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An Experimental Tool to Estimate the Probability of a Nucleotide Presence in the Crystal Structures of the Nucleotide–Protein Complexes

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Abstract

A correlation between the ligand–protein affinity and the identification of the ligand in the experimental electron density maps obtained by X-ray crystallography has been tested for a number of RNA-binding proteins. Bacterial translation regulators ProQ, TRAP, Rop, and Hfq together with their archaeal homologues SmAP have been used. The equilibrium dissociation constants for the N-methyl-anthraniloyl-labelled adenosine and guanosine monophosphates titrated by the proteins have been determined by the fluorescent anisotropy measurements. The estimated stability of the nucleotide–protein complexes has been matched with a presence of the nucleotides in the structures of the proposed nucleotide–protein complexes. It has been shown that the ribonucleotides can be definitely identified in the experimental electron density maps at equilibrium dissociation constant <10 μM. At KD of 20–40 μM, long incubation of the protein crystals in the nucleotide solution is required to obtain the structures of the complexes. The complexes with KD value higher than 50 μM are not stable enough to survive in crystallization conditions.

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Abbreviations

ssRNA:

Single-stranded RNA

AMP:

Adenosine-5′-monophosphate

GMP:

Guanosine-5′-monophosphate

Mant-AMP:

2′/3′-(N-Methyl-anthraniloyl)-adenosine-5′-monophosphate

Mant-GMP:

2′/3′-O-(N-Methyl-anthraniloyl)-guanosine-5′-monophosphate

KD :

Equilibrium dissociation constants

SmAP:

Sm-like archaeal protein

SsoSmAP2:

Sm-like archaeal protein from Sulfolobus solfataricus

MjaSmAP:

Sm-like archaeal protein from Methanococcus jannaschii

EcoHfq:

Protein Hfq from Escherichia coli

PaeHfq:

Protein Hfq from Pseudomonas aeruginosa

aIF2γ:

Gamma subunit of archaeal initiation factor aIF2γ from S. solfataricus

Lsm:

Sm-like proteins

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Acknowledgements

This work was supported by RSF grant #14-14-00496.

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Authors and Affiliations

  1. Institute of Protein Research RAS, Pushchino, Russian Federation

    Maria Nemchinova, Vitaly Balobanov, Ekaterina Nikonova, Natalia Lekontseva, Alisa Mikhaylina, Svetlana Tishchenko & Alexey Nikulin

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  1. Maria Nemchinova
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  2. Vitaly Balobanov
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  3. Ekaterina Nikonova
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Correspondence to Alexey Nikulin.

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Nemchinova, M., Balobanov, V., Nikonova, E. et al. An Experimental Tool to Estimate the Probability of a Nucleotide Presence in the Crystal Structures of the Nucleotide–Protein Complexes. Protein J 36, 157–165 (2017). https://doi.org/10.1007/s10930-017-9709-y

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