Thermodynamics of protein-peptide interactions in the ribonuclease S system studied by titration calorimetry

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. Residue 13 in the peptide is methionine. According to the X-ray structure of the complex of S-protein and S-peptide (1-20), this residue is almost fully buried. We have substituted Met-13 with seven other hydrophobic residues ranging in size from glycine to phenylalanine and have determined the thermodynamic parameters associated with the binding of these analogues to S-protein by titration calorimetry at 25 degrees C. These data should provide useful quantitative information for evaluating the contribution of hydrophobic interactions in the stabilization of protein structures.     

Heat capacity changes for protein-peptide interactions in the ribonuclease S system.

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. We have substituted the wild-type residue Met-13 with six other hydrophobic residues ranging in size from alanine to phenylalanine and have determined the thermodynamic parameters associated with binding of these analogues to S-protein by titration calorimetry in the temperature range 5-25 degrees C. The heat capacity change (delta Cp) associated with binding was obtained from a global analysis of the temperature dependences of the free energies and enthalpies of binding. The delta Cp's were not correlated in any simple fashion with the nonpolar surface area (delta Anp) buried upon binding.     

Ribonuclease S-peptide as a carrier in fusion proteins.

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).     

Ribonuclease S-peptide. A model for molecular recognition.

The relationship of structure to function in the recognition of ribonuclease S-peptide by S-protein was studied by several methods. Liquid phase peptide synthesis was employed to generate analogs of S-peptide in which from 1 to 8 residues were deleted from the NH2-terminal end of the S-peptide. Additional derivatives were made by substitutions in the NH2-terminal three amino acids or by modifying the S-peptide analogs by trifluoroacetylation. The analogs were generated in the following way. S-Peptide was cleaved with chymotrypsin. The fragment obtained, RNase(9-20), was purified and lengthened step by step using liquid phase peptide synthesis. A second set of analogs were prepared by cleavage of CF3CO-S-peptide with elastase and the resulting CF3CO-RNase(7-20), similarly lengthened. The various analogs of S-peptide were tested in their capacity to combine with S-protein and regenerate biological activity as measured by Vmax and Kb. This work shows a positive contribution of every one of the first 8 NH2-terminal residues of S-peptide to the molecular recognition of S-protein in the presence of RNA substrate. Substitution of the first 3 residues by alanine or blocking of the free amino groups decreases recognition, indicating that the original primary structure is the most favorable one.     

15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding.     

Conformationally driven protease-catalyzed splicing of peptide segments: V8 protease-mediated synthesis of fragments derived from thermolysin and ribonuclease A.

We have studied the conformation as well as V8 protease-mediated synthesis of peptide fragments, namely amino acid residues 295-316 (TC-peptide) of thermolysin and residues 1-20 (S-peptide) of ribonuclease A, to examine whether "conformational trapping" of the product can facilitate reverse proteolysis. The circular dichroism study showed cosolvent-mediated cooperative helix formation in TC-peptide with attainment of about 30-35% helicity in the presence of 40% 1-propanol and 2-propanol solutions at pH 6 and 4 degrees C. The thermal melting profiles of TC-peptide in the above cosolvents were very similar. V8 protease catalyzed the synthesis of TC-peptide from a 1:1 mixture of the non-interacting complementary fragments (TC295-302 and TC303-316) in the presence of the above cosolvents at pH 6 and 4 degrees C. In contrast, V8 protease did not catalyze the ligation of S1-9 and S10-20, although S-peptide could assume helical conformation in the presence of the cosolvent used for the semisynthetic reaction. V8 protease was able to synthesize an analog of S-peptide (SA-peptide) in which residues 10-14 were substituted (RQHMD-->VAAAK). While S-peptide exhibited helical conformation in the presence of aqueous propanol solutions, SA-peptide displayed predominantly beta-sheet conformation. SA-peptide showed enhanced resistance to proteolysis as compared with S-peptide. Thus, failure of semisynthesis of S-peptide may be a consequence of high flexibility around the 9-10 peptide bond due to its proximity to the helix stop signal. The results suggest that protease-mediated ligations may be achieved by design and manipulation of the conformational aspects of the product.     

The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S-peptide analogues with enhanced helical stability

Recent work has shown that, with synthetic analogues of C-peptide (residues 1-13 of ribonuclease A), the stability of the peptide helix in H2O depends strongly on the charge on the N-terminal residue. We have asked whether, in semisynthetic ribonuclease S reconstituted from S-protein plus an analogue of S-peptide (1-15), the stability of the peptide helix is correlated with the Tm of the reconstituted ribonuclease S. Six peptides have been made, which contain Glu9----Leu, a blocked alpha-COO- group (-CONH2), and either Gln11 or Glu11. The N-terminal residue has been varied; its charge varies from +2 (Lys) to -1 (succinyl-Ala). We have measured the stability of the peptide helix, the affinity of the peptide for S-protein (by C.D. titration), and the thermal stability of the reconstituted ribonuclease S. All six peptide analogues show strongly enhanced helix formation compared to either S-peptide (1-15) or (1-19), and the helix content increases as the charge on the N-terminal residue changes from +2 to -1. All six peptides show increased affinity for S-protein compared to S-peptide (1-19), and all six reconstituted ribonucleases S show an increase in Tm compared to the protein with S-peptide (1-19). The Tm increases as the charge on residue 1 changes from +2 to -1. The largest increment in Tm is 6 degrees. The results suggest that the stability of a protein can be increased by enhancing the stability of its secondary structure.     

Amide proton exchange used to monitor the formation of a stable alpha-helix by residues 3 to 13 during folding of ribonuclease S

We make use of the known exchange rates of individual amide proton in the S-peptide moiety of ribonuclease S (RNAase S) to determine when during folding the alpha-helix formed by residues 3 to 13 becomes stable. The method is based on pulse-labeling with [3H]H2O during the folding followed by an exchange-out step after folding that removes 3H from all amide protons of the S-peptide except from residues 7 to 14, after which S-peptide is separated rapidly from S-protein by high performance liquid chromatography. The slow-folding species of unfolded RNAase S are studied. Folding takes place in strongly native conditions (pH 6.0, 10 degrees C). The seven H-bonded amide protons of the 3-13 helix become stable to exchange at a late stage in folding at the same time as the tertiary structure of RNAase S is formed, as monitored by tyrosine absorbance. At this stage in folding, the isomerization reaction that creates the major slow-folding species has not yet been reversed. Our result for the 3-13 helix is consistent with the finding of Labhardt (1984), who has studied the kinetics of folding of RNAase S at 32 degrees C by fast circular dichroism. He finds the dichroic change expected for formation of the 3-13 helix occurring when the tertiary structure is formed. Protected amide protons are found in the S-protein moiety earlier in folding. Formation or stabilization of this folding intermediate depends upon S-peptide: the intermediate is not observed when S-protein folds alone, and folding of S-protein is twice as slow in the absence of S-peptide. Although S-peptide combines with S-protein early in folding and is needed to stabilize an S-protein folding intermediate, the S-peptide helix does not itself become stable until the tertiary structure of RNAase S is formed.     

High affinity RNase S-peptide variants obtained by phage display have a novel "hot-spot" of binding energy.

Using phage display mutagenesis, high affinity variants of RNase S-peptide were produced that bind to RNase S-protein over 100-fold more tightly than the wild type S-peptide. The S-peptide: S-protein interface was further characterized using "biased" phage display libraries, where each targeted residue was constrained to be either polar or nonpolar. The use of these tailored libraries placed constraints on the type of interactions present during affinity maturation process and allowed more amino acids to be randomized simultaneously. These results, in conjunction with kinetic association and dissociation constants determined by surface plasmon resonance (SPR), highlight the role of a single mutation (A5W) in increasing S-peptide binding affinity. High affinity S-peptide variants were only identified when tryptophan was present in the phage display library at position 5, suggesting that this residue is a "hot-spot" of binding energy in the high affinity variants. Analysis of SPR data in the presence of denaturant suggests that the increased affinity is a result of increased hydrophobic interactions in the transition state rather than a stabilization of helical structure.     

Recombination of S-peptide with S-protein during folding of ribonuclease S. II. Kinetic characterization of a stable folding intermediate shown by S-protein at pH 1.7.

At pH 1.7 S-peptide dissociates from S-protein but S-protein remains partly folded below 30 °C. A folded form of S-protein, labeled I₃, is detected and measured by its ability to combine rapidly with S-peptide at pH 6.8 and then to form native ribonuclease S. The second-order combination reaction (k = 0.7 × 10⁶m^?1s^?1 at 20 °C) can be monitored either by tyrosine absorbance or fluorescence emission; the subsequent first-order folding reaction (half-time, 68 ms; 20 °C) is monitored by 2′CMP ² binding. Combination with S-peptide and folding to form native RNAase S is considerably slower for both classes of unfolded S-protein (see preceding paper).I₃ shows a thermal folding transition at pH 1.7: it is completely unfolded above 32 °C and reaches a limiting low-temperature value of 65% below 10 °C. The 35% S-protein remaining at 10 °C is unfolded as judged by its refolding behavior in forming native RNAase S at pH 6.8. The folding transition of S-protein at pH 1.7 is a broad, multi-state transition. This is shown both by the large fraction of unfolded S-protein remaining at low temperatures and by the large differences between the folding transition curves monitored by I₃ and by tyrosine absorbance.The fact that S-protein remains partly folded after dissociation of S-peptide at pH 1.7 but not at pH 6.8 may be explained by two earlier observations. (1) Native RNAase A is stable in the temperature range of the S-protein folding transition at pH 1.7, and (2) the binding constant of S-protein for S-peptide falls steadily as the pH is lowered, by more than four orders of magnitude between pH 8.3 and pH 2.7, at 0 °C. The following explanation is suggested for why folding intermediates are observed easily in the transition of S-protein but not of RNAase A. The S-protein transition is shifted to lower temperatures, where folding intermediates should be more stable: consequently, intermediates in the folding of RNAase A which do not involve the S-peptide moiety and which are populated to almost detectable levels can be observed at the lower temperatures of the S-protein transition.     

Nuclear magnetic resonance titration curves of histidine ring protons. Ribonuclease S-peptide and S-proteins.

The histidine C-2 proton NMR titration curves of ribonuclease S-peptide (residues 1 to 20) and S-protein (residues 21 to 124) are reported. Although S-protein contains 3 histidine residues, four discrete resonances are observed to titrate. One of these arises from the equivalent histidine residues of unfolded S-protein. The variation in area of the four resonances indicate that there is a reversible pH-dependent equilibrium between the folded and unfolded forms of S-protein, with some unfolded material being present at most pH values. Two of the resonances of the folded S-protein can be assigned to 2 of the histidine residues, 48 and 105, from the close similarity of their titration curves to those in ribonuclease. These similarities indicate a homology of portions of the folded conformation of S-protein to that of ribonuclease in solution. These results indicate that the complete amino acid sequence is not required to produce a folded conformation similar to the native globular protein, and they appear to eliminate the possibility that proteins fold from their NH2 terminus during protein synthesis. The low pH inflection present in the titration curve assigned to histidine residue 48 in ribonuclease is absent from this curve in S-protein. This is consistent with our previous conclusion that this inflection arises from the interaction of histidine 48 with aspartic acid residue 14, which is also absent in S-protein. The third titrating resonance of native S-protein is assigned to the remaining histidine residue at position 119. The properties of this resonance are not identical with either of the titration curves of the active site histidine residues 12 and 119 of ribonuclease. The resonance assigned to histidine 119 is the only one significantly affected on the addition of sodium phosphate to S-protein, indicating that some degree of phosphate binding occurs. In both the absence and presence of phosphate this curve also lacks the low pH inflection observed in the histidine 119 NMR titration curve in ribonuclease. This difference presumably arise from a conformational between ribonuclease and the folded S-protein involving a carboxyl group.     

S-peptide as a potent peptidyl linker for protein cross-linking by microbial transglutaminase from Streptomyces mobaraensis

We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation.     

Recombination of S-peptide with S-protein during folding of ribonuclease S. I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein.

The refolding kinetics of ribonuclease S have been measured by tyrosine absorbance, by tyrosine fluorescence emission, and by rapid binding of the specific inhibitor 2′CMP ² to folded RNAase S. The S-protein is first unfolded at pH 1.7 and then either mixed with S-peptide as refolding is initiated by a stopped-flow pH jump to pH 6.8, or the same results are obtained if S-protein and S-peptide are present together before refolding is initiated. The refolding kinetics of RNAase S have been measured as a function of temperature (10 to 40 °C) and of protein concentration (10 to 120 μm). The results are compared to the folding kinetics of S-protein alone and to earlier studies of RNAase A. A thermal folding transition of S-protein has been found below 30 °C at pH 1.7; its effects on the refolding kinetics are described in the following paper (Labhardt &; Baldwin, 1979).In this paper we characterize the refolding kinetics of unfolded S-protein, as it is found above 30 °C at pH 1.7, together with the kinetics of combination between S-peptide and S-protein during folding at pH 6.8. Two classes of unfolded S-protein molecules are found, fast-folding and slow-folding molecules, in a 20: 80 ratio. This is the same result as that found earlier for RNAase A; it is expected if the slow-folding molecules are produced by the slow cis-trans isomerization of proline residues after unfolding, since S-protein contains all four proline residues of RNAase A.The refolding kinetics of the fast-folding molecules show clearly that combination between S-peptide and S-protein occurs before folding of S-protein is complete. If combination occurred only after complete folding, then the kinetics of formation of RNAase S should be rather slow (5 s and 100 s at 30 °C) and nearly independent of protein concentration, as shown by separate measurements of the folding kinetics of S-protein, and of the combination between S-peptide and folded S-protein. The observed folding kinetics are faster than predicted by this model and also the folding rate increases strongly with protein concentration (apparent 1.6 order kinetics). The fact that RNAase S is formed more rapidly than S-protein alone is sufficient by itself to show that combination with S-peptide precedes complete folding of S-protein. Computer simulation of a simple, parallel-pathway scheme is able to reproduce the folding kinetics of the fast-folding molecules. All three probes give the same folding kinetics.These results exclude the model for protein folding in which the rate-limiting step is an initial diffusion of the polypeptide chain into a restricted range of three-dimensional configurations (“nueleation”) followed by rapid folding (“propagation”). If this model were valid, one would expect comparable rates of folding for RNAase A and for S-protein and one would also expect to find no populated folding intermediates, so that combination between S-peptide and S-protein should occur after folding is complete. Instead, RNAase A folds 60 times more rapidly than S-protein and also combination with S-peptide occurs before folding of S-protein is complete. The results demonstrate that the folding rate of S-protein increases after the formation, or stabilization, of an intermediate which results from combination with S-peptide. They support a sequential model for protein folding in which the rates of successive steps in folding depend on the stabilities of preceding intermediates.The refolding kinetics of the slow-folding molecules are complex. Two results demonstrate the presence of folding intermediates: (1) the three probes show different kinetic progress curves, and (2) the folding kinetics are concentration-dependent, in contrast to the results expected if complete folding of S-protein precedes combination with S-peptide. A faster phase of the slow-refolding reaction is detected both by tyrosine absorbance and fluorescence emission but not by 2′CMP binding, indicating that native RNAase S is not formed in this phase. Comparison of the kinetic progress curves measured by different probes is made with the use of the kinetic ratio test, which is defined here.     

An equilibrium folding intermediate detected in the thermal unfolding transition of ribonuclease S by circular dichroism

The thermal-denaturation transition of ribonuclease S (RNAase S) is measured by circular dichroism at 225 nm. Only conformational transitions involving the S-peptide–S-protein complex are detected at this wavelength. Different pathways of thermal unfolding at high and low concentrations are apparent: at low concentrations the temperature of half-completion of denaturation (T_m) varies with concentration. Above a total enzyme concentration of 50 μM, T_m remains constant. The observed data can be explained on the basis of a model where the association–dissociation step occurs between S-peptide and thermally (at least partly) unfolded S-protein. The complex as a whole undergoes a major folding–unfolding transition in the course of which the S-peptide μ-helix appears to be formed. The unfolded complex is well populated in the unfolding transition region for enzyme concentrations of 100 μM or more. The model succeeds in deducing thermodynamic parameters from the thermal denaturation curves in various different ways. The values thus obtained are fully self-consistent and, moreover, consistent with the values for the apparent association constant and apparent association enthalpy as measured in enzyme-dilution experiments and by batch calorimetry.     

Hydrogen exchange from identified regions of the S-protein component of ribonuclease as a function of temperature,pH, and the binding of S-peptide

A medium resolution hydrogen exchange method (Rosa & Richards, 1979) has been used to measure the average rates of amide hydrogen exchange for known segments of the S-protein portion of ribonuclease-S. The analytical procedure permitted exchange rates to be monitored for seven S-protein fragments distributed throughout the structure, including regions of α-helix and β-sheet. Kinetics were measured as a function of pH, temperature and S-peptide binding.The pH dependence of exchange from isolated S-protein between pH 2·8 and pH 7·0 was found to deviate significantly from a first-order dependence on hydroxide ion concentration. The protection against exchange with increasing pH appeared to be closely related to the electrostatic stabilization of S-protein. It is suggested that such favorable electrostatic interactions result in increased energy barriers to the conformational fluctuations that provide solvent access to the time-average crystallographic structure. This explanation of the observed correlation between stability and exchange kinetics is also consistent with the calculated apparent activation energies for exchange from S-protein between 5·5 and 20 °C.S-peptide binding dramatically slows exchange from many S-protein sites, even those distant from the area of S-peptide contact. Interestingly, the effects of complex formation are not evenly propagated throughout S-protein. The most significantly perturbed sites (≥10³-fold reduction in exchange rate constants) lie within fragments derived from regions of secondary structure. Exchange from several other fragments is not significantly affected. The S-peptide—S-protein dissociation constant at neutral pH is so small that the measured exchange must have occurred from the complex and not from the dissociated parts.     

Conformational properties of the N-terminal residues of S-peptide. II. The guanidine hydrochloride–water–trifluoroethanol system

The Conformational properties of synthetic S-peptide analogs, in which the residues in the N-terminal sequence 1–6 were progressively deleted or replaced with amino acids of lower helical propensity, were studied by CD. Increasing the concentration of guanidine hydrochloride and decreasing the temperature were found to produce progressive destruction of ordered conformations, in the parallel with the increasing solubility of the peptide unit, while increasing the concentration of trifluoroethanol and decreasing the temperature produced the opposite effect. The maximum helicity determined in the these sets of experiments is found equal to or greater than that determined in the formation of the ribonuclease S′ complexes. With some peptides the maximum value of predicted helical conformation is reached, and the tendency of tertiary structure to reduce the maximum possible helicity is evident. We discuss the validity of the procedure by which conformational information, drawn from measurements in helicogenic solvents, is related to the state in native protein.     

Substrate specificity of Bacillus subtilis intracellular serine protease. Hydrolysis of insulin beta-chain, native ribonuclease A and p-nitroanilide peptide substrates

Intracellular serine protease, termed ISP-103, was isolated from Bacillus subtilis, strain 103. The substrate specificity of the enzyme was compared to that of secretory subtilisins. Similar to subtilisins, ISP-103 cleaves a single peptide bond Ala20-Ser21 within the native pancreatic ribonuclease A, which results in the accumulation of trypsin-sensitive ribonuclease S, consisting of a non-covalently bound S-peptide (20 amino acid residues) and S-protein (104 amino acid residues). The enzyme hydrolyzes a single peptide bond Leu15-Tyr16 of the B-chain of oxidized bovine insulin, in contrast to the subtilisins cleaving four additional bonds. ISP prefers Leu rather than Phe in the P1 binding site of the rho-nitroanilide peptide substrates and shows a more strict dependence of the activity on the presence of the hydrophobic residues in the P2 and P3 sites. The data obtained indicate that the substrate specificity of ISP, being within the borders of subtilisin specificity, is nevertheless much more restricted.     

Microinjected ribonuclease A as a probe for lysosomal pathways of intracellular protein degradation

There are multiple pathways of intracellular protein degradation, and molecular determinants within proteins appear to target them for particular pathways of breakdown. We use red cell-mediated microinjection to introduce radiolabeled proteins into cultured human fibroblasts in order to follow their catabolism. A well-characterized protein, bovine pancreatic ribonuclease A (RNase A), is localized initially in the cytosol of cells after microinjection, but it is subsequently taken up and degraded by lysosomes. This lysosomal pathway of proteolysis is subject to regulation in that RNase A is taken up and degraded by lysosomes at twice the rate when serum is omitted from the culture medium. Subtilisin cleaves RNase A between residues 20 and 21, and the separated fragments are termed RNase S-peptide (residues 1–20) and RNase S-protein (residues 21–124). Microinjected RNase S-protein is degraded in a serum-independent manner, while RNase S-peptide microinjected alone shows a twofold increase in degradation in response to serum withdrawal. Furthermore, covalent linkage of S-peptide to other proteins prior to microinjection causes degradation of the conjugate to become serum responsive. These results show that recognition of RNase A and certain other proteins for enhanced lysosomal degradation during serum withdrawal is based on some feature of the amino-terminal 20 amino acids. The entire S-peptide is not required for enhanced lysosomal degradation during serum withdrawal because degradation of certain fragments is also responsive to serum. We have identified the essential region to be within residues 7–11 of RNase S-peptide (Lys-Phe-Glu-Arg-Gln; KFERQ). To determine whether related peptides exist in cellular proteins, we raised antibodies to the pentapeptide. Affinity-purified antibodies to KFERQ specifically precipitate 25–35% of cellular proteins, and these proteins are preferentially degraded in response to serum withdrawal. Computer analyses of known protein sequences indicate that proteins degraded by lysosomes at an enhanced rate in response to serum withdrawal contain peptide regions related, but not identical, to KFERQ. We suggest two possible peptide motifs related to KFERQ and speculate about possible mechanisms of selective delivery of proteins to lysosomes based on such peptide regions.     

Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7-11 as the essential pentapeptide

We have identified a pentapeptide region of microinjected ribonuclease A that is required for enhanced degradation of this protein during serum withdrawal. We introduced reductively methylated [3H]ribonuclease A, [3H]ribonuclease S-protein (residues 21-124), and [3H]ribonuclease S-peptide (residues 1-20) into the cytosol of human fibroblasts by red cell-mediated microinjection and osmotic lysis of pinosomes. The degradative rates of ribonuclease A and ribonuclease S-peptide are increased 2-fold upon withdrawal of serum, while catabolism of ribonuclease S-protein is not regulated in this manner. Certain fragments of ribonuclease S-peptide are also degraded in a serum-dependent fashion (residues 1-14 and 4-13), while other fragments are not (residues 1-10 and 2-8). [3H]Ribonuclease S-peptide is cleaved into two smaller radioactive peptides during loading into red cell ghosts. We tentatively identified the larger fragment as residues 7-11 based on its molecular weight determined by Sephadex chromatography in the presence of 8 M urea combined with sequential Edman degradation to identify the position of radioactive lysines. The smaller peptide fragment appears to be the amino-terminal dipeptide, Lys-Glu, and/or residues 7-8, Lys-Phe. After microinjection into fibroblasts, the pentapeptide is degraded at an enhanced rate in the absence of serum, while degradation of the dipeptide is not affected. We confirmed that residues 7-11 constitute the larger hydrolysis product of S-peptide by synthesizing this pentapeptide and radiolabeling it by reductive methylation. It migrated at the expected position after Sephadex chromatography in 8 M urea and was further hydrolyzed only slightly during loading into red cells. Finally, degradation of this pentapeptide after injection into fibroblasts was enhanced 2-fold upon serum withdrawal. These results, combined with our other recent studies (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993), suggest that the pentapeptide, Lys-Phe-Glu-Arg-Gln, targets microinjected ribonuclease A to lysosomes for enhanced degradation during serum deprivation.