A Solid Phase Synthetic Study of Structure-Function Relationships in the Amino-terminal Region of Staphylococcal Nuclease

Abstract Synthetic peptides corresponding to residues (6 through 47), (9 through 47), (10 through 47), (11 through 47), (12 through 47), (16 through 47), and (18 through 47) in staphylococcal nuclease were prepared by the Merrifield solid phase method. Whereas synthetic-(6–47) and synthetic-(9–47) are about equally effective in generating both RNase and DNase activity upon mixing with native nuclease tryptic fragment nuclease-T-(49–149), synthetic-(10–47) is only partially effective and synthetic-(11–47) through synthetic-(18–47) generate essentially no activity. Synthetic-(6–47) is also the most capable of forming a stable complex with nuclease-T-(49–149), as judged by the resistance of the DNase activity of this semisynthetic nuclease-T species to destruction by trypsin in the presence of deoxythymidine 3',5'-diphosphate and Ca++, as well as by the extent of the shift and intensification of the fluorescence emission spectrum of the tryptophanyl residue in nuclease-T-(49–149). By these latter criteria, synthetic-(9–47) and synthetic-(10–47) form progressively weaker nuclease-T complexes than does synthetic-(6–47). On the other hand, all of the synthetic peptides through synthetic-(18–47) are able to bind, at least slightly, with nuclease-T-(49–149) as judged by the ability to enhance the binding of 125I-[nuclease-T-(49–149)] to antinuclease antibody. The results for these peptides, considered together with structural features in the 2 A model of nuclease elucidated by Dr. F. A. Cotton and his associates, suggest important structural functions for residues lysine 9 and glutamic acid 10 in both nuclease-T and nuclease.

Whereas synthetic-(6-47) and synthetic-(9-47) are about equally effective in generating both RNase and DNase activity upon mixing with native nuclease tryptic fragment nuclease-T-(49-149), synthetic-(10-47) is only partially effective and synthetic-(1 l-47) through synthetic-(1 8-47) generate essentially no activity.  is also the most capable of forming a stable complex with nuclease-T-(49-149), as judged by the resistance of the DNase activity of this semisynthetic nuclease-T species to destruction by trypsin in the presence of deoxythymidine 3',5'-diphosphate and Ca++, as well as by the extent of the shift and intensification of the fluorescence emission spectrum of the tryptophanyl residue in nuclease-T-(49-149). By these latter criteria, synthetic-(9-47) and synthetic-(10-47) form progressively weaker nuclease-T complexes than does synthetic-(6-47).
On the other hand, all of the synthetic peptides through synthetic-(18-47) are able to bind, at least slightly, with nuclease-T-  as judged by the ability to enhance the binding of 1251- [nuclease-T-(49-149)] to antinuclease antibody.
The results for these peptides, considered together with structural features in the 2 A model of nuclease elucidated by Dr. F. A. Cotton and his associates, suggest important structural functions for residues lysine 9 and glutamic acid 10 in both nuclease-T and nuclease. lacking the first 5 residues at the amino terminus (2). This derivative is completely active and has physical characteristics that are extremely similar to those of the native enzyme. Further tryptic digestion of nuclease or nuclease-(6-149) yields two smaller fragments, nuclease-T-(6-48) (residues 6 through 48) and nuclease-T-(49-149) (residues 49 or 50 through 149), which are, themselves, structureless and inactive but can combine noncovalently to form nuclease-T, a species having about 8 to 10% of the activity of nuclease and a structure that also appears to be very similar to that of the native enzyme (2,3).2 Based on the properties of both nuclease- (6-149) and nuclease-T, at least the first 5 amino acid residues at the amino terminus of nuclease are unessential to enzymic structure and function.
Solid phase synthesis of a fragment corresponding to residues 6 through 47 has been accomplished (4) and applied to studies of the function of certain amino acids in nuclease-T-(6-48)3 in the formation of active nuclease-T (5-7).
Crude synthetic-(6-47) can combine with nuclease-T-  to yield a partially active semisynthetic nuclease-T (4).  is about as effective as synthetic-  in forming the active complex (5)) suggesting that residues 6 to 8 are unessential for the formation of active nuclease-T.
On the other hand, synthetic-(18-47) does not form an active complex with nuclease-T-(49-149) (5). We have now prepared and characterized several other solid phase synthetic truncated fragments of nuclease-T-(6-48) in order to elucidate further the structural requirements, at the amino terminus of nuclease, for enzymic activity and conformation.

MATERIALS AND METHODS
Synthetic peptides were prepared by the solid phase procedure of Merrifield (8 carbonyl proline to the Merrifield resin and the repetitive amino acid coupling routine have been described previously (4). The procedures for deblocking and isolation of the crude completed peptides by HF treatment of peptidyl resin, followed by piperidine treatment of the resin-cleaved, partially deblocked peptide and final Sephadex G-25 fine fractionation, have also been described (4)(5)(6)(7).
Amino acid compositions were determined with a Spinco model 120B automatic amino acid analyzer on samples hydrolyzed under reduced pressure with 6 N HCI, in the presence of 1 ~1 of mercaptoethanol and 10 ~1 of 5% phenol, at 110' for 24 hours. Amino acid analyses of peptides previously subjected to nitrous acid treatment (9) prior to acid hydrolysis yielded the amount of residual e-trifluoroacetyl lysine remaining due to incomplete deblocking by piperidine (4). Peptide maps were prepared as described previously (7).
Nuclease activity against both heat-denatured salmon sperm DNA and yeast RNA was determined spectrophotometrically (10). Fluorescence emission spectra were determined with an Aminco-Bowman recording spectrophotofluorometer (11) on samples excited at 295 rnp (12). 1251 content in liquid samples was determined by counting these samples in 10 ml of Bray's solution (13) with a Nuclear-Chicago model Mark II scintillation counter on the carbon 14 channel.

RESULTS
Synthetic peptides were prepared which consisted of residues (6 through 47), (9 through 47), (10 through 47), (11 through 47), (12 through 47), (16 through 47), and (18 through 47) of nuclease. The amino acid compositions for these peptides, shown in Table  I, correspond closely to the expected values based on the structure of nuclease-T-(6-48), as presented in Fig. 1. The synthetic peptide preparations were further examined by correlation of tryptic peptide maps with the map for nuclease-T-(6-48) (2), as shown for synthetic-(g-47), -(ll-47), and -(18-47) in Fig. 2. All three peptide maps contain ninhydrin-and Pauly-positive Components I and II (cross-hatched), shown previously to correspond to the tryptic peptides 36 to 47 and 25 to 28, respectively (2, 7). As expected, however, only the map for synthetic-(9-47) contains the component (III) corresponding to tryptic Peptide 10 to 16. The tryptic Peptide 11 to 16, which should be present for synthetic-(11-47), has not been identified but appears to be migrating in the abnormally large dense area in which Component II is located.
In addition, synthetic-(9-47) and synthetic- (11-47) show a component (IV) corresponding to tryptic The DNase and RNase activities generated by all of the synthetic peptides upon mixing with nuclease-T-(49-149) were measured.
For DNase activity, 0.01 pmole of each synthetic peptide was incubated with 0.01 bmole of nuclease-T-(49-149) in 600 ~1 of 0.05 M Tris buffer, pH 8, for 1 hour at room temperature before activity assays were carried out. A similar incubation, except in a total of 60 ~1, was carried out prior to RNase activity assays. As shown in Table II, synthetic-(6-47) and synthetic-(9-47) generate the highest levels of activity, while synthetic-(10-47) is much less effective. All other synthetic peptides effect no significant activity.
In addition, the relative amount of enzymic activity generated for each peptide, as compared with that effected for synthetic-(6-47), is similar for both DNA and RNA substrates.
Clearly, an abrupt transition in ability to promote enzymic activity upon addition to nuclease-T-(49-149) occurs upon loss of residues 9 and 10. As found previously (4,5,7), the DNase activity generated for crude synthetic- (6-47) is about 4 '% that effected with nuclease-T-(6-48). Since it is apparent that synthetic-(6-47), -(Q-47), and -(lo-47) can effect enzymically active complexes with nuclease-T-(49-149) (Table II), the stability of these complexes to tryptic digestion was studied and compared with that found for native nuclease-T After incubation at room temperature for 1 hour, 1 ~1 of 1% trypsin was added to each sample and incubation continued. The DNase activities of the trypsin-treated peptide mixtures, as well as of the untreated controls, were measured at various times after trypsin addition.
On the other hand, as shown in Table III, the activities generated by all synthetic peptides upon incubation with nuclease-T-  are protected, to differing extents, against proteolytic destruction by inhibitor and calcium ions. Such protection corresponds to the effect found for the mixture of nuclease-T-(6-48) with nuclease-T-(49-149). It has been shown previously (20) that nuclease-T-(6-48), when added to nuclease-T-(49-149), causes a distinct blue shift and intensification of the fluorescence emission spectrum of the tryptophan residue in nuclease-T-(49-149), this effect being interpreted as due to the burying of the tryptophanyl side chain in a hydrophobic environment during the folding of the native fragments to form nuclease-T.

Issue of September
This effect was studied for the series of analogues from synthetic-(6-47) through synthetic-(U-47).
Nuclease-T-(49-149) (9 x 1O-3 pmole) was incubated with each of the crude synthetic peptides (9 x 10e2 pmole, or a IO-fold molar excess over nuclease-T-(49-149)) in 0.4 ml 0.05 M Tris buffer, pH 8, containing 0.01 M CaCl? and 0.0001 M deoxythymidine 3',5'-diphosphate. After incubation for 1 hour at room temperature, the fluorescence emission spectrum of each sample, as well as of nuclease-T-  alone, was recorded. The spectrum for nuclease-T-  alone was corrected for the slight emission found for the buffer; the spectrum for each (synthetic peptide)- [nuclease-T-(49-149)] mixture was corrected for the spectrum shown for the synthetic peptide alone in buffer. The corrected spectra are shown in Fig. 3. Clearly, the intensification and shift of the spectral maximum from that of nuclease-T-  alone were greatest with synthetic-(6-47) and progressively less for synthetic-(9-47) through synthetic-(12.47).
Synthetic-( 16-47) and synthetic-( 18-47) produced no significant effect on the fluorescence of nuclease-T-(49-149). The relative ability of each fragment to effect the shift and intensification of fluorescence corresponds quite closely to the relative abiliby of each peptide to produce enzymic activity (Table  II).
As a further means of characterizing the binding of nuclease-T-  with each of the synthetic peptides, advantage was taken of the ability of nuclease-T-  to enhance the binding of Y- [nuclease-T-(49-149)] to antinuclease antibody (15), probably as a result of formation of antigenic sites specified by conformat,ional features of nuclease-T. Previous results (15) have shown that synthetic-(6-47), -(g-47), and -(l&47) are all effective in enhancing 1251- [nuclease-T-(49-149)] binding. With the use of a procedure similar to that described previously (15) Samples were prepared and spectra were measured as described in the text.  mpmole) were added to each sample and incubation continued at 4" for an additiona, 24 hours. Then, 50 ~1 of sheep antirabbit immunoglobulin G antiserum were mixed with each sample. After 48.hour incubation at 4", the precipitate in each sample was centrifuged, washed with cold NaCl buffer solution, dissolved in 1 ml of 0.1 N NaOH, and tested for radioactivity content.
For controls, normal rabbit immunoglobulin G was used instead of antinuclease antibody, in an amount necessary to produce a similar amount of precipitate as judged by the Azso of solutions of the final, dissolved precipitates.
Each trial with antinuclease was corrected for the radioactivity nonspecifically bound to precipitate in the appropriate control (about 5 to 7 y. of the total radioactivity bound to antinuclease). The results are summarized in Table IV. As expected from previous studies on synthetic-(6-47), synthetic-(g-47), a,nd synthetic-(18.47) (15), all of the synthetic peptides are able to effect an enhancement of l'%[nuclease-T-(49-149)] binding to antinuclease. These data are discussed further below.

DISCUSSION
Synthetic analogues, differing from synthetic-(6-47) by deletions of residues at the amino-terminal end, have been tested for the extent to which they interact with nuclease-T-(49-149). Based on the ability to generate nuclease activity with the latter 4722 Functional Role of  and synthetic-(9-47) are the most effective in forming a specifically folded semisynthetic enzyme complex corresponding to native nuclease-T. On the other hand, deletion of residue 9 (lysine) to yield synthetic-(10-47) results in a marked decrease in the ability to effect activity.
Further deletion of residue 10 (glutamic acid) to give synthetic-(ll-47) results in essentially complete loss in this property.
The activity generated by each of synthetic-(6.47), -(9.47), and -(lo-47) appears to be due to the specific formation of a semisynthetic nuclease-T complex as indicated by the resistance of this activity to tryptic inactivation in the presence of inhibitor and Ca* (Table III).
On the other hand, the tightness of the semisynthetic nuclease-T complexes formed probably decreases with decreasing chain length from synthetic-  to synthetic-(lo-47), based on the differences in tryptic stability shown.
The ability of the peptide fragments to form a complex with nuclease-T-  having a conformation essential for activity is generally paralleled by a shift and intensification of the fluorescence emission spectrum of the tryptophanyl residue of nuclease-T-(49-149).
Thus, the extent of shift and intensification shown in Fig. 3 is greatest for synthetic-(6-47), which effects the greatest activity, and insignificant for two of those peptides, synthetic-  and synthetic-(l&47), which effect no activity.
A significant decrease in fluorescence intensification and spectral shift is effected with the loss of residues 6 through 8 (from synthetic-  to synthetic-(9-47)) without a corresponding change in enzymic activity (Table II).
This spectral difference suggests that synthetic-(9-47) forms a somewhat looser but essentially equally active complex with nuclease-T-(49-149), a conclusion also suggested by the decreased tryptic stability of the semisynthetic nuclease-T complex formed by synthetic-(9-47) ( Table III).
It seems reasonable to assume that the enhancement of 1251- [nuclease-T-(49-149)] binding can occur if the synthetic peptide can at least partially join in complex with nuclease-T-(49-149) to generate some, if not all, of the conformation-dependent antigenic sites of nuclease-T.
In addition, as has been suggested from studies on the immunological properties of fragments of myoglobin (21), antibody against a specific antigenic site may combine with the fragment only when it assumes its native conformational state. The antibody may thus "trap" a particular conformational variant of the system in question, in this instance, semisynthetic nuclease-T.
In this light, it may be suggested that synthetic-(11-B), -(12-47), -(16-47), and -(18-47), although they generate no active complex with nuclease-T-(49-149), can bind with this fragment well enough to effect, at least partially, the formation of a semisynthetic complex that is conformationally related to nuclease-T. That synthetic-(11-47) and synthetic-  can bind loosely to nuclease-T-(49-149) is, in fact, also suggested by the small fluorescence shift and intensification effected by these peptides (Fig. 3). The data for synthetic-  are in general agreement not only with previous immunochemical data (15), but with the previous demonstration that synthetic -(18-47) can bind to Sepharose to which nuclease-T-(49-149) has been covalently bound (5).
Based on the properties shown above for the shortened peptides synthetic-(6-47) through synthetic-(l&47), it may be concluded that residues 9 and 10 appear to be critical in allowing synthetic-(6-47) to form an active semisynthetic complex with nuclease-T-(49-149).
Loss of residue 10 causes essentially complete loss of the ability to effect enzymic activity.
On the other hand, synthetic-(11-47) through synthetic-(G-47), although they generate no activity, appear to be partially effective in forming a complex with nuclease-T-  with at least some of the conformational features of nuclease-T. 4 The importance of residues 9 and 10 in maintaining nuclease structure is suggested by the localization of these residues in the three-dimensional structure of nuclease.s Although neither lysine 9 nor glutamic acid 10 is close to the catalytic or substrate binding regions, both residues appear to be involved in interactions which could stabilize the nuclease structure by linking a significant part of the large pleated sheet structure (residues 13 to 35) to other parts of the folded nuclease molecule.
Further investigations of the crystal structure of nuclease, now in progress at the Massachusetts Institute of Technology, as well as the synthesis of other peptide analogues in the nuclease-T-(6-48) region, will allow a more detailed description of the participation of residues 9 and 10 in nuclease structure and function.  also has been prepared and shown to be able to form an active semisynthetic complex with nuclease-T-(49-149). This complex has properties similar to those for the complex with synthetic-(6-47) but appears to be somewhat less stable. Based on the earlier observation that nuclease-(B-149) is as active as nuclease itself (2), it may be suggested that, although the NH,terminal 5 residues of nuclease serve no essential function, the existence of these residues in synthetic-(l-47) partially destabilizes the normal folding with nuclease-T-(49-149).
The mechanism for interference has not been investigated.