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Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7014-7018, October 1987 Conformational studies of a-globin in 1-propanol: Propensity of the to limit the sites of proteolytic cleavage (semisynthesis/a-helical conformation/protein structure/organic cosolvents/segmental flexibility) K. SUBRAMONIA IYER AND A. SEETHARAMA ACHARYA The Rockefeller University, 1230 York Avenue, New York, NY 10021 Communicated by Christian B. Anfinsen, June 15, 1987

ABSTRACT Selective condensation of the unprotected The protease-catalyzed reverse proteolysis for the semi- fragments ofa-globin-namely, a1..30 and a31.141-is catalyzed synthesis of noncovalent analogues of proteins has been by Staphylococcus aureus V8 protease in the presence of 25% emerging as a useful procedure to prepare covalent variants 1-propanol. The propensity of 1-propanol to induce the a- of proteins (7). The presence of organic cosolvents is crucial helical conformation and to generate a "native-like" topology for the protease-catalyzed ligation of the complementary for the polypeptide chain has been now investigated in an fragments of the fragment-complementing systems, and so attempt to understand the molecular basis of this - far glycerol has been found to be the only good cosolvent for catalyzed stereospecific condensation. Removal of heme from these investigations (5). However, during the course of our the a-chain decreases the overall a-helical conformation of the semisynthetic studies of a-chain from the complementary protein considerably. A significant amount of the a-helical fragments, we found that 1-propanol was a better organic conformation is restored in the presence of25% 1-propanol and cosolvent compared with glycerol for the V8 protease- the ofa-globin by V8 protease becomes more selective catalyzed reformation ofGlu-30-Arg-31 peptide bonds in an concomitant with the increase in helicity. V8 protease digestion equimolar mixture of fragments a1_30 and a31.47 as well as ofa-globin at pH 6.0 and 40C occurs at Glu-30, Asp47, Glu-27, a1_30 and a31.141 (8, 9). Efficient semisynthesis occurred even and Glu-23 in the absence of 1-propanol. In the presence of when the concentration of 1-propanol was as low as 25% and 25% 1-propanol, the digestion is selective to the peptide bond with the heme-free globin fragments. Furthermore, the ofGlu-30. This selectivity appears to be a characteristic feature semisynthesis was very specific for the Glu-30 and Arg-31 of the native conformation of a-chain (polypeptide chain with peptide bond. V8 protease did not induce the peptide bond bound heme). 1-Propanol induces the a-helical conformation formation in shorter amino-terminal fragments with carbox- into RNase S. peptide also. However, this increased helical yl-terminal glutamic acid-i.e., a1.23, a1.27, and a31.141. The conformation did not protect the RNase S peptide from V8 selectivity in the reverse proteolysis in the fragment-com- protease digestion at the Glu-9-Arg-10 peptide bond. RNase plementing system is generally considered as a reflection of S is in an a-helical conformation in RNase an the "native-like" ordered structure in the mixture of com- peptide S, plementary fragments (7). However with a-chain, the remov- interacting fragment-complementing system of S protein and S al of heme to prepare the apoprotein (globin) results in the peptide. S peptide is resistant to V8 protease in this loss of "native conformation" of the polypeptide chain. conformation. Thus, the resistance of a peptide bond in a Therefore, it appeared that 1-propanol may be inducing a segment of a protein to protease digestion appears to be a native-like conformation in the globin fragments or globin consequence of the secondary structure as well as the tertiary under the conditions used for semisynthesis. In an interactions of this segment with the rest of the molecule. The attempt to begin to answer these aspects, we have now results suggest that the 1-propanol induces a-helical confor- investigated the influence of 1-propanol on the overall con- mation into segments of a-globin as well as packing of these formation of a-globin using far-ultraviolet circular dichroism helices in a native-like topology. (far-UV CD) and susceptibility of the polypeptide chain to proteolysis in the presence of 1-propanol. There has been considerable interest in recent years in the structure of proteins in organic (1). The low- MATERIALS AND METHODS temperature studies ofenzymes, cryoenzymology, as well as low-temperature crystallography have necessitated the use of Hemoglobin A (HbA) and a-chain of HbA that reacted with organic solvents in these protein structural studies (2). In p-hydroxymercuribenzoate (pHMB) were prepared as we have described (10). Globin from a-chain was prepared by the addition, many enzyme-catalyzed reactions are also being method of Rossi-Fanelli et al. (11). Staphylococcus aureus investigated in organic media to enhance the industrial V8 protease was obtained from Miles Laboratories (Naper- applications, specifically to take advantage of the high ville, IL). selectivity and specificity of the enzyme (3). With proteases, CD Spectra. The CD spectra was measured in an Aviv it has been possible to achieve "reverse proteolysis"-i.e., 6ODS spectropolarimeter fitted with a thermostated cell use proteases to catalyze the synthesis of peptide bonds by holder. Ellipticity values at 222 nm were converted to mean incorporating large amounts of organic solvents (i.e., like molar residue ellipticity values using the equation: [10222 = , glycerol, etc.) in the reaction mixtures (4). Mr/100LC, where 6 = degrees, L = cell path length (dm), C The reformation of ribonuclease A from RNase S (5) and = concentration (g/ml), and Mr = mean residue molecular nuclease A from nuclease T (6) are the classical examples of weight. this reverse proteolysis approach for semisynthesis of pro- Digestion by V8 Protease. pHMB a-chain (in the carbon- teins (7). monoxy form) and a-globin at a concentration of 1 mg/ml in 10 mM NH4OAc at pH 6 or pH 8 was incubated with V8 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: pHMB, p-hydroxymercuribenzoate; RP-HPLC, re- in accordance with 18 U.S.C. §1734 solely to indicate this fact. versed-phase HPLC.

7014 Downloaded by guest on September 27, 2021 Biochemistry: lyer and Acharya Proc. Natl. Acad. Sci. USA 84 (1987) 7015 protease for 24 hr and the substrate-to-enzyme ratio was from the unprotected globin fragments a1l30 and a31_141 maintained at 200:1. After digestion at the desired tempera- suggest that the conformation of a-globin in 25% 1-propanol ture, the digests were lyophilized and analyzed by reversed- at pH 6.0 is probably native-like-i.e., the topology of the phase HPLC (RP-HPLC) (12). helical segments of a-globin may resemble that of a-chain. The digestion of a-globin by V8 protease at pH 6.0 was RESULTS carried out in the presence and absence of 1-propanol and compared with that of a-chain to determine whether the 1-Propanol-Induced Increase in the Helicity of a-Globin. overall tertiary interactions of a-globin in 1-propanol have The high helical content of a-chain is a consequence of the any native-like features. A 24-hr digestion was carried out so cooperative noncovalent interaction ofthe polypeptide chain that the small differences in the activity of V8 protease [in the and heme (13). Removal ofheme results in a considerable loss presence and absence of 1-propanol (25%)] do not complicate of a-helical conformation of polypeptide chain (Fig. 1). CD the interpretation of the results. At pH 6.0, the a-globin was spectra of a-globin in the absence of 1-propanol (Fig. 1) digested extensively by V8 protease both at 25°C and 37°C reveals that >50% of the molar ellipticity of the a-chain was (Fig. 2C). On the other hand, when the temperature was lost on removal of heme. 0222 of a-globin at pH 6.0 and 40C was only about 40% of the a-chain. However, when 1- lowered to 4°C, the digestion was significantly limited. The propanol was incorporated into the system, the helical four components eluting around 30, 32, 36, and 45 min content of a-globin was significantly increased. 6222 of a- represent the fragments a1l23, a1.27, a1l30, and a3147, respec- globin in 25% 1-propanol was nearly 80% of that of a-chain. tively (15), and represent the four small molecular weight Thus, the loss of secondary structure of the a-chain that components (Fig. 2A) generated as the major cleavage occurs concomitant with the removal ofheme is compensated to a considerable degree on inclusion of25% 1-propanol in the buffer. The propensity of the apoprotein to take up the more a-helical conformation is a function of 1-propanol concen- tration (Fig. 1 Inset). However, the increase in the ellipticity of the a-globin at 0222 is not directly related to the concen- tration of the alcohol. In the range of 0-10%, a very small change in the secondary structure of the protein was ob- served. On the other hand, in the range of 10-25% 1- propanol, a significant increase in the value of 0222 occurred. Further increase in the concentration of 1-propanol induced only a small change in the value of 0222. Trifluoroethanol is an organic solvent that is generally used to determine the propensity of polypeptide fragments of protein to take up an a-helical conformation (14). Therefore, it was of interest to determine the relative efficiency of trifluoroethanol and 1-propanol to induce the a-helical con- formation to a-globin. 0222 of a-globin in 25% and 50% trifluoroethanol was nearly 85% and 90%, respectively, of that of a-chain and was slightly higher than that obtained in the presence of 25% and 50% 1-propanol. Thus, the propen- sity of 1-propanol to induce the a-helical conformation in a-globin appears to be comparable to that oftrifluoroethanol. Influence of 1-Propanol on the Digestibility of a-Globin by V8 Protease at 40C. The sharp increase in the helical content of a-globin in the range of 10-25% 1-propanol as well as the efficient V8 protease-catalyzed semisynthesis of a-globin

10 20 30 40 50 60 70 Time, min FIG. 2. Influence of 1-propanol on the digestibility of a-globin by V8 protease. The V8 protease digestion was carried out at pH 6.0 X, nm with an enzyme-to-substrate ratio of 1:200 for 24 hr. The digests were subjected to RP-HPLC on a Whatman Partisil 10-ODS-3 column, FIG. 1. Far-UV CD spectra of a-globin and a-chain. The spectra using a linear gradient of 5-50% acetonitrile, both containing 0.1% were taken in 10 mM NH4OAc at pH 6.0 and 4°C. Protein concen- trifluoroacetic acid over 60 min. The elution of peptides was tration was 0.1 mg/ml. A, a-chain; B, a-globin; C, a-globin in 25% monitored at 210 nm. (A) a-Globin at 40C. (B) a-Globin in 25% 1-propanol; D, a-globin in 25% trifluoroethanol. (Inset) Dependence 1-propanol at 4°C. (C) a-Globin at 37°C without 1-propanol. (D) Of 6222 of a-globin on the concentration of 1-propanol. a-Globin at 370C in the presence of 25% 1-propanol. Downloaded by guest on September 27, 2021 7016 Biochemistry: Iyer and Acharya Proc. Natl. Acad. Sci. USA 84 (1987) products. With a-globin, a1_30 and a31_47 were the two major V8 protease digestion products (Fig. 2A). Thus, the peptide bonds of Glu-30 and Asp-47 are the two readily susceptible sites for proteolysis in a-globin at 4TC. However, when the digestion of a-globin was carried out in the presence of 25% 1-propanol, only a1l30 was generated. The digestion was limited to the peptide bond of Glu-30 of a-globin (Fig. 2B). Thus, it is clear that the 1-propanol-induced conformation of a-globin at 4TC affords a selective proteolysis to the peptide bond of Glu-30. Influence of 1-Propanol on the Digestion ofa-Globin at 250C and 370C. The propensity of1-propanol to increase the helical content of the globin is not limited to 4TC. Although the 0222 of a-globin at 25°C and 37°C (in the absence of propanol) was considerably lower than that seen at 4°C, in the presence of 1-propanol, helical conformation of a-globin increased at 25°C and 37°C. However, these values are much lower than the value obtained with a-globin in 25% 1-propanol at 4°C. Nevertheless, the digestibility ofa-globin at 25°C and 37°C in N the presence of 25% 1-propanol was very selective to the 1-27 31-O047 B peptide bond of Glu-30 and Arg-31 (Fig. 2D). Thus, the V8 1-0 protease digestibility of a-globin does not appear to be a Heme direct correlate of the overall secondary structure of the globin. Influence of Glycerol on the V8 Protease Digestibility of a-Globin. Polyhydric , like ethylene glycol and glycerol, have been reported to stabilize the protein structure (; ref. 16), and in some cases these have been shown to limit the proteolytic digestion of proteins (17). A Furthermore, glycerol is the widely used solvent to induce proteases to reform peptide bonds in the fragment-comple- I-30 Heme menting systems (7). Therefore, in an attempt to establish whether the limited proteolysis of a-globin is unique to 1-propanol, or common to other alcohols such as glycerol, the influence of25% glycerol on the V8 protease digestibility of a-globin was also investigated. The RP-HPLC pattern of the V8 protease digest of a-globin was not influenced by the presence of 25% glycerol during the digestion. Thus, the A influence of 1-propanol on the conformation of a-globin 20 40 60 80 appears to be unique to this alcohol. Time, min Digestion of a-Chain at 4°C. The propensity of 1-propanol the apoprotein to FIG. 3. Influence of pH and temperature on the V8 protease to limit the V8 protease digestion of digestion of a-chain. Conditions used for the digestion were similar Glu-30-Arg-31 at 4C and 37°C prompted the investigation of to that described in Fig. 2. The digest was subjected to RP-HPLC the susceptibility of a-chain to V8 protease digestion at low analysis on a Brownlee Lab Aquapore RP-300 column. An acetoni- temperature to determine whether the 1-propanol-induced trile gradient (in 0.1% trifluoroacetic acid) of 5-50% over 100 min was conformation of a-globin is relevant to that of a-chain used to elute the digest, and the flow rate was 1 ml/min. (A) a-Chain (heme-induced conformation) maintained by the native ter- at pH 6.0 and 40C. (B) a-Chain at pH 6.0 and 370C. (C) a-Chain at tiary interactions. The peptide bond of Glu-30 and Arg-31 of pH 8.0 and 370C. (D) a-Chain at pH 8.0 and 40C. a-chain is in fact one of the readily accessible bonds of the polypeptide chain for V8 protease digestion at pH 6.0 and The digestion of a-chain at pH 8.0 and 37TC was also 37°C (15). However, on longer incubations of the type used investigated to determine the influence of pH on the acces- in the present studies (24 hr), extensive digestion takes place sibility ofthe peptide bonds ofa-chain for digestion (Fig. 3C). (Fig. 3B). On the other hand, at 4°C, the V8 protease digestion A significant decrease in the susceptibility of the peptide ofa-chain was selective to the peptide bond ofGlu-30 and the bond of Asp-47 was seen when digestion was carried out at digestion did not occur at any of the other peptide bonds that 370C and pH 8.0, instead of pH 6.0. Furthermore, at this are susceptible at 37°C (Fig. 3A). higher pH, digestion at Glu-23 and Glu-27 was also signifi- digestion cantly inhibited. Again, at pH 8.0, when the digestion was V8 Protease Digestion of a-Chain at pH 8.0. The carried out at 4TC the peptide bonds of Glu-23, Glu-27, and ofa-chain at pH 6.0 at 4°C is selective to peptide bond 30-31, Asp-47 became completely resistant to proteolysis. The and nearly the same digestion pattern was obtained at 25°C cleavage now occurred exclusively at Glu-30. (results not shown). The peptide bonds ofGlu-23, Glu-27, and The CD spectra of a-chain at 370C were nearly the same, Asp-47 became accessible only at 37°C. These additional sites whether they were taken at pH 6.0 or pH 8.0. Thus, the ofdigestion apparently represent the regions ofthe chain that observed differences in the V8 protease digestion pattern of exhibit increased segmental flexibility when the temperature a-chain at pH 6.0 and pH 8.0 at 37°C cannot be directly was increased. However, when 25% 1-propanol was incor- correlated with the changes in the overall secondary struc- porated, the digestion of the a-chain also became limited to tural features ofa-chain. Apparently, the increased digestion the peptide bond of Glu-30 and Arg-31 (results not shown). at 37°C and pH 6 represents the increased segmental flexi- Presumably, the presence of 1-propanol decreases the seg- bility ofthe polypeptide chain in the region of a given peptide mental flexibility of the a-chain, just as in the case of bond. a-globin. Influence of 1-Propanol on the CD Spectra and the V8 Downloaded by guest on September 27, 2021 Biochemistry: lyer and Acharya Proc. Natl. Acad. Sci. USA 84 (1987) 7017 Protease Digestion of RNase S Peptide. In an attempt to DISCUSSION determine whether the propensity of 1-propanol to limit the V8 protease digestion of a-globin is unique or common to The limited proteolysis ofthe globular proteins is expected to other peptide fragments, influence of 1-propanol on the CD occur at either the surface loops, the random segments of spectra and the V8 protease digestion ofRNase S peptide (18) polypeptide chains, or the flexible joints between the do- was examined. This amino-terminal 20-residue fragment of mains rather than at internal segments or rigid elements of RNase A contains the segment that forms one of the helical secondary structure such as a-helices. Fontana et al. (20) regions of the native protein (19). The CD spectra of RNase have demonstrated that the limited proteolysis as well as S peptide (Fig. 4) clearly show that the presence of 25% autolysis ofthermolysin leads to hydrolysis ofa small number 1-propanol increased the value of 6222 of S peptide. There- of peptide bonds located in the exposed surface segments of fore, the propensity of 1-propanol to induce a-helical con- the polypeptide chains characterized by highest segmental formation to polypeptides is not limited to a-globin but seems mobility. They have demonstrated that a close correlation to be true of peptide fragments of proteins as well. exists between the segmental flexibility of the protein as V8 protease digestion of S peptide at pH 6.0 and 40C revealed by x-ray crystallography and the sites of limited produced the two fragments representing residues 1-9 and proteolysis of thermolysin (20). Thus, the regions of a-chain 10-20, respectively (Fig. 4 Inset A). The presence of 25% that become accessible for V8 protease digestion on disso- 1-propanol did not protect the S peptide from being digested ciation of the chain from the a2,82 tetrameric structure and at the peptide bond of Glu-9; nearly the same digestion when the pH of the isolated chain is decreased, as well as on pattern was obtained (Fig. 4, trace B) in the presence of dehemming of the isolated chain, could be considered as a 1-propanol as well. Thus, the protection of a-globin at the reflection of a progressive unfolding of the molecule. The peptide bonds of Glu-23, Glu-27, and Asp-47 from protease accessibility of the region of the Glu-30 and Arg-31 peptide digestion in the presence of 1-propanol appears to be a bond ofthe polypeptide chain ofthe a-subunit of hemoglobin consequence of the overall conformational aspects of the to V8 protease digestion increases as a consequence of its polypeptide chain in the presence of 1-propanol and not a dissociation from the p-subunit; the segmental flexibility of direct consequence of increased a-helical conformation of this region increases. On removal of heme, the region around the polypeptide chain. Asp-47 also shows increased accessibility to proteolysis. We Only the isolated S peptide is digested by V8 protease. have observed that HbA is very resistant to V8 protease When it is part of the intact functional protein (RNase A or digestion (17). In the tetrameric Hb none of the glutamyl/ RNase S) this digestion does not occur. RNase S and RNase aspartyl peptide bonds of a- or P-chain is accessible to V8 A are completely resistant to V8 protease digestion at 4°C. protease digestion. On the other hand, isolated a-chain is Thus, the a-helical conformation ofthe Glu-9-Arg-10 region digested very selectively at the peptide bond 30-31 at pH 8.0 by itself is not sufficient to protect the Glu-9 and Arg-10 and 4°C. If the temperature of a-chain digestion is increased peptide bond from V8 protease digestion. The differential to 37°C, some amount of accessibility is rendered to the accessibility of this bond apparently is a consequence of peptide bond of Asp-47 and cleavage also occurs to some with the S extent at the peptide bond of Glu-27. When the pH of the tertiary interactions of the helical S peptide region a-chain is lowered to 6.0 and the temperature is maintained protein part of RNase A. at 37°C, the peptide bond of Asp-47 is readily accessible for Influence of 1-Propanol on the Digestibility of a-Globin by hydrolysis. The results suggest that lowering the pH of . The 1-propanol-induced stabilization ofthe a-globin a-chain at 37°C from 8.0 to 6.0 increases the segmental conformation toward V8 protease digestion could be unique flexibility of the a-chain in the region around Asp-47. If the to this enzyme or general to other proteases. Accordingly, temperature is now lowered to 4°C, even at pH 6.0, the trypsin digestion of a-globin at 4°C in the presence and flexibility of this region of Asp-47 of the chain appears to absence of 25% 1-propanol was investigated. a-Globin was decrease significantly, since the peptide bond ofAsp-47 is not digested well at 4°C by trypsin even at a low enzyme accessible for digestion at 4°C. concentration of 1:2000. However, if 25% 1-propanol was At the lower temperature (4°C) and pH 6.0, the region present, very little tryptic digestion of a-globin occurred. around Asp-47 of the polypeptide chain could again be Thus, it is clear that the conformation of a-globin induced on rendered susceptible to proteolysis by removing the heme. inclusion of 25% 1-propanol limits the tryptic digestion of the Concomitant with this accessibility of Asp-47, the peptide polypeptide chain. Thus, it appears that 1-propanol indeed bonds of Glu-23 and Glu-27 also become accessible to induces the overall stabilization of the a-globin structure, proteolysis. This accessibility of the peptide bonds of P-helix with an overall topology comparable to that ofnative a-chain. and of the CD corner of a-chain to V8 protease is accompa-

FIG. 4. Far-UV CD spectra of RNase S peptide. Conditions are the same as those used in Fig. 1. A, S peptide; B, S peptide in 25% 1-propanol. (Inset) Influence of 1-propanol on the V8 protease digest- ibility of the RNase S peptide at pH 6.0 and 4°C. RNase peptide was digested at a concentration of 0.2 mg/mi. The 24-hr digest was subjected to RP-HPLC on a Whatman Partisil 10-ODS-3 col- umn. A linear gradient of 5-50% acetonitrile (in 230 0.1% trifluoroacetic acid) over 100 min was used. A, X, nm without 1-propanol; B, with 25% 1-propanol. Downloaded by guest on September 27, 2021 7018 Biochemistry: Iyer and Acharya Proc. Natl. Acad, Sci. USA 84 (1987) nied by a significant loss of a-helical structure (nearly fragments of a-globin comparable to that in a-chain. A 55-60%). Inclusion of 25% 1-propanol in the medium in- native-like topology of the polypeptide chain would provide creases the a-helical content of the a-globin closer to that of the proximity for the a-carboxyl group (of a1_30) and a-amino a-chain, with a concomitant induction of resistance to the group (of a31_141), the condensation of which has to be peptide bond of Asp-47 of the polypeptide chain to V8 catalyzed by V8 protease. Furthermore, the overall a-helical protease digestion. A selective digestion of the polypeptide content of a mixture of the complementary fragments (in 25% chain (a-globin) occurs at the peptide bond 30-31 in the 1-propanol) is lower than that of a-globin (in 25% 1-propa- presence of 1-propanol. Thus, the propanol-induced confor- nol). Thus, the introduction of "continuity" in the polypep- mation in a-globin exhibits native-like features, as reflected tide chain also increases the propensity of the molecule to in its accessibility of peptide bonds to V8 protease cleavage. take up a native-like topology in the presence of 1-propanol- Fronticelli and Gold (21) have studied the helical content of i.e., the stability of the helical segments increases. Such an fragments off3-globin-namely, P1-30 and p155--as a function increase in the stability could function as a "molecular trap" of concentration. Over the range of 0-90% meth- for shifting the equilibrium of the hydrolytic reaction in favor anol, the helical content of the fragments showed a progres- of proteosynthesis. Further studies are needed to establish sive increase with the increase in the concentration of relative roles of 1-propanol-induced proximity of a-amino alcohol. On the other hand, 1-propanol-induced formation of and a-carboxyl groups in the mixture of complementary a-helical conformation in a-globin appears to exhibit a certain fragments and the increased stability of the helical segments degree of cooperativity. When the generation of a-helical (in 1-propanol) on the introduction of the continuity in the conformation is studied as a function of the organic solvent, polypeptide chain to facilitate the synthesis of a-globin. The most of the structural changes occur in a narrow range of facile condensation of al30 and a31_47 in the presence of 10-25% propanol. This implies that the induction of an 1-propanol suggests that the latter may play a significant role a-helical structure to a segment of the polypeptide chain in the V8 protease-catalyzed synthesis of al47 as well as potentiates a-helical structure to other segments-i.e., gen- al-141* eration of a-helical structure in a-globin is cooperative. This is suggestive of the packing of the helices in a native-like This work was supported by National Institutes of Health Grant topology once they are induced, thereby stabilizing each HL-27183 and a Grant-in-Aid from the American Heart Association, this leads to a limiting of the New York City Affiliate to A.S.A. The Aviv 6ODS CD spectrometer other. Apparently, stabilization was purchased with funds from National Science Foundation Grant digestion of the a-globin chains to the regions of high PCM-84-00268. A.S.A. is an Established Fellow of the American segmental flexibility. Heart Association, New York City Affiliate. We thank Dr. J. M. The increase in the a-helical conformation of S peptide in Manning for the facilities provided, Dr. G. Sahni for the V8 protease the presence of 25% 1-propanol is considerably lower than digestion experiments with RNase S, and Judith A. Gallea for helping the theoretical value expected when the segment 2-13 of the in the preparation of the manuscript. peptide is in helical conformation. This suggests that the stability of the a-helix induced into S peptide by 1-propanol 1. Fink, A. L. & Petsko, G. A. (1981) Adv. Enzymnol. Relat. Areas is lower as compared with that of the same region in RNase Mol. Biol. 52, 177-246. induc- 2. Fink, A. L. & Cartwright, S. J. (1981) CRC Crit. Rev. Biochem. 12, A. The V8 protease digestion studies suggest that the 145-207. tion of the a-helical conformation to a segment of protein by 3. Zaks, A. & Klibnov, A. M. (1985) Proc. Natl. Acad. Sci. USA 82, itself is not enough to afford resistance to a glutamyl peptide 3192-3196. bond to V8 protease digestion. The resistance of S peptide 4. Kullman, W. (1985) J. Protein Chem. 4, 1-22. region in RNase S and in RNase A to V8 protease digestion 5. Homandberg, G. A. & Laskowski, M., Jr. (1979) Biochemistry 18, is an apparent reflection of the overall conformation of S 586-592. 6. Homandberg, G. A. & Chaiken, I. M. (1980) J. Biol. Chem. 255, peptide in the protein (RNase A or RNase S) involving the 1406-1412. secondary structure of S peptide and the tertiary interaction 7. Chaiken, I. M. (1981) CRC Crit. Rev. Biochem. 11, 255-301. of the segment with the rest of the molecule. The conse- 8. Seetharam, R. & Acharya, A. S. (1986) J. Cell. Biochem. 30, 87-99. quence of these secondary and tertiary interactions of S 9. Acharya, A. S., Khan, S. A. & Seetharam, R. (1985) Proc. Am. peptide region of RNase A is the stabilization of the a-helical Pept. Symp. 9th, 335-338. segment of the peptide and this leads to the differential 10. Acharya, A. S. & Manning, J. M. (1980) J. Biol. Chem. 255, accessibility of the peptide bond of Glu-9 and Arg-10 of the 1406-1412. for V8 protease digestion. 11. Rossi-Fanelli, A., Antonini, E. & Caputo, A. (1964) Adv. Protein peptide Chem. 19, 73-222. Thus, the tryptic and V8 protease digestion pattern of 12. Acharya, A. S., Di Donato, A., Manjula, B. N., Fischetti, V. A. & a-globin in the presence of 1-propanol can be considered as Manning, J. M. (1983) Int. J. Pept. Protein Res. 22, 78-82. the reflection of the native-like topology of the helical 13. Yip, Y. K., Waks, M. & Beychok, S. (1972) J. Biol. Chem. 247, segments of a-globin. Whether this is a general property of 7237-7244. apoproteins of other heme proteins, if not of all proteins, is 14. Johnson, C. W., Jr. (1985) Methods Biochem. Anal. 31, 61-163. a subject worthy of further investigation. 15. Seetharam, R., Dean, A., Iyer, k. S. & Acharya, A. S. (1986) On the basis ofthe results ofthe present study, it is possible Biochemistry 25, 5949-5955. to speculate as to the mechanism(s) by which the 1-propanol- 16. Gekko, K. & Timashiff, S. N. (1981) Biochemistry 20, 4667-4676. induced conformation of a-globin could modulate the enzy- 17. Slobin, L. I., Clark, R. V. & Olson, M. 0. J. (1981) Biochemistry 20, 5761-5767. mic condensation of the fragments of a-globin. Our prelim- 18. Richards, F. M. & Vithayathil, P. J. (1959) J. Biol. Chem. 234, inary studies have shown that the overall a-helical content of 1459-1465. an equimolar mixture of a1l30 and a31_141 increases consid- 19. Richards, F. M. & Wyckoff, H. W. (1971) Enzyme 4, 647-806. erably in the presence of 1-propanol. It is conceivable that 20. Fontana, A., Fassina, G., Vita, C., Dalzoppo, D., Zamai, M. & this increased helicity results in a native-like topology of the Zambonin, M. (1986) Biochemistry 25, 1847-1851. polypeptide chains in this mixture of the complementary 21. Fronticelli, R. & Gold, R. (1976) J. Biol. Chemn. 251, 4968-4972. Downloaded by guest on September 27, 2021