The Protein Journal

, Volume 36, Issue 3, pp 157–165 | Cite as

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

  • Maria Nemchinova
  • Vitaly Balobanov
  • Ekaterina Nikonova
  • Natalia Lekontseva
  • Alisa Mikhaylina
  • Svetlana Tishchenko
  • Alexey Nikulin
Article

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.

Keywords

Nucleotide–protein interactions RNA–protein interactions Equilibrium dissociation constant Fluorescence anisotropy Protein crystallography Transcription factors 

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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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Institute of Protein Research RASPushchinoRussian Federation

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