Nucleotidefree

Nucleotidefree is a term used in biochemistry and molecular biology to describe a molecule, solution, or biological state that lacks nucleotides. Nucleotides include the monomer units of nucleic acids (adenine, cytosine, guanine, thymine or uracil) as well as energy-rich cofactors such as ATP and GTP. In many contexts, nucleotidefree implies an apo or ligand-free state, in contrast to a nucleotide-bound (holo) form.

In structural and enzymology studies, nucleotidefree often refers to the state of a protein that has no bound nucleotide. This apo form can be compared with holo forms to understand how nucleotide binding influences conformation, stability, and activity. The concept is especially important for ATPases, GTPases, kinases, and motor proteins, where binding and hydrolysis of nucleotides drive function.

Obtaining nucleotidefree samples typically involves removing bound nucleotides from proteins or complexes. Common approaches include extended dialysis or gel filtration to separate bound from free nucleotides, use of nucleotide-exchange or affinity purification methods, and treatment with enzymes such as apyrase to hydrolyze residual nucleotides. In cellular or in vitro systems, nucleotide pools can be depleted by metabolic inhibitors, competition with non-hydrolyzable analogs, or enzymatic degradation, though complete removal can be challenging and may affect stability.

Applications of nucleotidefree states include studying binding sites, conformational changes, and mechanism of action; guiding structural determination by X-ray crystallography, NMR, or cryo-EM; and assessing nucleotide affinity and exchange kinetics.