G protein-coupled receptors (GPCRs) exhibit a wide variety of signaling behaviors in response to different ligands. Using a small label on the cytosolic interface of TM6 (Cys-265), 19F NMR spectra of the β2 adrenoreceptor, β2AR, reconstituted in maltose–neopentyl glycol detergent micelles, revealed two distinct inactive states, an activation intermediate state en route to activation, and, in the presence of a G protein mimic, a predominant active state. An analysis of spectra as a function of temperature reveals that for all ligands, the activation intermediate is entropically favored and enthalpically disfavored. β2AR enthalpy changes toward activation are notably lower than those observed with rhodopsin – a likely consequence of basal activity and the fact that the ionic lock and other interactions stabilizing the inactive state of β2AR, are weaker. Positive entropy changes toward activation likely reflect greater mobility (configurational entropy) in the cytoplasmic domain, confirmed through an order parameter analysis. Ligands greatly influence overall changes in enthalpy and entropy of the system and corresponding changes in population and amplitude of motion of given states, suggesting a complex landscape of states and substates.

We present an off-resonance carbon R(1rho) NMR experiment utilizing weak radiofrequency fields and selective polarization transfers for quantifying chemical-exchange processes in nucleic acids. The experiment extends the range of accessible time scales to approximately 10 ms, and its time-saving feature makes it possible to thoroughly map out dispersion profiles and conduct measurements at natural abundance. The experiment unveiled microsecond-to-millisecond exchange dynamics in a uniformly labeled A-site rRNA and in unlabeled, damaged DNA that would otherwise be difficult to characterize by conventional methods.

We present an NMR strategy for characterizing picosecond-to-nanosecond internal motions in uniformly 13C/15N-labeled RNAs that combines measurements of R1, R1rho, and heteronuclear 13C{1H} NOEs for protonated base (C2, C5, C6, and C8) and sugar (C1') carbons with a domain elongation strategy for decoupling internal from overall motions and residual dipolar coupling (RDC) measurements for determining the average RNA global conformation and orientation of the principal axis of the axially symmetric rotational diffusion. TROSY-detected pulse sequences are presented for the accurate measurement of nucleobase carbon R1 and R1rho rates in large RNAs. The relaxation data is analyzed using a model free formalism which takes into account the very high anisotropy of overall rotational diffusion (Dratio approximately 4.7), asymmetry of the nucleobase CSAs and noncollinearity of C-C, C-H dipolar and CSA interactions under the assumption that all interaction tensors for a given carbon experience identical isotropic internal motions. The approach is demonstrated and validated on an elongated HIV-1 TAR RNA (taum approximately 18 ns) both in free form and bound to the ligand argininamide (ARG). Results show that, while ARG binding reduces the amplitude of collective helix motions and local mobility at the binding pocket, it leads to a drastic increase in the local mobility of "spacer" bulge residues linking the two helices which undergo virtually unrestricted internal motions (S2 approximately 0.2) in the ARG bound state. Our results establish the ability to quantitatively study the dynamics of RNAs which are significantly larger and more anisotropic than customarily studied by NMR carbon relaxation.

Many recently discovered non-coding RNAs do not fold into a single native conformation, but rather, sample many different conformations along their free energy landscape to carry out their biological function. Unprecedented insights into the RNA dynamic structure landscape are provided by solution-state NMR techniques that measure the structural, kinetic, and thermodynamic characteristics of motions spanning picosecond to second timescales at atomic resolution. From these studies a basic description of the RNA dynamic structure landscape is emerging, bringing new insights into how RNA structures change to carry out their function as well as applications in RNA-targeted drug discovery and RNA bioengineering.

The HIV-1 transactivation response element (TAR) RNA binds a variety of proteins and is a target for developing anti-HIV therapies. TAR has two primary binding sites: a UCU bulge and a CUGGGA apical loop. We used NMR residual dipolar couplings, carbon spin relaxation (R(1) and R(2)), and relaxation dispersion (R(1rho)) in conjunction with molecular dynamics and mutagenesis to characterize the dynamics of the TAR apical loop and investigate previously proposed long-range interactions with the distant bulge. Replacement of the wild-type apical loop with a UUCG loop did not significantly affect the structural dynamics at the bulge, indicating that the apical loop and the bulge act largely as independent dynamical recognition centers. The apical loop undergoes complex dynamics at multiple timescales that are likely important for adaptive recognition: U31 and G33 undergo limited motions, G32 is highly flexible at picosecond-nanosecond timescales, and G34 and C30 form a dynamic Watson-Crick basepair in which G34 and A35 undergo a slow (approximately 30 mus) likely concerted looping in and out motion, with A35 also undergoing large amplitude motions at picosecond-nanosecond timescales. Our study highlights the power of combining NMR, molecular dynamics, and mutagenesis in characterizing RNA dynamics.