An Experimental Tool to Estimate the Probability of a Nucleotide Presence in the Crystal Structures of the Nucleotide-Protein Complexes.
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 KDof 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 KDvalue higher than 50 μM are not stable enough to survive in crystallization conditions.
Institute of Protein Research RAS, Pushchino, Russian Federation.
Maria
M
Nemchinova
Institute of Protein Research RAS, Pushchino, Russian Federation.
Vitaly
V
Balobanov
Institute of Protein Research RAS, Pushchino, Russian Federation.
Ekaterina
E
Nikonova
Institute of Protein Research RAS, Pushchino, Russian Federation.
Natalia
N
Lekontseva
Institute of Protein Research RAS, Pushchino, Russian Federation.
Alisa
A
Mikhaylina
Institute of Protein Research RAS, Pushchino, Russian Federation.
Svetlana
S
Tishchenko
Institute of Protein Research RAS, Pushchino, Russian Federation. nikulin@vega.protres.ru.
Alexey
0000-0001-5391-6184
A
Nikulin
D019843:Archaeal Proteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D008956:Models, Chemical; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry*; D011336:Probability; D012269:Ribosomal Proteins / Q000737:chemistry*
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D009711:Nucleotides; D012269:Ribosomal Proteins
Equilibrium dissociation constant; Fluorescence anisotropy; Nucleotide–protein interactions; Protein crystallography; RNA–protein interactions; Transcription factors