“Active” conformation of an inactive semi-synthetic ribonuclease-S

doi:10.1016/0022-2836(81)90305-3

Journal of Molecular Biology

Volume 149, Issue 2, 25 June 1981, Pages 313-317

https://doi.org/10.1016/0022-2836(81)90305-3 Get rights and content

Abstract

We have studied the integrity of folded structure of a fully active semi-synthetic ribonuclease-S which lacks amino acid residues 16 through 20, and an inactive one with the same residues deleted and 4-fluoro-l-histidine substituted for active site histidine 12. Using “Y” form crystals, we obtained X-ray structural data to a resolution of 2·6 Å and, incorporating phase information calculated from refined ribonuclease-S coordinates, prepared several types of electron density maps. These showed that the overall backbone structure and active site configuration of both analogues do not differ noticeably from those of the native protein. Structural homology extends to the catalytically relevant side-chain at position 12; 4-F-His^† assumes the same position as does His in active ribonuclease-S. This supports the view that the 4-F-Hisl2 analogue is inactive due to a change in histidine 12 imidazole basicity, rather than to any significant conformational distortion within the active site.

References (17)

I.M. Chaiken et al.
J. Biol. Chem
(1977)
B.M. Dunn et al.
J. Biol. Chem
(1974)
B. Gutte
J. Biol. Chem
(1975)
B. Gutte
J. Biol. Chem
(1977)
F.M. Richards et al.
H.C. Taylor et al.
J. Biol. Chem
(1977)
H.W. Wyckoff et al.
J. Biol. Chem
(1970)
C.A. Deakyne et al.
J. Amer. Chem. Soc
(1979)

There are more references available in the full text version of this article.

Cited by (32)

Semisynthesis of Human Ribonuclease-S
2021, Bioconjugate Chemistry
Since its conception, the ribonuclease S complex (RNase S) has led to historic discoveries in protein chemistry, enzymology, and related fields. Derived by the proteolytic cleavage of a single peptide bond in bovine pancreatic ribonuclease (RNase A), RNase S serves as a convenient and reliable model system for incorporating unlimited functionality into an enzyme. Applications of the RNase S system in biomedicine and biotechnology have, however, been hindered by two shortcomings: (1) the bovine-derived enzyme could elicit an immune response in humans, and (2) the complex is susceptible to dissociation. Here, we have addressed both limitations in the first semisynthesis of an RNase S conjugate derived from human pancreatic ribonuclease and stabilized by a covalent interfragment cross-link. We anticipate that this strategy will enable unprecedented applications of the “RNase–S” system.
Current applications of <sup>19</sup>F NMR to studies of protein structure and dynamics
2012, Progress in Nuclear Magnetic Resonance Spectroscopy
Citation Excerpt :
Phenylalanine in particular is often buried within the protein hydrophobic core and has been used extensively in studies of protein folding where it has been implicated in the formation and stabilization of folding intermediates of both native and nonnative character [74,75]. Tyrosine and histidine are often located within enzyme active sites where their role may be structural, for example through metal coordination [76] and hydrogen bonding [6], or mechanistic, such as in examples of acid–base catalysis [77]. Furthermore, fluorination alters the side chain pKa of these residues, allowing for the examination of pH dependent properties.
Control of function of a small peptide by a protein
2006, Bioorganic and Medicinal Chemistry Letters
Solution-state NMR investigations of triosephosphate isomerase active site loop motion: Ligand release in relation to active site loop dynamics
2001, Journal of Molecular Biology
Product release is partially rate determining in the isomerization reaction catalyzed by Triosephosphate Isomerase, the conversion of dihydroxyacetone phosphate to d-glyceraldehyde 3-phosphate, probably because an active-site loop movement is necessary to free the product from confinement in the active-site. The timescale of the catalytic loop motion and of ligand release were studied using ¹⁹F and ³¹P solution-state NMR. A 5′-fluorotryptophan was incorporated in the loop N-terminal hinge as a reporter of loop motion timescale. Crystallographic studies confirmed that the structure of the fluorinated enzyme is indistinguishable from the wild-type; the fluorine accepts a hydrogen bond from water and not from a protein residue, with minimal perturbation to the flexible loop stability. Two distinct loop conformations were observed by ¹⁹F NMR. Both for unligated (empty) and ligated enzyme samples a single species was detected, but the chemical shifts of these two distinct species differed by 1.2 ppm. For samples in the presence of subsaturating amounts of a substrate analogue, glycerol 3-phosphate, both NMR peaks were present, with broadened lineshapes at 0°C. In contrast, a single NMR peak representing a rapid average of the two species was observed at 30°C. We conclude that the rate of loop motion is less than 1400 s⁻¹ at 0°C and more than 1400 s⁻¹ at 30°C. Ligand release was studied under similar sample conditions, using ³¹P NMR of the phosphate group of the substrate analogue. The rate of ligand release is less than 1000 s⁻¹ at 0°C and more than 1000 s⁻¹ at 30°C. Therefore, loop motion and product release are probably concerted and likely to represent a rate limiting step for chemistry.
Evaluation of subtilisin cleavage specificity for RNase A peptide bonds by high-performance liquid chromatography and mass spectrometric analysis
2000, Analytical Biochemistry
Affinity maturation of phage-displayed peptide ligands
1996, Methods in Enzymology
When the fitness being selected for is affinity for a target receptor molecule, the foregoing program is called “affinity maturation,” the term coined by immunologists for the interspersed rounds of selective stimulation by antigen and somatic mutation of antibody genes that is thought to give rise to antibodies, with increasing affinity in the course of an immune response. This chapter discusses the affinity maturation from random peptide libraries displayed on phage. The procedures and underlying principles are discussed in the context of a specific exemplar experiment, in which ligands for a model receptor were selected from a library of random 15-mers. The model receptor is S-protein, a 104-residue fragment of bovine ribonuclease prepared by partial digestion, with subtilisin; the other fragment, S-peptide, corresponds to the N-terminal 20 amino acids. Neither fragment alone is enzymatically active, but when they are mixed S-peptide binds strongly to S-protein, restoring the enzyme activity.

View all citing articles on Scopus

^☆: The work at Duke was supported by contracts from the National Institutes of Health, NIH Grant GM 15000 and RCDA GM 00041 (to D.C.R.).

^†: Abbreviation used: 4-F-His, 4-fluoro-l-histidine.

View full text

Journal of Molecular Biology

Letter to the editor“Active” conformation of an inactive semi-synthetic ribonuclease-S☆

Abstract

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Amer. Chem. Soc

Letter to the editor
“Active” conformation of an inactive semi-synthetic ribonuclease-S☆