.0 mg) in the middle of a 0-50% sucrose gradient containing 1% carrier ampholine (pH 8-9.5).

Circular Dichroism Spectra

The CD spectra were measured on a J-720 automatic spectropolarimeter (Japan Spectroscopic Co., Ltd.) at the indicated temperature. Proteins were dissolved in 10 mM Gly-HCl buffer (pH 3.0) in the absence or presence of 0.15 M NaCl, or in 10 mM sodium acetate buffer (pH 5.5) in the absence or presence of 3 M GdnHCl. For the measurement of the far-ultraviolet (UV) CD spectra (200-260 nm), the protein concentration was approximately 0.1 mg/ml, and a cell with an optical pathlength of 2 mm was used. For the measurement of the near-UV CD spectra (250-320 nm), the protein concentration was 0.5-1.0 mg/ml and a cell with an optical pathlength of 10 mm was used. The mean residue ellipticity ([], degreesbullet cm^2bulletdmol) was calculated by using an average amino acid molecular weight of 110.

Denaturation Experiments

Thermal, chemical, and acid denaturations of the proteins were analyzed by using CD. Proteins were dissolved to a concentration of 0.1 mg/ml in 10 mM Gly-HCl buffer (pH 3.0) for the thermal denaturation experiments, in 20 mM sodium acetate buffer (pH 5.5) containing the appropriate concentration of GdnHCl for the GdnHCl denaturation experiments, and in 10 mM Gly-HCl buffer (pH 1.7-3.2) for the acid denaturation experiments. The GdnHCl and acid denaturation experiments were carried out at 25 °C and 10 °C, respectively.

The reversibility of the denaturation of the nicked protein in GdnHCl was examined by the following procedures. The protein was dissolved to a concentration of 4-5 mg/ml in 20 mM sodium acetate buffer (pH 5.5) containing 3 M GdnHCl, and then diluted by 20-fold with 10 mM Gly-HCl buffer (pH 3.0), 10 mM sodium acetate buffer (pH 5.5), and 10 mM Tris-HCl buffers (pH 7.8 and 9.4). These solutions were further diluted by 10-fold with 20 mM sodium phosphate buffer (pH 6.5) and were subjected to cation-exchange HPLC using an ES502C column to determine the amount of the reconstituted protein. This second dilution with 20 mM sodium phosphate buffer (pH 6.5) did not seriously affect the yield of the reconstitution, probably because the concentrations of the N- and C-fragments were too low to permit effective fragment association.

Thermal and GdnHCl denaturation curves, as well as the parameters characterizing these denaturations, such as the midpoint of the thermal transition (T), the midpoint of the transition in the GdnHCl concentration ([D]), the free energy change in H(2)O (DeltaG[H(2)O]), and the measurement of the dependence of DeltaG on the GdnHCl concentration (m), were determined as described previously (Kimura et al., 1992), by monitoring the change in the CD value at 220 nm.

Fragment Association Experiments

The N- and C-fragments, which were isolated by reverse-phase HPLC and lyophilized, were dissolved in 10 mM Gly-HCl buffer (pH 3.0) to a concentration of 150 µM (1.5 mg/ml for the N-fragment and 1.2 mg/ml for the C-fragment). These solutions were diluted by 10-fold with an appropriate buffer, mixed in a 1:1 ratio in volume, and kept at 20 °C for 1 h. The fraction of the reconstituted nicked protein was estimated by subjecting this solution to cation-exchange HPLC, using an ES502C column. Elution of the protein from the ES502C column was performed as described for the separation of the intact and nicked proteins. The fraction of the reconstituted nicked protein could be estimated from far-UV CD spectra as well, because they represent the sum of those of the N-fragment, the C-fragment, and the nicked K86A protein. The CD value at a given wavelength for the nicked K86A protein ([]) is available. The sum ([]) of those for the N-fragment ([](N)) and the C- fragment ([](C)) can be calculated by the equation: [] = [](N) 87/155 + [](C) 68/155. If the fraction of the reconstituted nicked protein were f, the CD value of the mixture ([]) at a given wavelength should be identical with those calculated by the equation: [] = f [] + (1 - f) [].

ANS Binding

Binding of ANS to the protein was analyzed by measuring the fluorescence on a Hitachi F-4000 fluorescence spectrophotometer at 10 or 55 °C. The excitation wavelength was 376 nm, and the emission was monitored from 400 to 600 nm. The spectra obtained in the absence of the protein was used as a blank. The concentration of the protein was 1 µM, and that of the ANS was 50 µM.

Gel Filtration

Gel filtration was performed at room temperature using a column (0.6 50 cm) of Sephadex G-75, which was equilibrated either with 10 mM Gly-HCl buffer (pH 3.0) or with 10 mM sodium acetate buffer (pH 5.5) in the absence or presence of 3 M GdnHCl. The flow rate was 7 ml/h, and fractions of 300 µl were collected. Elution of the protein was detected by measuring the absorbance of each fraction at 280 nm or by applying an aliquot of each fraction to reverse-phase HPLC.


RESULTS

Properties of the Nicked K86A Protein

In the nicked K86A protein molecule, the peptide bond is cleaved in the basic protrusion (Fig. 1), which is important for the substrate binding (Kanaya et al., 1991). Determination of the kinetic parameters for the hydrolysis of the M13 DNA/RNA hybrid indicates that the cleavage of the peptide bond between Lys and Arg results in a large increase in the K value, along with a large decrease in the V(max) value (Table 1). This means that the introduction of the nick into the basic protrusion considerably weakened the affinity of the enzyme for the substrate. The pI value of the nicked K86A protein is lower than that of the intact molecule by 0.5 pH unit (Table 1). The cleavage of the peptide bond generates both amino and carboxyl groups. However, only the carboxyl group, which may be fully ionized in the region where the positive charges are clustered, contributes to reduce the pI value of the protein. The far-UV CD spectrum of the nicked K86A protein, as well as that of the intact K86A protein, were identical with that of the wild-type protein (data not shown). In contrast, a considerable difference in the near-UV CD spectra was observed between the nicked K86A and wild-type proteins (Fig. 2). The near-UV CD spectrum of the intact K86A protein was similar to that of the wild-type protein (Fig. 2). Chemical, thermal, and acid denaturations of the nicked K86A protein will be described below.




Figure 2: CD spectra of the wild-type and K86A proteins. The near-UV CD spectra of the wild-type (thinline), intact K86A (brokenline), and nicked K86A (thickline) proteins are shown. These spectra were measured at pH 5.5 in the absence of GdnHCl, as described under ``Experimental Procedures.''



Chemical Denaturation

Upon chemical (GdnHCl) denaturation, the CD spectra of the wild-type, intact K86A, and nicked K86A proteins were dramatically changed, so that the broad trough with a minimum [] value of -13,000 at 216 nm almost completely disappeared (data not shown). Consequently, the CD value at 220 nm increased from -12,000 to -2,000. This means that the protein in a chemically denatured state is almost fully unfolded. The GdnHCl-denatured state is therefore designated as the U state. The elution profile of the nicked protein in the U state from a column (0.6 50 cm) of Sephadex G-75 equilibrated with 10 mM sodium acetate buffer (pH 5.5) containing 3 M GdnHCl is shown in Fig. 3. Because the N- and C-fragments eluted after the wild-type protein, the N- and C-fragments from the nicked protein were dissociated in the presence of 3 M GdnHCl. When the reversibility of the GdnHCl denaturation of the nicked protein was examined by dilution, as described under ``Experimental Procedures,'' this protein was refolded with a yield of 35.3% at pH 3.0, 72.7% at pH 5.5, 43.3% at pH 7.8, and 2.6% at pH 9.4. This means that the nicked protein is most effectively reconstituted from the N- and C-fragments at pH 5.5. The far-UV CD spectrum and the enzymatic activity of the reconstituted protein, which was purified by cation-exchange HPLC using an ES502C column, were identical with those of the nicked protein in the native state. The denaturations of the wild-type and intact K86A proteins were fully reversible in GdnHCl at pH 5.5.


Figure 3: Elution profile of the nicked protein from a Sephadex G-75 column. The nicked K86A protein was applied to a Sephadex G-75 column (0.6 50 cm), which was previously equilibrated with 10 mM sodium acetate buffer (pH 5.5) containing 3 M GdnHCl. The column was eluted with the same buffer at a flow rate of 7 ml/h. Fractions (300 µl) were collected, and an aliquot of each fraction was subjected to reverse-phase HPLC to determine the concentrations of the N-fragment (up triangle, filled) and C-fragment () simultaneously. Arrows indicate the positions at which blue dextran (1), wild-type RNase HI (2), and NaCl (3) eluted from the column.



The GdnHCl denaturation curves of the nicked and intact K86A proteins are shown in Fig. 4. The replacement of Lys by Ala does not seriously affect the stability of the protein. However, the introduction of the nick into this mutant protein dramatically destabilizes the protein, by 1.07 M in [D] (Table 2). The fragment dissociation might be the reason for the dramatic decrease in the stability of the nicked protein.


Figure 4: GdnHCl-induced denaturation curves of the intact and nicked K86A proteins. The apparent fraction of unfolded protein, which was calculated as described previously (Kimura et al., 1992), is shown as a function of GdnHCl concentration. , intact K86A protein; bullet, nicked K86A protein. GdnHCl denaturation curves were determined at pH 5.5 and 25 °C by monitoring the change in the CD value at 220 nm.





Thermal Denaturation

The thermal denaturation of the nicked protein was analyzed at pH 3.0, instead of pH 5.5, because the thermal denaturation of the intact protein is reversible at pH 5.5 only under the conditions, in which the nicked protein is chemically denatured. The far- and near-UV CD spectra of the nicked protein at 10 °C represent those of the nicked protein in the native state (Fig. 5). The far-UV CD spectrum of the nicked protein changed at 55 °C, so that it exhibited a trough with a minimum [] value of -12,000 at 202 nm, which was accompanied by a shoulder with a [] value of -5,500 at 220 nm (Fig. 5a). Likewise, the near-UV CD spectrum of the nicked protein changed at 55 °C, so that the positive signals almost completely disappeared (Fig. 5b). These results indicate that the nicked protein in a thermally denatured (D(T)) state is partially folded, but has little tertiary structure.


Figure 5: CD spectra of the thermally denatured nicked protein. The far-UV (a) and near-UV (b) CD spectra of the nicked K86A protein in the native (thinline), D(T) (thickline), and D(T)` (brokenline) states are shown. The CD spectra of the nicked protein in the native and D(T)` states were measured at pH 3.0 and 10 °C, and that in the D(T) state was measured at pH 3.0 and 55 °C.



The far- and near-UV CD spectra of the nicked protein in the D(T) state were identical with those of the wild-type and intact K86A proteins in the D(T) state. In contrast to the wild-type and intact K86A proteins, however, the thermal denaturation of the nicked protein was not reversible. The far- and near-UV CD spectra of the nicked protein, which was thermally denatured at 55 °C and then refolded by lowering the temperature to 10 °C, were similar to those of the nicked protein in the D(T) state (Fig. 5). The conformation of this nicked protein is therefore designated as the D(T)` state. The far-UV CD spectrum of the nicked protein in the D(T)` state exhibited a trough with a minimum [] value of -13,500 at 202 nm, which is accompanied by a shoulder with a [] value of -6,500 at 220 nm (Fig. 5a). Gel filtration of the nicked protein in the D(T)` state with Sephadex G-75 indicates that the N- and C-fragments are dissociated from each other at pH 3.0 upon thermal denaturation and remained in monomeric forms even when the temperature was lowered to 10 °C (data not shown).

The thermal denaturation curves of the wild-type, nicked K86A, and intact K86A proteins are shown in Fig. 6. Because the thermal denaturation of the nicked protein is not reversible, only its apparent T value is compared with those of the wild-type and intact K86A proteins (Table 2). Consistent with the results obtained in the GdnHCl denaturation experiments, the Lys Ala mutation did not seriously affect the thermal stability of the protein, but the introduction of the nick dramatically reduced it, by 16.4 °C in T.


Figure 6: Thermal denaturation curves of the wild-type and K86A proteins. The apparent fraction of unfolded protein, which was calculated as described previously (Kimura et al., 1992), is shown as a function of temperature. , wild-type protein; , intact K86A protein; bullet, nicked K86A protein. Thermal denaturation curves were determined at pH 3.0 by monitoring the change in the CD value at 220 nm.



Acid Denaturation

The pH dependence of the far-UV CD spectrum of the nicked K86A protein indicates that this protein was fully converted to an acid-denatured (D(A)) state at pH 2.42 (Fig. 7a). In contrast, the intact K86A protein was not fully denatured, even at pH 1.70 (Fig. 7b). The resemblance between the far-UV CD spectrum of the intact protein at pH 1.70 and that of the nicked protein at pH 2.63 suggests that the intact protein molecules exist in both the native and D(A) states at pH 1.70. The intact protein was not fully denatured at a pH below 1.7. Instead, the CD value at 220 nm increased as the pH decreased below 1.5 (data not shown), suggesting that the secondary structure content in this protein increased as the pH decreased below 1.5. Gel filtration of the nicked protein in the D(A) state with Sephadex G-75 indicates that the N- and C-fragments are dissociated from each other upon acid denaturation and remain in monomeric forms (data not shown).


Figure 7: pH dependence of the CD spectrum of the nicked protein. The far-UV CD spectra of the nicked (a) and intact (b) K86A proteins, which were measured at 10 °C in 10 mM Gly-HCl buffer at the pH indicated in the figure, are shown.



ANS is a fluorescent dye that binds to hydrophobic regions of proteins, and it is used as a probe for the detection of the molten globular state of proteins. ANS effectively bound to the nicked protein in either the D(A) or D(T)` state, whereas it did not bind to the nicked protein in the D(T) state (data not shown). It bound to the nicked protein in the native state, but to a much lesser extent. Because the CD spectrum of the nicked protein in the D(A) state (Fig. 7a) is identical with that of the nicked protein in the D(T)` state (Fig. 5a), the conformation of the nicked protein in the D(A) state might be identical with that of the nicked protein in the D(T)` state.

Conformations of the N- and C-fragments

The conformations of the N- and C-fragments, which were isolated by reverse-phase HPLC, were analyzed at 20 °C by CD. The far-UV CD spectra of the N- and C-fragments in 10 mM Gly-HCl buffer (pH 3.0) indicate that the N-fragment is partially folded, but the C-fragment is almost fully disordered (Fig. 8). The far-UV CD spectrum of the N-fragment at pH 3.0 exhibited a trough with a minimum [] value of -13,000 at 205 nm, which is accompanied by a shoulder with a [] value of -7,600 at 220 nm. Upon a pH shift from 3.0 to 5.5, the spectrum of the N-fragment was significantly changed, so that it exhibited a broad trough with two minimum [] values of -13,000 at 208 nm and -11,000 at 222 nm (Fig. 8a). A similar spectral change was observed when 0.15 M NaCl was added at pH 3.0. These results indicate that the helical content in the N-fragment increases, either by raising the pH from 3.0 to 5.5 or by adding salt at pH 3.0. The helical content in the N-fragment in the absence of salt at pH 3.0 was calculated as 21.2%, and that at pH 5.5 as 34.1%, by the method of Wu et al.(1981). In contrast to the spectrum of the N-fragment, that of the C-fragment only marginally changed when the pH was shifted from 3.0 to 5.5 or when salt was added (Fig. 8b). The helical content in the C-fragment was low (8.0%) at either pH. The far-UV CD spectrum of the C-fragment was basically unchanged in the pH range from pH 3.0 to 9.0, and in the NaCl concentration range from 0 to 1.0 M, indicating that the predominant conformation of the C-fragment is similar to that of random peptides under these conditions.


Figure 8: CD spectra of the N- and C-fragments. The far-UV CD spectra of the N-fragment (a) and C-fragment (b) were measured in 10 mM Gly-HCl buffer (pH 3.0), 10 mM Gly-HCl buffer (pH 3.0) containing 0.15 M NaCl, and 10 mM sodium acetate buffer (pH 5.5) at 20 °C. Because the spectra at pH 3.0 in the presence of 0.15 M NaCl were almost identical with those at pH 5.5, only the spectra at pH 3.0 in the absence of salt (brokenline) and at pH 5.5 (solidline) are shown.



The near-UV CD spectra of the N- and C-fragments at either pH 3.0 or 5.5 were similar to that of the nicked protein in the D(T) state, shown in Fig. 5b (data not shown). These results suggest that the N- and C-fragments have little tertiary structure at pH 3.0 and pH 5.5. When the thermal denaturation of the N-fragment was examined, either at pH 5.5 or at pH 3.0 in the presence of 0.15 M NaCl, by monitoring the change in the CD value at 220 nm, no transition was observed in the range from 10 to 70 °C. ANS bound to the N-fragment much more effectively than to the C-fragment, as shown in Fig. 9. The intensity of the fluorescence of the N-fragment at pH 3.0 was similar to that of the nicked protein in the D(A) state. This result indicates that the ANS binding site resides in the N-fragment. The intensity of the fluorescence of the N-fragment at pH 5.5 was lower than that at pH 3.0 (Fig. 9). The formation of an additional alpha-helix in the N-fragment probably affects the conformation of the ANS binding site. In contrast, the intensity of the fluorescence of the C-fragment at pH 5.5 was slightly higher than that at pH 3.0 (Fig. 9). The conformation of the C-fragment slightly changed at pH 5.5, so that a weak ANS binding site was created.


Figure 9: ANS fluorescence spectra of the N- and C-fragments. The ANS fluorescence spectra of the N-fragment in 10 mM Gly-HCl buffer (pH 3.0) (thickline) and 10 mM sodium acetate buffer (pH 5.5) (thinline), and those of the C-fragment in 10 mM Gly-HCl buffer (pH 3.0) (thickbrokenline) and 10 mM sodium acetate buffer (pH 5.5) (thinbrokenline) are shown. These spectra were measured at 10 °C, as described under ``Experimental Procedures.''



To examine whether the N- and C-fragments exist in monomeric forms, they were applied to a Sephadex G-75 column at pH 3.0 and pH 5.5. The C-fragment was eluted from the column at the position at which a monomeric form of this fragment elutes, at either pH. The N-fragment was eluted from the column at the position at which a monomeric form of the N-fragment elutes, at pH 3.0. In contrast, the N-fragment was not recovered from the column at pH 5.5. It seems unlikely that the N-fragment aggregates to form a precipitate at pH 5.5, because it can associate with the C-fragment to form the nicked protein with a high yield (80%), as described below. The N-fragment may exist either in a monomeric or oligomeric form at pH 5.5. In either case, the N-fragment may have a hydrophobic surface and, therefore, may irreversibly adsorb to Sephadex G-75 at pH 5.5.

Fragment Association

The association of the N- and C-fragments was investigated at 20 °C by CD. If these fragments were associated to form the nicked protein, the far-UV CD spectrum of the mixture of these peptides would be different from the sum of the two spectra of these peptides as individually measured. As expected, little difference was observed between these spectra at pH 3.0 in the absence of salt (Fig. 10a). In contrast, a marked difference was observed between these spectra, either at pH 5.5 (Fig. 10b) or at pH 3.0 in the presence of 0.15 M NaCl (Fig. 10c). These results indicate that the N- and C-fragments are associated to form the nicked protein at either pH 5.5 or at pH 3.0 in the presence of salt, but not at pH 3.0 in the absence of salt. It is notable that the spectrum of the mixture of the N- and C-fragments at pH 3.0 is identical with that of the nicked protein in either the D(A) state (Fig. 7a) or the D(T)` state (Fig. 5a). This means that the conformations of the isolated N- and C-fragments at pH 3.0 reflect those in the D(A) state. The fractions of the nicked protein reconstituted from the N- and C-fragments were calculated from the far-UV CD spectra of the mixtures of these peptides as roughly 80% at pH 5.5 and 30% at pH 3.0 in the presence of 0.15 M NaCl, as described under ``Experimental Procedures.'' The formation of the nicked protein was confirmed by HPLC, using the ES502C column (Fig. 11). The far-UV CD spectrum and the enzymatic activity of the nicked protein, which was reconstituted from the N- and C-fragments and was purified by HPLC, using the ES502C column, were identical with those of the nicked protein in the native state.


Figure 10: CD spectra of the mixtures of the N- and C-fragments. The far-UV CD spectra of the mixture of the N- and C-fragments (solidline), in which equimolar amounts of these fragments (15 µmol/liter) are mixed, are shown in comparison with the sum of the spectra of these fragments (brokenline). These spectra were measured at 20 °C in the following buffers: a, 10 mM Gly-HCl buffer (pH 3.0); b, 10 mM sodium acetate buffer (pH 5.5); c, 10 mM Gly-HCl buffer (pH 3.0) containing 0.15 M NaCl. The sum of the spectra of the N- and C-fragments was obtained from the spectra of these fragments, which were individually measured under the conditions mentioned above, as described under ``Experimental Procedures.''




Figure 11: Elution of the reconstituted nicked protein from the ES502C column. Elution profiles of the mixtures of the N- and C-fragments, which were prepared either at pH 3.0 (thinline) or at pH 5.5 (thickline) in the absence of salt, are shown. The equimolar amounts of the N- and C-fragments (15 µmol/liter) were mixed, as described under ``Experimental Procedures.'' The concentrations of these peptides in the resultant solution were 7.5 µM (0.075 mg/ml for the N-fragment and 0.06 mg/ml for the C-fragment), if they were not associated with each other. An aliquot (100 µl) of this solution was withdrawn, diluted by 10-fold with 20 mM sodium phosphate buffer (pH 6.5), and subjected to cation-exchange HPLC using ES502C. Elution of the reconstituted nicked protein from the ES502C column was performed as described for the separation of the intact and nicked proteins. The brokenline represents the concentration of Na(2)SO(4).




DISCUSSION

Conformation of the Nicked Protein

Comparison of the far- and near-UV CD spectra of the nicked protein with those of the intact protein suggests that the introduction of the nick into the basic protrusion does not seriously affect the secondary structure of the protein, but alters the tertiary structure in the vicinity of aromatic residues. Because the basic protrusion contains a cluster of Trp residues, which may be important for maintaining its conformation, the introduction of the nick into this region probably modifies this Trp cluster and thereby leads to a serious conformational change in the basic protrusion. This conformational change may be the reason why the nicked protein has lower substrate binding affinity than the intact protein. Similar changes in the near-UV CD spectra and the kinetic parameters for the enzymatic activity were observed when either one of the Trp residues in the basic protrusion was replaced by Ala (Kanaya et al., 1991).

Denaturation of the Nicked Protein

The nicked protein dissociates into the N- and C-fragments upon thermal, chemical, and acid denaturations. The identity in the far- and near-UV CD spectra between the nicked protein in the D(T) state and the intact protein in the D(T) state indicates that the conformations of the N- and C-fragments in the D(T) state are identical with those of the corresponding regions in the intact protein in the D(T) state. Likewise, the conformations of these peptides in the U state are identical with those of the corresponding regions in the intact protein in the U state.

Are the conformations of these peptides in the D(A) state identical with those of the corresponding regions in the intact protein in the D(A) state as well? Unlike the nicked protein, the intact protein is not fully converted to the D(A) state, even at an extremely low pH. Upon acid titration, the unfolding of the intact K86A protein to the D(A) state begins around pH 2.5 (Fig. 7b). The fraction of the protein in the D(A) state increases as the pH decreases below 2.5. However, prior to the completion of this acid denaturation process, the protein refolds into a molten globular state at a pH below 1.5, because of an increase in the ionic strength by the addition of HCl. (^2)This acid-induced refolding also has been observed for other proteins, such as apomyoglobin, beta-lactamase, and cytochrome c (Goto et al., 1990; Harada et al., 1994). Dabora and Marqusee(1994) previously defined the conformation of the Cys-free mutant protein of E. coli RNase HI at pH 1.0 as an acid state (A state), which may represent a refolding state of this protein induced by acid. Thus, it seems likely that the CD spectrum of the intact protein at pH 1.70 (Fig. 7b) represents a mixture of the proteins in the native and D(A) states. The molten globular state (A state) may exist as well at this pH, but to a small extent. The pH dependence of the far-UV CD spectrum of the intact protein gave the same isodichroic point at 208 nm, with a [] value of -11,000, as that of the nicked protein (Fig. 7). This result strongly suggests that the CD spectra of the intact and nicked proteins in the D(A) states are identical with each other, and therefore, the conformations of the N- and C-fragments in the D(A) state are identical with those of the corresponding regions in the intact protein in the D(A) state.

The nicked and intact proteins in the D(A) state were less unfolded than that in the U state. Generally, proteins are positively charged at around pH 2, because most of the carboxylates are protonated at this pH. Fink et al.(1994) have suggested that the resulting electrostatic repulsion leads to unfolding to the D(A) state, but it is not sufficient to overcome interactions, such as hydrophobic forces and hydrogen bonds, which favor protein folding.

Renaturation (Reconstitution) of the Nicked Protein

The N- and C-fragments in the nicked protein, which were dissociated in the presence of GdnHCl (>1 M), were most effectively reassociated at pH 5.5, when the concentration of GdnHCl was decreased to less than 0.3 M by dilution. The yields of this reconstitution at pH 7.8 and 9.4 are lower than that at pH 5.5, probably because the solubility of the N-fragment at a pH above 5.5 is lower than that at pH 5.5. At pH 3.0, the solubility of the N-fragment is higher than that at pH 5.5. However, the N- and C-fragments are more positively charged at pH 3.0 than at pH 5.5, and the resulting intermolecular electrostatic repulsion may suppress the interaction between these fragments. It is notable that the N- and C-fragments are not reassociated at all at pH 3.0 in the absence of salt. These fragments are reassociated at pH 3.0 in the presence of 0.3 M GdnHCl, because GdnHCl acts as a salt, instead of a denaturant, at this concentration.

The addition of salt at pH 3.0, or a shift of the pH from 3.0 to 5.5 is required for the association of the N- and C-fragments (Fig. 10). Analyses of the conformations of these fragments by CD (Fig. 8) suggest that the additional formation of an alpha-helix in the N-fragment facilitates the folding of the C-fragment, which alone is almost fully disordered, and thereby facilitates the association of these fragments to form the nicked protein. The conformation of the C-fragment, either at pH 5.5 or at pH 3.0 in the presence of 0.15 M NaCl, which is designated as the D(A)` state, is basically the same as that in the D(A) state (Fig. 8b). In contrast, the conformation of the N-fragment either at pH 5.5 or at pH 3.0 in the presence of 0.15 M NaCl differs from that in the D(A) state in the content of the secondary structure (Fig. 8a). This conformation is defined as a molten globular (M) state (Kuwajima, 1989), for the following reasons. 1) It has high (native-like) secondary structure and little tertiary structure, 2) it lacks cooperative thermal unfolding transition, and 3) it has the ability to bind ANS.

Schemes for the denaturation and renaturation (reconstitution) of the nicked K86A protein described above are summarized in Fig. 12.


Figure 12: Schemes for denaturation and renaturation (reconstitution) of the nicked K86A protein. N bullet C represents the nicked K86A protein in the native state. N, N, N, and N represent the N-fragment in the U, D(T), D(A), and M states, respectively. Likewise, C, C, C, and C represent the C-fragment in the U, D(T), D(A), and D(A)` states. The N-fragment in the M state (N) and the C-fragment in the D(A)` state (C) are shown in parentheses, because they are stable only when they are separated from each other. The D(T)` state is not shown in these schemes, because it is identical with the D(A) state.



Folding Pathway

The conformation of the N-fragment in the M state is of special interest, because its formation seems to be responsible for the reconstitution of the nicked protein. The helical contents in the N-fragment in the D(A) and M states were calculated as 21.2 and 34.1%, respectively, whereas that of the C-fragment in either the D(A) or D(A)` state was calculated as 8.0%. The helical contents in the N-terminal (1-87) and C-terminal (88-155) segments of the intact protein in the native state are 38 and 44%, respectively. These results suggest that the alpha-helices are partially formed in the N-fragment in the D(A) state and are almost fully formed in the N-fragment in the M state, but are poorly formed in the C-fragment in either the D(A) or D(A)` state. This then evokes the question as to which alpha-helix is additionally formed in the N-fragment when its conformation is changed from the D(A) to the M state.

The N-terminal segment (1-87) of the intact protein in the native state forms three alpha-helices (alphaI, alphaII, and alphaIII), as shown in Fig. 1. The contents of these helices in the N-terminal segments are 18% for alphaI, 10% for alphaII, and 10% for alphaIII. Among them, the alphaI-helix is located at the center of the protein molecule in the hydrophobic core region. This helix forms a coiled-coil-like structure with the alphaIV-helix, and also contacts the beta-sheet. Analyses for the conformations of the small peptides by CD suggested that the alphaI-helix folds independently, but the alphaII- and alphaIII-helices do not. (^3)In addition, refolding studies with NMR measurements of hydrogen exchange suggested that the alphaI-helix is the first to fold. (^4)These results allow us to propose that the alphaI-helix is the initiation site for the folding of the intact RNase HI molecule. The alphaI-helix might be at least partially formed in the N-fragment in the D(A) state and the complete formation of the alphaI-helix in the N-fragment (M state) may facilitate the formation of the alphaIV-helix in the C-fragment and thereby facilitate the fragment association. Alternatively, the alphaI-helix may be completely formed in the N-fragment in the D(A) state, and may be able to induce the formation of the alphaIV-helix in the C-fragment only at a pH higher than 3.0, at which the intermolecular electrostatic repulsion between the N- and C-fragments is suppressed.

The conformations of the N-fragment in the D(A) and M states may represent a folding intermediate of the E. coli RNase HI molecule. Structural analyses of this fragment in the D(A) and M states will therefore provide valuable information on the folding process of this protein. Because the first four beta-strands and the alpha-helix connecting the third and fourth beta-strands (Fig. 1), which reside in the corresponding regions of proteins with the RNase H-like fold, are arranged identically in these protein structures, and because all the catalytically essential carboxylates, which are Asp, Glu, and Asp in E. coli RNase HI (Kanaya et al., 1990a), are located in the regions corresponding to the N-fragment, the folding topology consisting of these secondary structure elements (Fig. 13) may be evolutionarily conserved in the protein structures with an RNase H-like fold. Katayanagi et al. (1990) have previously shown that the crystal structure of E. coli RNase HI has a similarity with that of DNase I (Suck and Oefner, 1986) in the folding of a beta-sheet. The similar folding topology has also been observed in the E. coli exonuclease III structure (Mol et al., 1995). However, these proteins may not be evolutionarily related with E. coli RNase HI, because the folding topology shown in Fig. 13is not conserved in these protein structures.


Figure 13: A topology conserved in the RNase H-like fold. A possible folding topology, which is evolutionarily conserved in the protein structures with the RNase H-like fold, is shown. Arrows represent the beta-strands, and the cylinder represents the alpha-helix.




FOOTNOTES

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

§
To whom correspondence should be addressed: Dept. of Material and Life Science, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan. Tel.: 81-6-877-5111 (ext. 3307); Fax: 81-6-879-7936.

(^1)
The abbreviations used are: ANS, 1-anilino-8-naphthalenesulfonic acid; GdnHCl, guanidine hydrochloride; HPLC, high performance liquid chromatography.

(^2)
M. Oobatake, personal communication.

(^3)
E. Kanaya and S. Kanaya, personal communication.

(^4)
K. Yamasaki, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. K. Yamasaki, M. Oobatake, K. Nishikawa, and M. Ikehara for helpful discussions. We also thank C. Nakai and Y.-Y. Liu for technical assistance.


REFERENCES

  1. Ariyoshi, M., Vassylyev, D. G., Iwasaki, H., Nakamura, H., Shinagawa, H., and Morikawa, K. (1994) Cell 78,1063-1072 [Medline] [Order article via Infotrieve]
  2. Beals, J. M., Haas, E., Krausz, S., and Scheraga, H. A. (1991) Biochemistry 30,7680-7692 [Medline] [Order article via Infotrieve]
  3. Bierzynski, A., Kim, P. S., and Baldwin, R. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,2470-2474 [Abstract]
  4. Chaffotte, A., Guillou, Y., Delepierre, M., Hinz, H.-J., and Goldberg, M. E. (1991) Biochemistry 30,8067-8074 [Medline] [Order article via Infotrieve]
  5. Dabora, J. M., and Marqusee, S. (1994) Protein Sci. 3,1401-1408 [Abstract/Free Full Text]
  6. Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R., and Davies, D. R. (1994) Science 266,1981-1981 [Medline] [Order article via Infotrieve]
  7. Dyson, H. J., Merutka, G., Waltho, J. P., Lerner, R. A., and Wright, P. E. (1992) J. Mol. Biol. 226,795-817 [Medline] [Order article via Infotrieve]
  8. Fink, A. L., Calciano, L. J., Goto, Y., Kurotsu, T., and Palleros, D. R. (1994) Biochemistry 33,12504-12511 [Medline] [Order article via Infotrieve]
  9. Fontana, A. (1990) in Peptides: Chemistry, Structure and Biology (Rivier, J. E., and Marshall, G. R., eds) pp. 557-565, ESCOM, Leiden, The Netherlands
  10. Gay, G. P., and Fersht, A. R. (1994) Biochemistry 33,7957-7963 [Medline] [Order article via Infotrieve]
  11. Gay, G. P., Ruiz-Sanz, J., Davis, B., and Fersht, A. R. (1994a) Proc. Natl. Acad. Sci. U. S. A. 91,10943-10946 [Abstract/Free Full Text]
  12. Gay, G. P., Ruiz-Sanz, J., and Fersht, A. R. (1994b) Biochemistry 33,7964-7970 [Medline] [Order article via Infotrieve]
  13. Girons, I. S., 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]
  14. Goodwin, T. W., and Morton, R. A. (1946) Biochem. J. 40,628-632
  15. Goto, Y., Calciano, L. J., and Fink, A. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,573-577 [Abstract]
  16. Griko, Y. V., Gittis, A., Lattman, E. E., and Privalov, P. L. (1994) J. Mol. Biol. 243,93-99 [CrossRef][Medline] [Order article via Infotrieve]
  17. Harada, D., Kidokoro, S., Fukada, H., Takahashi, K., and Goto, Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,10325-10329 [Abstract/Free Full Text]
  18. Holm, L., and Sander, C. (1994) Proteins Struct. Funct. Genet. 19,165-173 [Medline] [Order article via Infotrieve]
  19. Jennings, P. A., and Wright, P. E. (1993) Science 262,892-896 [Medline] [Order article via Infotrieve]
  20. Kanaya, S., and Crouch, R. J. (1983) J. Biol. Chem. 258,1276-1281 [Abstract/Free Full Text]
  21. Kanaya, S., Kohara, A., Miyagawa, M., Matsuzaki, T., Morikawa, K., and Ikehara, M. (1989) J. Biol. Chem. 264,11546-11549 [Abstract/Free Full Text]
  22. Kanaya, S., Kohara, A., Miura, Y., Sekiguchi, A., Iwai, S., Inoue, H., Ohtsuka, E., and Ikehara, M. (1990a) J. Biol. Chem. 265,4615-4621 [Abstract/Free Full Text]
  23. Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990b) Biochem. J. 271,59-66 [Medline] [Order article via Infotrieve]
  24. Kanaya, S., Katsuda-Nakai, C., and Ikehara, M. (1991) J. Biol. Chem. 266,11621-11627 [Abstract/Free Full Text]
  25. Kanaya, S., Oobatake, M., Nakamura, H., and Ikehara, M. (1993) J. Biotechnol. 28,117-136 [CrossRef][Medline] [Order article via Infotrieve]
  26. Katayanagi, K., Miyagawa, M., Matsushima, M., Ishikawa, M., Kanaya, S., Ikehara, M., Matsuzaki, T., and Morikawa, K. (1990) Nature 347,306-309 [CrossRef][Medline] [Order article via Infotrieve]
  27. Katayanagi, K., Miyagawa, M., Matsushima, M., Ishikawa, M., Kanaya, S., Nakamura, H., Ikehara, M., Matsuzaki, T., and Morikawa, K. (1992) J. Mol. Biol. 223,1029-1052 [Medline] [Order article via Infotrieve]
  28. Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., and Kanaya, S. (1992) J. Biol. Chem. 267,21535-21542 [Abstract/Free Full Text]
  29. Kippen, A. D., Sancho, J., and Fersht, A. R. (1994) Biochemistry 33,3778-3786 [Medline] [Order article via Infotrieve]
  30. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24,946-950 [CrossRef]
  31. Kuwajima, K. (1989) Proteins Struct. Funct. Genet. 6,87-103 [Medline] [Order article via Infotrieve]
  32. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  33. Matouschek, A., Kellis, J. T., Serrano, L., Bycroft, M., and Fersht, A. R. (1990) Nature 346,440-445 [CrossRef][Medline] [Order article via Infotrieve]
  34. Miller, J. H. (ed) (1972) Experiments in Molecular Genetics , p. 433, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  35. Miranker, A., Robinson, C. V., Radford, S. E., Aplin, R. T., and Dobson, C. M. (1993) Science 262,896-900 [Medline] [Order article via Infotrieve]
  36. Mol, C. D., Kuo, C.-F., Thayer, M. M., Cunningham, R. P., and Tainer, J. A. (1995) Nature 374,381-386 [CrossRef][Medline] [Order article via Infotrieve]
  37. Neri, D., Billeter, M., Wider, G., and Wüthrich, K. (1992) Science 257,1559-1563 [Medline] [Order article via Infotrieve]
  38. Oas, T. G., and Kim, P. S. (1988) Nature 336,42-48 [CrossRef][Medline] [Order article via Infotrieve]
  39. Osterhout, J., Jr., Baldwin, R. L., York, E. J., Stewart, J. M., Dyson, H. J., and Wright, P. E. (1989) Biochemistry 28,7059-7064 [Medline] [Order article via Infotrieve]
  40. Peng, Z., and Kim, P. S. (1994) Biochemistry 33,2136-2141 [Medline] [Order article via Infotrieve]
  41. Richards, F. M., and Vithayathil, P. J. (1959) J. Biol. Chem. 234,1459-1465 [Free Full Text]
  42. Sancho, J., and Fersht, A. R. (1992) J. Mol. Biol. 224,741-747 [Medline] [Order article via Infotrieve]
  43. Sancho, J., Neira, J. L., and Fersht, A. R. (1992) J. Mol. Biol. 224,749-758 [Medline] [Order article via Infotrieve]
  44. Shiba, K., and Schimmel, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,1880-1884 [Abstract]
  45. Shortle, D., and Meeker, A. K. (1989) Biochemistry 28,936-944 [Medline] [Order article via Infotrieve]
  46. Staley, J. P., and Kim, P. S. (1990) Nature 344,685-688 [Medline] [Order article via Infotrieve]
  47. Suck, D., and Oefner, C. (1986) Nature 321,620-625 [Medline] [Order article via Infotrieve]
  48. Taniuchi, H., and Anfinsen, C. B. (1971) J. Biol. Chem. 246,2291-2301 [Abstract/Free Full Text]
  49. Tasayco, M. L., and Carey, J. (1992) Science 255,594-597 [Medline] [Order article via Infotrieve]
  50. Vesterberg, O. (1971) Methods Enzymol. 22,389-412 [CrossRef]
  51. Waltho, J. P., Feher, V. A., Merutka, G., Dyson, H. J., and Wright, P. E. (1993) Biochemistry 32,6337-6347 [Medline] [Order article via Infotrieve]
  52. Wu, C.-S. C., Ikeda, K., and Yang, J. T. (1981) Biochemistry 20,566-570 [Medline] [Order article via Infotrieve]
  53. Yang, W., and Steitz, T. (1995) Structure 3,131-134 [Medline] [Order article via Infotrieve]
  54. Yang, W., Hendrickson, W. A., Crouch, R. J., and Satow, Y. (1990) Science 249,1398-1405 [Medline] [Order article via Infotrieve]

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