An in vitro peptide folding model suggests the presence of the molten globule state during nascent peptide folding

Bo Zhou, Kegui Tian and Guozhong Jing1

National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, People's Republic of China


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Result
 Discussion
 References
 
Although molten globules have been widely accepted as a general intermediate in protein folding, there is no clear evidence to show their presence during nascent peptide folding. This paper concentrates on whether the molten globule state occurs, and if it does, when does it form during nascent peptide folding, by comparing the changes in conformation during peptide chain extension of staphylococcal nuclease R. The results show that a large N-terminal fragment of staphylococcal nuclease, SNR121, which already contains more than 80% amino acid sequence of the nuclease, is found to fulfill all the criteria for the molten globule state, suggesting that the molten globule should occur at a later stage of peptide elongation. At this stage the hydrophobic collapse of the polypeptide chain occurs driven by the hydrophobic force, which leads to the formation of a solvent-accessible non-polar core, characterized by the high ANS-binding fluorescence. The nascent peptide folding of the nuclease is a hierarchical process that at the very least includes the following steps: secondary structure accumulation, pre-molten globule state, molten globule state, post-molten globule state and finally the native state. Constant conformation adjustment is necessary for correct folding and active expression of the protein.

Keywords: molten globule state/nascent peptide folding/N-terminal fragment/staphylococcal nuclease


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Result
 Discussion
 References
 
It is known that proteins are synthesized from the N-terminus in vivo and that the information contained in the amino acid sequence encodes the three-dimensional structure of the biologically active protein; as a result, many proteins spontaneously fold into their correct conformation (Prat Gay et al., 1995Go). Although a number of molecular chaperones have been found in recent years which assist the folding of polypeptides, the final folded structure of a protein is still dictated by its amino acid sequence. Furthermore, certain auxiliary proteins interact with incompletely folded nascent polypeptides. The interaction may possibly be dictated by the structural characteristics of the growing chain as it emerges from the ribosome (Langer et al., 1992Go; Frydman et al., 1994Go; Hartl et al., 1994Go). Thus, study of the conformational features of a series of N-terminal fragments with different chain lengths will reveal a dynamic process of nascent peptide folding during the elongation of peptide chains and provide further understanding of the pathway of nascent peptide folding. Molten globules have been extensively studied as a general intermediate in protein folding and their presence is widely accepted (Ptitsyn et al., 1990Go; Jennings and Wright, 1993Go; Ptitsyn, 1995aGo,bGo; Creighton, 1997Go). It may be assumed that a nascent polypeptide chain during and immediately after its biosynthesis is in the molten globule state (Bychkova and Ptitsyn, 1993Go). However, there is no clear evidence to show that the molten globule state occurs during nascent peptide folding, or if it does, when it occurs. Here, a family of N-terminal fragments of staphylococcal nuclease R were used as an in vitro nascent peptide folding model to detect the presence of the molten globule state in the physiological state, to evaluate what role it plays during nascent peptide folding and to explore the possible pathway of nascent peptide folding.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Result
 Discussion
 References
 
Materials

Staphylococcal nuclease R (SNase R) is an analogue of staphylococcal nuclease (SNase A, EC 3.1.4.7), in which a hexapeptide (DPTVYS) is appended to the N-terminal alanine of SNase A. The additional residues of SNase R have no discernible effect on conformation of the remainder of the molecule or its activity (Evans et al., 1989Go; Jing et al., 1992Go). According to the crystal structure of the nuclease, the overall tertiary structure is composed of a highly twisted five-strand ß-barrel (residue range: 10–19, 22–27, 30–36, 71–76 and 88–95) and three {alpha}-helices (residue range: 54–68, 98–106 and 121–135) (Loll and Lattman, 1989Go; Hynes and Fox, 1991Go). The seven N-terminal fragments of SNase R—SNR52, SNR79, SNR102, SNR110, SNR121, SNR135 and SNR141—which extend from residues –6–52, –6–79, –6–102, –6–110, –6–121, –6–135 and –6–141, respectively, were produced in Escherichia coli DH5{alpha} cells harboring an appropriate recombinant plasmid, and purified to be a single band on an SDS polyacrylamide gel as described before (Jing et al., 1995Go; Tian et al., 1998Go). In this case, SNR141 contains the residues for formation of all the secondary structures in the native nuclease. SNR135 contains all the residues forming the ß-barrel and three {alpha}-helices. Compared with SNR135, the residues forming the third {alpha}-helix were removed from SNR121 and SNR110. The difference between SNR121 and SNR110 is that 11 more residues (residue range 111–121), which form the left side of the nucleotide binding pocket (Hynes and Fox, 1991Go), were removed in the case of SNR110. SNR102 just contains the residues for formation of the ß-barrel and the first {alpha}-helix. SNR79 contains the residues forming the first four ß-strands and the first {alpha}-helix, while SNR52 contains the residues forming the first three ß-strands.

Protein concentrations of the fragments were determined by the method of Goodwin and Morton (1946). It was noteworthy that all the fragments have very good solubility: there were no intermolecular aggregation or oligomerization to be found for the fragments in the working concentrations checked by the method described by Shortle et al. (1989).

ANS (1-anilinonaphthalene-8-sulfonate; Sigma) was used as a hydrophobic fluorescence probe for monitoring the molten globule state. Guanidine hydrochloride (GdnHCl, ultra pure) was purchased from Life Technologies. pdTp (thymidine 3',5'-bisphosphate), a competitive inhibitor of the nuclease, was purchased from Pharmacia. All other reagents were of analytical grade.

Circular dichroism spectra

Circular dichroism (CD) spectra were recorded on a Jasco J-500A Spectro-polarimeter at 20°C. A quartz cuvette with 1.0 mm path length was used for far-UV (250–200 nm) measurements and a quartz cuvette with 10.0 mm path length for near-UV (310–250 nm) measurements. Protein concentration was 0.4 mg/ml in 20 mM Tris–HCl buffer (pH 7.4) for far-UV and 1.0 mg/ml for near-UV measurements.

Size-exclusion chromatography on protein I60 column

The molecular sizes of SNase R and its N-terminal fragments were compared by measuring the retention times of standard globular proteins (ribonuclease A, ß-lactoglobulin, {alpha}-lactalbumin, cytochrome C), SNase R and its N-terminal fragments on protein I60 size-exclusion chromatography. The protein I60 column (7.0 mmx30 cm) was used with a Waters 994 system. All the buffers and samples used in the chromatography were filtered through a 0.22 µm sterile filter. Each sample was dissolved in mobile phase buffer (0.1 M KH2PO4, 0.2 M NaCl, pH 7.0) to give a final concentration of 1.0 mg/ml. Each sample (50.0 µg) was injected into the column equilibrated with the mobile phase. The chromatography was performed at a flow-rate of 0.5 ml/min at 20°C. The effluent was monitored at 280 nm. S20,w (sedimentation coefficient at 20°C in water) of SNase R and its N-terminal fragments was obtained from the standard curve of S20,w of the standard globular proteins versus their retention times on the protein I60 column. Because S20,w is proportional to the square of the Stokes radius (Rs) when the solvent and temperature are not changed, the relative Stokes radii of the N-terminal fragments were obtained by comparing their S20,w with that of SNase R. These relative sizes were used to compare the molecular size of SNase R and its N-terminal fragments in this paper.

ANS-binding fluorescence

ANS was used as a hydrophobic fluorescence probe to study the change of surface hydrophobicity of the N-terminal fragments and to monitor the formation of the molten globule state during elongation of the peptide. ANS-binding fluorescence spectra of the N-terminal fragments of SNase R were measured in 20 mM Tris–HCl (pH 7.4) using a Hitachi F4010 Spectro-fluorometer at 25°C. Protein and ANS concentrations were 8 and 80 µM, respectively. The excitation wavelength was 345 nm, and the slit width was 10 nm.

Nuclease activity assay

The activity of SNase R and its N-terminal fragments for hydrolysis of single-stranded DNA was measured with a Shimadzu UV-250 spectrophotometer according to the method described by Cautrecasas et al. (1967).


    Result
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 Abstract
 Introduction
 Materials and methods
 Result
 Discussion
 References
 
According to Ptitsyn (1987), the essential characteristics of the molten globule state are substantial secondary structure, little or no tertiary structure (reflecting highly mobile side chains), compact size and a more exposed hydrophobic surface area than in the native state (Fink et al., 1993Go). In this paper, the conformational features of staphylococcal nuclease R and its seven N-terminal fragments are compared using far- and near-ultraviolet circular dichroism spectra (CD), ANS-binding fluorescence and molecular size-exclusion chromatography. The results show that an N-terminal fragment, SNR121, fulfills all the criteria described above and adopts the features of the molten globule state not observed with the longer and shorter N-terminal fragments.

SNR121 has substantial secondary structure, but no tertiary structure

Figure 1Go shows the relative values of the mean residue ellipticity of SNase R and the N-terminal fragments at 222 nm ([{theta}]222), which represent changes in secondary structure content, especially the characteristics of the {alpha}-helical conformation, during elongation of the N-terminal peptide. It can be seen that the secondary structures of the N-terminal fragments tend to increase with growth of the peptide chain. The largest fragment SNR141, in which eight amino acid residues are deleted from the C-terminus of SNase R, has almost the same [{theta}]222 value as that for SNase R, indicating that the deletion does not significantly affect the secondary structure of SNase R. However, compared with other fragments, SNR121 has a higher value of [{theta}]222 than even SNR135, and the relative value of [{theta}]222 for SNR121 is 57% of that of SNase R, though SNR121 has 14 more amino acid residues deleted from the C-terminal of SNase R than SNR135 does, including the amino acid residues for formation of the third {alpha}-helix in the native nuclease. The {alpha}-helix content in a stable acid-induced molten globule-like state of the nuclease is 64% of that in the native state (Fink et al., 1993Go). Thus, there is a pronounced secondary structure in the N-terminal fragment SNR121. In addition, Fourier transform infrared spectrum (FTIR) was used to analyze the secondary structures of the N-terminal fragments of the nuclease. The FTIR results also showed that SNR121 has more secondary structure content than other N-terminal fragments of the nuclease except the largest fragment SNR141 (Jing et al., 1995Go; Tian et al., 1998Go).



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Fig. 1. Relative [{theta}]222 of SNase R and its N-terminal fragments. (A) Original far-ultraviolet CD spectra of SNase R and its N-terminal fragments. 1, SNase R; 2, SNR141; 3, SNR121; 4, SNR135; 5, SNR110; 6, SNR102; 7, SNR79; 8, SNR52; 9, unfolded SNR52 and SNR79. (B) Relative [{theta}]222 of SNase R and its N-terminal fragments, calculated from (A) using the formula: Relative [{theta}]222 (%) = [{theta}]222F/[{theta}]222Sx100, where [{theta}]222F is the mean residue ellipticity of the peptide fragment at 222 nm and [{theta}]222S is the mean residue ellipticity of SNase R at 222 nm.

 
It is known that the ellipticity in the near-UV CD region, particularly around 277 nm, reflects the asymmetry of the environment of aromatic groups and is usually considered as an index of the uniqueness of protein tertiary structure. There are nine potential aromatic residues responsible for near-UV CD signal, they are Y–2, Y27, Y54, Y85, Y91, Y93, Y113, Y115 and W140 in SNase R. For both SNR135 and SNR 121, the W140 residue has been removed. Therefore, SNR135 and SNR121 are comparable. Figure 2Go shows the relative values of [{theta}]277 for SNase R and its N-terminal fragments, which are indicators of the environment of the aromatic residues in polypeptides and represent changes in the tertiary structure during elongation of the N-terminal peptide. Compared with SNase R in the native state and in the unfolded state (in 6 M GdnHCl), SNR141 has almost the same value of [{theta}]277 as that of SNase R, which reflects the presence of a rigid asymmetrical environment of aromatic side chains, i.e. the presence of a rigid tertiary structure corresponding well to the fact that both SNase R and SNR141 have the same unfolding and refolding patterns with a highly cooperative transition during unfolding and refolding in GdnHCl as monitored by fluorescence (Zhou and Jing, 1996Go). SNR135 has a larger value of [{theta}]277 than the other shorter N-terminal fragments, showing that SNR135 still contains some residual tertiary structure elements, though the rigid tertiary structure does not appear in SNR135 as indicated by its unfolding behavior in GdnHCl without a cooperative transition (Zhou and Jing, 1998Go). Compared with SNR135, the near-UV CD signal at 277 nm for SNR121 is apparently reduced and closed to that of the denatured SNase R, indicating that there is little or no tertiary structure in SNR121 and the other fragments except for SNR141.



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Fig. 2. Relative [{theta}]277 of SNase R and its N-terminal fragments. (A) Original near-ultraviolet CD spectra of SNase R and its N-terminal fragments. From bottom to top: SNase R, SNR141, SNR135, SNR102, SNR121, SNR110, SNase R in 6 M GdnHCl, SNR79 and SNR52. (B) Relative [{theta}]277 of SNase R and its N-terminal fragments, calculated from (A) using the formula: Relative [{theta}]277 (%) = [{theta}]277F/[{theta}]277Sx100, where [{theta}]277F is the mean residue ellipticity of the peptide fragment at 277 nm and [{theta}]277S is the mean residue ellipticity of SNase R at 277 nm.

 
SNR121 still has relatively compact size

One of the most important properties of the molten globule state is its relatively compact size. The molecular sizes of SNase R and its N-terminal fragments have been determined using molecular exclusion chromatography on a protein I60 column with a Waters 994 system. Figure 3Go shows the relative Stokes radii of SNase R and the N-terminal fragments SNR102, SNR110, SNR121, SNR135 and SNR141. It can be seen that the molecular size of the N-terminal fragments is not proportional to their peptide chain length. Although the N-terminal fragments were regarded as partially unfolded molecules with certain amounts of residual structure (Shortle and Meeker, 1989Go; Jing et al., 1995Go), the relative Stokes radii of the fragments are different. The relative Stokes radius of SNR102 is much greater than that of SNase R and the other fragments, suggesting that SNR102 has a much more open and extended structure. The Stokes radius of SNR135 is also relatively great, indicating that its conformation is relatively expanded, although it contains almost all the amino acid residues forming the major secondary structure observed in SNase R and retains the capacity to fold into a native-like conformation in the presence of pdTp and Ca2+ (Zhou and Jing, 1998Go). The smaller Stokes radius of SNR141 indicates that SNR141 is more compact than SNase R, which may be due to the more compact ß-strands existing in SNR141 (Jing et al., 1995Go). Finally, the relative Stokes radii of SNR121 and SNR110 are both smaller than those of SNase R and the other fragments, suggesting that SNR121 and SNR110 still have a very compact molecular size.



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Fig. 3. Relative Stokes radii (relative RS) of SNase R and its N-terminal fragments. The relative Stokes radii were calculated using the formula: Relative RS = RSF/RSS, where RSF is the Stokes radius of the peptide fragment and RSS is the Stoked radius of SNase R.

 
SNR121 has unique hydrophobic surface area as indicated by the much stronger affinity of a fluorescent hydrophobic probe (ANS) to the fragment

Ptitsyn (1995b) suggested that the semi-flexible nature of the molten globule state permits some internal non-polar groups to become exposed to water thus making the surface of this state more hydrophobic than that of the native state. One of the clear manifestations of this phenomenon is that the molten globule state can bind non-polar molecules from solution much more strongly than the native state. A typical example is that ANS, which binds to solvent-accessible clusters of non-polar atoms in the native state (Stryer, 1965Go), is bound more strongly to molten globule state proteins (Semisotnov et al., 1987Go, 1991Go; Fink et al., 1993Go). Therefore, ANS provides a particularly convenient, sensitive test for the molten globule state as its binding leads to a large increase in its fluorescence (Ptitsyn, 1995bGo).

Figure 4Go shows the relative intensities of ANS-binding fluorescence of SNase R and its N-terminal fragments. SNR121 has the highest ANS-binding fluorescence. In addition, the fluorescence maximum for SNR121 is blue shifted compared with that of SNase R and the other fragments. These results demonstrate that a solvent-accessible non-polar core for SNR121 has been formed, which greatly increases the affinity of ANS to the fragment. In contrast with SNR121, the shorter and larger fragments have much weaker ANS-binding fluorescence than SNR121. The regular change of the ANS-binding fluorescence from SNR52 to SNase R as shown in Figure 4Go is apparently due to conformational changes of the polypeptide chain during its elongation. The reason for the ANS-binding fluorescence decrease for other fragments will be discussed later. The unique ANS-binding fluorescence of SNR121, i.e. the existence of a maximum of the ANS affinity to SNR121 during peptide elongation from SNR52 to SNase R, provides clear evidence that SNR121 is in the molten globule state.



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Fig. 4. Relative intensities of ANS-binding fluorescence of SNase R and its N-terminal fragments. (A) Original fluorescence emission spectra for ANS in the presence of SNase R and its N-terminal fragments. From bottom to top: ANS itself, SNR52, SNR79, SNase R, SNR102, SNR141, SNR110, SNR135, SNR121. (B) Relative intensity of ANS-binding fluorescence (at 500 nm), calculated from (A) using the formula: Relative fluorescence (%) = FF/FSNR121x100, where FF is the intensity of ANS-binding fluorescence of the peptide fragment and FSNR121 is the intensity of ANS-binding fluorescence of the fragment SNR121.

 
Molten globule state may not be beneficial to protein activity expression

It has been noted that the activities of the large N-terminal fragments of SNase R differ depending on their peptide length. Compared with SNase R, SNR102 and SNR110 have very little activity, while the activities of SNR121, SNR135 and SNR141 are 0.8, 40.6 and 79.2% of SNase R, respectively. Therefore, higher activity occurs only when the polypeptide reaches a certain length. However, like SNase R, SNR141 and SNR121 are also activated in 0.1 M GdnHCl, but such activation is not observed for SNR135, SNR110 and SNR102. The difference in activity between SNR121 and SNR135 may be due to the further deletion of amino-acid residues from the C-terminus, which could damage the conformational adjustment ability of the polypeptide and could affect the interaction of the enzyme active site with its substrate. This suggestion has been proved by the observation that a competitive inhibitor of SNase R, pdTp, can make the far-UV CD spectrum of SNR135 look like that of SNase R with negative peaks at 208 and 222 nm in the presence of Ca2+. However, the presence of pdTp and Ca2+ has little effect on the far-UV CD spectrum of SNR121, suggesting that SNR135 has a more flexible conformation than SNR121 (Jing et al., 1995Go). ANS-binding fluorescence spectra also show that the ligand binding does not change the surface hydrophobicity of SNR121 (Figure 5AGo). However, in 0.1 M GdnHCl, the relative ANS-binding fluorescence intensity decreases greatly, and a clear blue shift of the emission maximum is observed in the presence of the ligands (Figure 5BGo). The phenomenon does not appear in the ANS-binding fluorescence spectra for the other N-terminal fragments given the same conditions (data not shown). It is very interesting that it is at 0.1 M GdnHCl that SNR121 is activated as described above. The changes in intensity and emission maximum of ANS-binding fluorescence shown in Figure 5Go indicate that the conformation (surface hydrophobicity) of SNR121 and its ability to bind the substrate are altered at 0.1 M GdnHCl. The activation for SNR121 by GdnHCl could be caused by the transformation of the conformation at its active site to a shape more favourable for expression of the activity, suggesting that the SNR121 molten globule state is not beneficial to its activity expression.



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Fig. 5. Fluorescence emission spectra of ANS for SNR121 under different conditions. The concentration was 8 µM for SNR121, 10 mM for Ca2+ and 80 µM for ANS. A, Fluorescence spectra of ANS with SNR121 in 20 mM Tris–HCl, pH 7.4 (solid line) and in the presence of pdTp and Ca2+ (dashed line). B, Fluorescence spectra of ANS with SNR121 in 20 mM Tris–HCl, pH 7.4 containing 0.1 M GdnHCl (solid line) and in the presence of pdTp and Ca2+ (dashed line). C, Fluorescence spectra of ANS in 20 mM Tris–HCl, pH 7.4 (solid line) and in 20 mM Tris–HCl, pH 7.4 containing 0.1 M GdnHCl (dashed line).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Result
 Discussion
 References
 
One important approach for understanding the mechanism of nascent peptide folding is to study the relationship between the formation process of the native conformation and the functional expression of peptides during the elongation of the peptide chain (Tsou, 1988Go). Previous experiments have demonstrated that the folding of nascent peptide chains may begin early in the synthesis process and that the amount of ordered structure increases with increasing peptide chain length. However, some structural adjustments are necessary for the newly synthesized polypeptide to attain its final native conformation (Jing et al., 1995Go; Tian et al., 1998Go; Zhou and Jing, 1998Go). This paper concentrates on whether the molten globule state occurs, and if it does, when does it form during nascent peptide folding. After characterization of the conformation features of the N-terminal fragments of SNase R, only SNR121 was found to fulfill all the criteria for the molten globule state. Since SNR121 already contains more than 80% amino acid sequence of the nuclease, the molten globule state should occur at a later stage of peptide elongation of the nuclease, at which hydrophobic collapse of the polypeptide chain occurs driven by the hydrophobic force, which leads to the formation of a solvent-accessible non-polar core, characterized by the high ANS-binding fluorescence. So the molten globule state is not only a kinetic intermediate in the protein folding pathway (Ptitsyn, 1995aGo,bGo; Creighton, 1997Go) but should also be a necessary stage during nascent peptide folding, which makes the nascent peptide become globularized and may generate an environment able to adopt a compact structure for the peptide chain and able to interact with some molecular chaperones, as stable association with the chaperonin is thought to require a critical length of polypeptide (Frydman et al., 1994Go; Hartl et al., 1994Go).

Now that the increase in ANS-binding fluorescence can be used to monitor the formation of the molten globule state, analysis of the changes in ANS-binding fluorescence during peptide chain elongation will contribute to our understanding of the process of the conformational changes during nascent peptide folding. Although SNR102 contains a certain amount of residual ordered secondary structure as described previously (Jing et al., 1995Go), as show in Figure 3Go, the relative size radius of SNR102 is larger than the other larger N-terminal fragments. This suggests that the overall conformation of the shorter fragment is much more expanded and closer to the unfolded state, which may explain why the ANS-binding fluorescence of SNR102 is lower. Compared with SNR102, SNR110 has a more ordered secondary structure (Figure 1Go), and a more compact size (Figure 3Go). However, the intensity of ANS-binding fluorescence for SNR110 is much lower than that for SNR121, suggesting that the solvent-accessible non-polar core for SNR110 has not formed, i.e. the hydrophobic collapse of the polypeptide has not yet occurred at this stage. The conformational stage of SNR110 can be characterized as pre-molten globule or pre-collapsed state (Ptitsyn, 1995bGo; Hilser and Freire, 1997Go). The pre-molten globule state should be a structural basis to form the molten globule state during peptide elongation. It can also be seen from Figure 4Go that the ANS-binding fluorescence intensity decreases from SNR121 to SNase R. The decrease in ANS-binding fluorescence for SNR135 indicates that ANS is being released from SNR135, which suggests that the non-polar core of the polypeptide is being screened from the solvent during peptide elongation. It is the conformational change in SNR135 that gives the polypeptide a greater ability to form a native-like conformation in the presence of the ligands and a much higher activity than SNR121. Although SNR135 has some properties of the molten globule state, such as relatively pronounced secondary structure and compact size, as well as a relatively higher ANS-binding fluorescence, the initial state of SNR135 does not represent the molten globule state but lies between the molten globule and the native state (Zhou and Jing, 1998Go), which can be characterized as the post-molten globule state. In this state, most residues responsible for the folding of the native backbone may occupy approximately native positions, which gives the polypeptide a greater ability for conformational adjustment in the presence of the substrate, but does not produce a tightly packed tertiary structure detected by near-UV CD spectrum and unfolding experiments (Zhou and Jing, 1998Go). The conformation of SNR135 represents a key transition from the molten globule state to the native conformation in nascent peptide folding of SNase R. Furthermore, as the polypeptide chain elongation takes place, it finally possesses the tightly packed tertiary structure shown in SNR141 with a conformation which is indistinguishable from the full-length nuclease, with almost the same secondary and tertiary structures (Figures 1 and 2GoGo), as well as the same unfolding and refolding behaviors (Jing et al., 1995Go; Zhou and Jing, 1996Go). However, since the activity of SNR141 is only 79.2% of the full-length enzyme, further conformational adjustment from SNR141 to the full-length enzyme is necessary for full expression of enzyme activity.

Since even SNR52 and SNR79 have a certain amount of residual ordered secondary structure and the amount of secondary structure increases from SNR52 to SNR102 (Tian et al., 1998Go), then there is a process of secondary structure accumulation before formation of the pre-molten globule stage. The N-terminal fragments with different chain lengths provide a good model in vitro to understand the nascent peptide folding pathway in physiological conditions. The nascent peptide folding of SNase R is a hierarchical process that at least includes secondary structure accumulation, pre-molten globule state, molten globule state, post-molten globule state and finally the native state. During nascent peptide folding, the peptide undergoes apparent conformational adjustments. The conformational pathway of the polypeptide chain of SNase R growing from its N-terminus in vitro seems to parallel that of the protein folding pathway as compared with the results described by Hilser and Freire (1997).


    Acknowledgments
 
This work was supported by grants (Nos 95-YU-15 and 39570170) from the China Committee for Science and Technology.


    Notes
 
1 To whom correspondence should be addressed; email: Jingenx{at}public.east.cn.net Back


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 Abstract
 Introduction
 Materials and methods
 Result
 Discussion
 References
 
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Received January 19, 1999; revised September 27, 1999; accepted October 11, 1999.