1058 Chemical Reviews, 1998, Vol. 98, No. 3
X. Ribonuclease S
The protease subtilisin prefers to cleave a single
peptide bond in native RNase A.304,305 The product of this cleavage, ribonuclease S (RNase S, where “S” refers to subtilisin), consists of two tightly associated fragments. These fragments are S-peptide, which derives from residues 1-20 of RNase A, and S- protein, which derives from residues 21-124. Al- though neither fragment alone has any ribonucle- olytic activity, RNase S has enzymatic activity similar to that of intact RNase A. The three-dimensional s t r u c t u r e o f c r y s t a l l i n e R N a s e S 3 0 6 - 3 0 8 w a s d e t e r - m i n e d s o o n a f t e r t h a t o f R N a s e A . 5 8 B e c a u reports on the structure of RNase A lacked detail58 (or were altogether incorrect309) early structural work on RNase S306,307 greatly stimulated interest in the enzyme.310 The structures of RNase S with bound s e i n i t i a l u r i d y l y l ( 3 ′ f 5 ′ ) - 5 ′ - d e o x y - 5 ′ - m e t h y l e n e a d e n o s i n e , 3 1 1 2 ′ - and cytidylyl(2′f5′)- deoxy-2′-fluoro-UpA,312 ApC,313 adenosine314 are also known .
A. S-Protein−S-Peptide Interaction
Only a low yield of native S-protein is isolable from the air oxidation of reduced S-protein.88 The recovery of native S-protein is complete, however, if the oxidation is performed in the presence of S-peptide, which presumably serves as a template for proper folding.315 A monoclonal antibody against native S-protein has been shown to have a similar effect, enhancing by 3.6-fold the yield of native S-protein. (In contrast to S-protein, the S-peptide portion of RNase A is not antigenic.317) 316
In addition to structural information, extensive thermochemical data have been acquired on the S-protein-S-peptide interaction. The value of Kd for RNase S is dependent on pH (ranging from 3.1 × 10-11 M at pH 8.3 to 1.1 × 10-6 M at pH 2.7318), temperature (ranging from 8.3 × 10-8 M at 30 °C to 9.2 × 10-6 M at 45 °C319), and ionic strength (increas- ing 7-fold as the concentration of NaCl is decreased from 0.5 M to 0.7 mM320). A complex of S-protein with only the 15 N-terminal amino acid residues of S-peptide (S15) is essentially identical in structure to that of RNase S.235 Isothermal titration calorim- etry has shown that the value of Kd for the S-protein‚ S15 complex is 1.1 × 10-7 M at 25 °C in 50 mM sodium acetate buffer, pH 6.0, containing NaCl (0.10 M). 321
B. New Technology
The S-peptide fragment of RNase A has had a singular role in the development of protein chemistry. Before molecular biologists were able to use recom- binant DNA techniques to explore protein structure- function relationships, chemists synthesized ana- logues of S-peptide and studied their complexes with S-protein. The preparation of RNase S322-326 by total synthesis occurred simultaneously with that of RNase A.11,71 In addition, work on RNase S provided the first three-dimensional structure of a protein-nucleic acid complex,327 as well as the first demonstration that a crystalline enzyme could be an active cata- lyst.328 (For reviews, see refs 15 and 16. For histori-
cal accounts, see refs 329 and 330.) These studies were successful in illuminating molecular aspects of enzymatic catalysis, protein-protein interactions, and protein-nucleic acid interactions, and were the harbinger of current work on proteins containing variant or nonnatural amino acid residues. Work on the structure and function of another semisynthetic
262,263 165,264,266,267,331-337 ribonuclease, RNase-(1-118)‚(111-124), made significant contributions. has also Re-
cently, the RNase S system has spawned or at least facilitated the development of many innovative tech- nologies.
1. Substrate−Leash Amplification
Chemical amplification takes place when a small chemical stimulus is magnified into a large chemical response.338 The RNase S system has provided the first example of one type of chemical amplification: “substrate-leash amplification”.339,340 Here, the S- peptide or S-protein fragment is immobilized on solid supports via a “leash” of poly(C) substrate. Each support releases its fragment when treated with the complementary enzyme fragment or with RNase A. The fragments released from a mixture of the two supports recombine to give RNase S activity. This system provides an amplification of activity that exceeds 104-fold. Such a cascade could serve as the basis for effective biosensors.
2. Sequence-Specific Ribonuclease
RNase S has been engineered to cleave only a specific sequence in an RNA molecule.341 This en- hanced specificity is attained by attaching a thiol- modified DNA oligonucleotide to the N-terminal cysteine residue of K1C S-peptide via a disulfide bond. The synthetic construct allows for the forma- tion of a hybrid RNase S that cleaves RNA with a specificity dictated by the DNA sequence. Analogous experiments have been performed with intact K1C RNase A. 36,268,342
3. Fusion Protein System
RNase S has served as the basis for a fusion protein system. Recombinant DNA technology has been used to produce a fusion protein in which S-peptide or S15 (also known as “S-TAG”) is attached covalently to a
The interaction of the S-
peptide portion of the fusion protein with immobilized S-protein allows for the facile purification of the fusion protein. Likewise, the interaction with soluble S-protein enables a sensitive ribonucleolytic assay to be used to detect the fusion protein either in
186,343-345 186,345 or after electrophoresis in a poly- solution acrylamide gel.
4. Antagonist from Phage Display
The RNase S system has produced a notable success in combinatorial chemistry. S-protein has been used to pan a filamentous phage library dis- playing hexapeptides of random sequence.346 The selected peptides have a sequence motif of (F/Y)NF- (E/V)(I/V)(L/V), which bears little resemblance to the sequence of S-peptide. One of the displayed peptides,