Abstract
The unique ribonuclease S (RNase S) system, derived from proteolytic cleavage of bovine ribonuclease A (RNase A), consists of a tight complex formed by a peptide (amino acids 1–20) and a protein (21–124) part. These fragments, designated as S-peptide and S-protein, can be separated by two purification steps. By addition of synthetic S-peptide derivatives to the S-protein, semisynthetic RNase S is reassembled with high efficiency. Based on this peptide–protein complementation noncanonical amino acids can be easily introduced into a protein host. Here we describe the preparation of the S-protein from RNase A as well as the characterization of the reassembled semisynthetic RNase S complex. Complex formation can be monitored by RNase activity, circular dichroism, or fluorescence polarization. Structure-based enzyme design of the RNase S scaffold is possible based on high-resolution crystal structures of RNase S and its semisynthetic variants.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Raines RT (1998) Ribonuclease A. Chem Rev 98:1045–1065
Marshall GR, Feng J, Kuster DJ (2008) Back to the future: ribonuclease A. Biopolymers 90: 259–277
Richards FM (1955) Titration of amino groups released during the digestion of ribonuclease by subtilisin. C R Trav Lab Carlsberg Chim 29:322–328
Richards FM, Vithayathil PJ (1959) The preparation of subtilisin-modified ribonuclease and separation of the peptide and protein components. J Biol Chem 234:1459–1465
Taylor HC, Komoriya A, Chaiken IM (1985) Crystallographic structure of an active, sequence-engineered ribonuclease. Proc Natl Acad Sci U S A 82:6423–6426
Genz M, Singer D, Hey-Hawkins E, Hoffmann R, Sträter N (2013) Crystal structure of Apo- and metalated thiolate containing RNase S as structural basis for the design of artificial metalloenzymes by peptide-protein complementation. Z Anorg Allg Chem 639: 2395–2400
Barnard EA (1969) Ribonucleases. Ann Rev Biochem 38:677–732
Imperiali B, Roy RS (1994) Coenzyme: amino acid chimeras—new residues for the assembly of functional proteins. J Am Chem Soc 116: 12083–12084
Hamachi I, Yamada Y, Matsugi T, Shinkai S (1999) Single- or dual-mode switching of semisynthetic ribonuclease S’ with an iminodiacetic acid moiety in response to the copper(II) concentration. Chem Eur J 5:1503–1511
Hamachi I, Hiraoka T, Yamada Y, Shinkai S (1998) Photoswitching of the enzymatic activity of semisynthetic ribonuclease S’ bearing phenylazophenylalanine at a specific site. Chem Lett 6:537–538
Dickman SR, Trupin K (1959) Ribonuclease assay based on uridine phosphate determination. Arch Biochem Biophys 82:355–361
Korn K, Greiner-Stoeffele T, Hahn U (2001) Ribonuclease assays utilizing toluidine blue indicator plates, methylene blue, or fluorescence correlation spectroscopy. Methods Enzymol 341:142–153
Lee CC, Trotman CNA, Tate WP (1983) A ribonuclease assay: separation of product from undegraded RNA substrate. Anal Biochem 135:64–68
Postek KM, LaDue T, Nelson C, Sandwick RK (1992) Spectrophotometric ribonuclease assays using dinucleoside monophosphate substrates. Anal Biochem 203:47–52
Greiner-Stoeffele T, Grunow M, Hahn U (1996) A general ribonuclease assay using methylene blue. Anal Biochem 240:24–28
Simons ER, Blout ER (1968) Circular dichroism of ribonuclease A, ribonuclease S, and some fragments. J Biol Chem 243:218–221
Bastos M, Pease JH, Wemmer DE, Murphy KP, Connelly PR (2001) Thermodynamics of the helix-coil transition: binding of S15 and a hybrid sequence, disulfide stabilized peptide to the S-protein. Proteins 42:523–530
Moerke NJ (2009) Fluorescence polarization (FP) assays for monitoring peptide-protein or nucleic acid: protein binding. Curr Protoc Chem Biol 1:1–15
Kim EE, Varadarajan R, Wyckoff HW, Richards FM (1992) Refinement of the crystal structure of ribonuclease S. Comparison with and between the various ribonuclease A structures. Biochemistry 31:12304–12314
Acknowledgment
This work was supported by the European Fund for Regional Structure Development (EFRE, European Unit). The authors like to thank the group of Prof. Dr. Ralf Hoffmann, especially Dr. David Singer for peptide synthesis as well as Dr. Daniel Knappe and Nicole Berthold, for introduction into fluorescence polarization.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Genz, M., Sträter, N. (2014). Posttranslational Incorporation of Noncanonical Amino Acids in the RNase S System by Semisynthetic Protein Assembly. In: Köhler, V. (eds) Protein Design. Methods in Molecular Biology, vol 1216. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1486-9_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1486-9_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1485-2
Online ISBN: 978-1-4939-1486-9
eBook Packages: Springer Protocols