Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific ¹H–¹³C Labeling and Fast Magic-Angle Spinning NMR

Abstract

Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize dynamics of phenylalanine residues using 1 H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling

Transition-State Compressibility and Activation Volume of Transient Protein Conformational Fluctuations

“…5 − 18 In fact, experimental characterization of ring flip rates did not progress much after the groundbreaking observations in the 1970s, partly because of limitations of the NMR technique, which required well-resolved signals in the slow-exchange regime. Recently, there has been a renaissance in these types of studies, 11 , 13 , 19 , 20 enabled by methodological advances, including site-selective isotope labeling of aromatic side chains, 21 − 27 relaxation dispersion experiments tailored to aromatic rings, 28 − 31 and improved analysis that takes into account the effects of strong 1 H– 1 H J -coupling within the aromatic ring. 16 …”

Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

“…Collectively, the absence of observable signals for most of the backbone sites in the loop, and the direct evidence from spin relaxation of D135 indicates that the loop undergoes µs motion. In an additional experiment that aims to detect the loop signals we exploited a specific and very sensitive isotopelabeling method of phenylalanines which we developed recently (50). In this isotope-labeling scheme, a single 1 H- 13 C pair in the para-position of the Phe ring is introduced in an otherwise fully deuterated sample.…”

“…We have recently achieved the resonance assignment of ca. 90 % of the backbone atoms, and about 70 % of the side-chain heavy atoms, as well as methyl groups of Ile, Leu and Val residues (41, 42) and Phe ring positions (43) in TET2, and developed an approach that uses medium-resolution cryo-EM data along with (primarily solid-state) NMR data to solve the structure of TET2 (41). With 353 residues per subunit, TET2 is among the largest proteins for which such near-complete assignment has been achieved, and the largest structure solved to date.…”

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

“…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] .…”