A Nearly Isosteric Photosensitive Amide-Backbone Substitution Allows Enzyme Activity Switching in Ribonuclease S

Abstract

psi[CS-NH]4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala4-Ala5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. psi[CS-NH](4)-RNase S was yielded by recombination of the S-protein and the respective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealed similar thermodynamic stability of the complex and an unperturbed enzymatic activity toward cytidine 2',3'-cyclic monophosphate (cCMP). Reversible photoisomerization with a highly increased cis/trans isomer ratio of the thioxopeptide bond of psi[CS-NH](4)-RNase S in the photostationary state occurred under UV irradiation conditions (254 nm). The slow thermal reisomerization (t(1/2) = 180 s) permitted us to determine the enzymatic activity of cis psi[CS-NH](4)-RNase S by measurement of initial rates of cCMP hydrolysis. Despite thermodynamic stability of cis psi[CS-NH](4)-RNase S, its enzymatic activity is completely abolished but recovers after reisomerization. We conclude that the thioxopeptide bond modified polypeptide backbone represents a versatile probe for site-directed photoswitching of proteins.

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction

“…Deuteration of amide protons destabilizes helical structures, but the effect is very small (14). A near isosteric change in the polypeptide backbone can be achieved by introducing a thioxopeptide bond, i.e., by replacing an amide oxygen by sulfur (15).…”

Time-Resolved Chiral Vibrational Spectroscopy

“…[48,49] It has recently been shown that this isomerization near the N-terminus of the S peptide in the protein complex ribonuclease S induces conformational changes sufficient to switch off enzymatic activity. [50] In many of these examples photo-excitation of a significant fraction of molecules is possible, resulting in large conformational changes and transient VCD signal levels can be similar to those observed in the Co(sp)Cl 2 model compound. In addition, the development of alternative detection schemes is about to further improve signal to noise levels in transient circular dichroism measurements.…”

Design, selection, and characterization of a split chorismate mutase

“…The N-terminal peptide comprising residues 1-20 (MjCM [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] ) was prepared by solid-phase peptide synthesis (SPPS) methods, and purified by HPLC. The C-terminal portion containing the additional mutation I77R to disfavor homodimerization 18 (MjCM * 22-93 ) was produced by heterologous expression in the CM-deficient Escherichia Coli strain KA13 27 with a C-terminal His 6 -tag and purified on a Ni-NTA column.…”

“…Solid-phase peptide synthesis mMjCM [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] was synthesized on a Wang resin preloaded with Fmoc-Leu (0.68 mmol/g) on a 433A peptide synthesizer (Applied Biosystems) on a 0.1 mmol scale with standard Fmoc-protected amino acids (Novabiochem). The peptide was cleaved from the resin and amino acid protecting groups were removed by incubation in 95% TFA, 2.5% H 2 O, and 2.5% TIS for 2 h. The resin was filtered off, excess TFA was evaporated, and the peptide was precipitated and washed with cold ether and dried under vacuum.…”

“…1,2 Split enzymes permit facile incorporation of synthetic peptide fragments into enzymes, which enabled structure-activity studies long before molecular cloning became a standard method for mutational analysis of proteins. 3 In addition, a wide variety of unnatural building blocks containing photoisomerizable groups, 4 environmentally sensitive dyes, 5 and modified backbones 6 could easily be incorporated into the peptide fragment of RNase S for biophysical studies.…”