Protein Side-Chain–DNA Contacts Probed by Fast Magic-Angle Spinning NMR

Abstract

Protein−nucleic acid interactions are essential in a variety of biological events ranging from the replication of genomic DNA to the synthesis of proteins. Noncovalent interactions guide such molecular recognition events, and protons are often at the center of them, particularly due to their capability of forming hydrogen bonds to the nucleic acid phosphate groups. Fast magicangle spinning experiments (100 kHz) reduce the proton NMR line width in solid-state NMR of fully protonated protein−DNA complexes to such an extent that resolved proton signals from sidechains coordinating the DNA can be detected. We describe a set of NMR experiments focusing on the detection of protein side-chains from lysine, arginine, and aromatic amino acids and discuss the conclusions that can be obtained on their role in DNA coordination. We studied the 39 kDa enzyme of the archaeal pRN1 primase complexed with DNA and characterize protein− DNA contacts in the presence and absence of bound ATP molecules.

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase

“…Proton-detected NMR experiments at fast MAS frequencies (>100 kHz) allow the identification of protons engaged in hydrogen bonds requiring only small amounts of protein in the order of 0.5 mg. The 1 H NMR chemical-shift value serves as a sensitive indicator for the formation of hydrogen bonds: a de-shielding effect is observed if protons are engaged in such interactions 44 , 45 , 58 , 69 . However, the chemical shift alone is not a sufficient criterion to prove hydrogen bonding.…”

“…Fast magic-angle spinning (MAS) nowadays provides sufficient spectral resolution for proton-detected sidechain studies 31 . Indeed, proton detection at fast MAS has become an important tool in structural biology in the past years for unraveling protein structures 32 – 41 , to characterize RNA molecules 42 and protein–nucleic acid interactions 43 – 45 , and to address protein dynamics 46 – 52 . A key advantage of solid-state NMR is the straightforward sample preparation, which simply consists of sedimentation from solution into the solid-state NMR rotor without requiring crystallization steps 53 , 54 yielding long-term stable protein samples 55 .…”

Virus Structures and Dynamics by Magic-Angle Spinning NMR

“…MAS frequencies of at least 8-10 kHz are necessary for recording well-resolved spectra for 13 C, 15 N, and 31 P nuclei. While this range of frequencies is sufficient for basic biological MAS NMR experiments, in practice the faster the specimen is spun, the higher the resolution and the sensitivity for the same rotor diameter.…”

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase

“…Proton-detected NMR experiments at fast MAS frequencies (> 100 kHz) allow the identification of protons engaged in hydrogen bonds requiring only small amounts of protein in the order of 0.5 mg. The 1 H NMR chemical-shift value serves as a sensitive indicator for the formation of hydrogen bonds: a deshielding effect is observed if protons are engaged in such interactions 42,43,54,67 . However, the chemical shift alone is not a sufficient criterion to prove hydrogen bonding.…”

“…The copyright holder for this preprint this version posted April 9, 2021. ; https://doi.org/10.1101/2021.04.08.438047 doi: bioRxiv preprint nowadays provides sufficient spectral resolution for proton-detected side-chain studies 29 . Indeed, proton detection at fast MAS has become an important tool in structural biology in the past years for unraveling protein structures [30][31][32][33][34][35][36][37][38][39] , to characterize RNA molecules 40 and proteinnucleic acid interactions [41][42][43] , and to address protein dynamics [44][45][46][47][48][49][50] . A key advantage of solidstate NMR is the straightforward sample preparation, which simply consists of sedimentation from solution into the solid-state NMR rotor without requiring crystallization steps [51][52][53] .…”