The use of isotopic substitution is a time-honoured method for simplifying the nuclear magnetic resonance spectra of biological macromolecules. For example, the biosynthetic incorporation of a heteronucleus such as 15N or 13C into a specific amino acid residue in a protein followed by direct observation of the 15N or 13C NMR spectrum could provide a means to specifically observe a given amino acid type in that protein. By observation of the chemical shift or relaxation properties as a function of pH, ligand concentration, etc. a number of important conclusions concerning the pKa values of specific residues, the affinity of the protein for various ligands, or dynamic properties of the protein can be deduced. (See Henry et al. 1986 a, b; 1987 for an elegant modern example). In such situations, direct observation of the heteronucleus is a powerful means to observe environmental changes (Niu et al. 1979) but often these measurements are not readily interpretable in terms of alterations of protein structure. Although proton-proton dipolar interactions (NOEs) typically provide the richest source of such structural information, these interactions are not monitored in most experiments which directly observe the heteronucleus.

Biosynthetic incorporation of 15N and 13C for assignment and interpretation of nuclear magnetic resonance spectra of 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).

https://onlinelibrary.wiley.com/doi/pdf/10.1002/pro.5560020307

Ribonuclease S‐peptide as a carrier in fusion proteins

Publisher Summary Bovine Ribonuclease has been a test protein in the study of a wide variety of chemical and physical methods of protein chemistry. It is widely employed in the course of the sequencing of RNA. This chapter discusses the usefulness of the cytoplasmic inhibitor of RNase to protect RNA in the course of the synthesis of complementary DNA by reverse transcriptase. It examines the use of the inhibitor to protect mRNA during the course of in vitro translation and during the preparation of rough microsomes and detached polysomes. The introduction of affinity chromatography has simplified the isolation of pure RNases by providing a highly efficient method for separating active enzymes from molecules that do not have an affinity for the coupled substrate analog. The technique can be used as an early step in purification or as a final step after ion exchange chromatography and gel filtration to isolate an RNase fraction of given charge and size.

12 Pancreatic Ribonuclease

Semisynthesis provides a flexible approach for using chemical synthesis to produce large, biologically active polypeptides and analogues. Currently developing improvements in the basic methods used, including polypeptide fragmentation, peptide synthesis, and reconstitution of synthetic and native components, make this overall approach applicable to a variety of species. Sequence modification through semisynthesis thus provides a flexible route to explore the code of rules whereby primary structure directs higher order properties of folded conformation and biological function of large peptides and proteins. The fruits of this endeavor, an understanding of how these macromolecules work, and therein, a basis for design of new structures that ultimately may be produced directly or by recombinant DNA methods, have begun to emerge.

Semisynthetic peptides and proteins

The solvent-accessibility-modified, Tanford-Kirkwood, discrete charge model for electrostatic effects is applied to both ribonuclease A and ribonuclease S. The behavior of individual titratable sites and the pH-dependent free energy of denaturation are correctly predicted. The use of the solvent-accessibility factor in reducing charge-site interactions introduces a higher Coulombic shielding for solvent-exposed sites. This shielding is interpreted as a higher local strength or alternatively a higher effective dielectric constant. Specific anion binding sites are determined by locating areas of high positive electrostatic potential at the protein solvent interface. The potential and thus the anion affinity of a given site are calculated and shown to vary with the pH-dependent charge array. pH-dependent anion binding constants are calculated for the ribonuclease S active site. These binding constants and the predicted response of the active-site histidine pK1/2 values to anion binding are shown to agree with experimental determinations.

Anion binding and pH-dependent electrostatic effects in ribonuclease

Specific peptide-protein interactions in the ribonuclease S' system studied by 13C nuclear magnetic resonance spectroscopy with selectively 13C-enriched peptides.

Direct observation of peptide exchange by stable isotope enrichment.

Ribonuclease S-protein exhibits a pH-dependent conformational transition between folded and unfolded states, and some unfolded S-protein persists up to pH 8. The histidine C2 proton resonance of the unfolded species was erroneously assigned by Bradbury et al. to histidine residue 119 of the folded species.

S Matsuura

Papers

Specific peptide-protein interactions in the ribonuclease S' system studied by 13C nuclear magnetic resonance spectroscopy with selectively 13C-enriched peptides.

Direct observation of peptide exchange by stable isotope enrichment.

Conformation of ribonuclease S-protein.