Protein-nucleic acidi nteractions play important roles not only in energy-providing reactions,s uch as ATPh ydrolysis, but also in reading, extending, packaging, or repairing genomes. Althought hey can often be analyzed in detail with X-ray crystallography, complementary methods are neededt ov isualize them in complexes, which are not crystalline. Here, we show how solid-stateN MR spectroscopy can detecta nd classify protein-nucleic interactions through site-specific 1 H-and 31 P-detected spectroscopicm ethods. The sensitivity of 1 Hc hemicalshift values on noncovalent interactions involved in these molecular recognition processes is exploiteda llowing us to probe directly the chemical bonding state, an information, which is not directly accessible from an X-ray structure. We show that these methods can characterize interactions in easy-to-prepare sediments of the 708 kDa dodecamericD naB helicasei nc omplex with ADP:AlF 4 À :DNA, and this despite the very challenging size of the complex.Nucleotide-protein interactions playacentral role in two major biological functions:i ne nergy-providing reactions, where ATPi sh ydrolyzed to yield energy to motor domains driving reactions [1,2] and in interactions with RNA or DNA, central in al arge variety of biomolecular functions. Binding of nucleotides, such as ATPa nd DNA, occurs throughn oncovalent interactions includingh ydrogen bonds, electrostatic (salt bridges), and van der Waals interactions [3,4] (the latter also comprising interactions between aromatic rings [5] ). These interactions have been typicallys tudied in the past by high-resolution X-ray crystallography. [4,6,7] Still, many of the scenarios described above involve protein complexes, which are difficult to crystallize, and when they do so, might reflect at insufficient resolution to clearly identify interactions.A lternative methods are therefore neededa nd can be providedt hrough solid-state NMR spectroscopy,w hich can access also large biomolecular complexes,a nd importantly in sample formats where the assemblies are simply sedimented into the NMR rotor. [8] And indeed, solid-state NMR spectroscopy has been used to identify residues at protein-RNA interfaces in smaller proteins. [9][10][11][12] Twoa pproaches are particularly promising to probe nucleotide interactions:p hosphorus-( 31 P) and proton-( 1 H) detected spectroscopy.D istances between 31 Ps pins of DNA and 15 N spins of ap rotein have been measuredb yu sing transferredecho, double-resonance (TEDOR) experiments. [9] Intermolecular information can also be obtained from 31 P-detected, heteronuclear correlation experimentsp robingt he spatial proximity of nucleotide 31 Pa nd protein 15 No r 13 Cn uclei. [9,13] Proton-detected solid-state NMR spectroscopy at fast MAS frequencies has emerged in the last years and needs today only af ew hundred micrograms of fully protonated protein sample. [14][15][16][17][18][19][20][21][22][23] Proton chemical-shift values are highly sensitivet oh ydrogen bonding [24][25][26][27] as shown in theoretical, [26,27] ...
