Alexandar L. Hansen

The Role of Ligands on the Equilibria Between Functional States of a G Protein-Coupled Receptor

G protein-coupled receptors exhibit a wide variety of signaling behaviors in response to different ligands. When a small label was incorporated on the cytosolic interface of transmembrane helix 6 (Cys-265), (19)F NMR spectra of the β2 adrenergic receptor (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. Analysis of the spectra as a function of temperature revealed 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, as confirmed through an order parameter analysis. Ligands greatly influence the overall changes in enthalpy and entropy of the system and the corresponding changes in population and amplitude of motion of given states, suggesting a complex landscape of states and substates.

Characterizing RNA dynamics at atomic resolution using solution-state NMR spectroscopy

Many recently discovered noncoding RNAs do not fold into a single native conformation but sample many different conformations along their free-energy landscape to carry out their biological function. Here we review solution-state NMR techniques that measure the structural, kinetic and thermodynamic characteristics of RNA motions spanning picosecond to second timescales at atomic resolution, allowing unprecedented insights into the RNA dynamic structure landscape. 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.

Extending the Range of Microsecond-to-Millisecond Chemical Exchange Detected in Labeled and Unlabeled Nucleic Acids by Selective Carbon R1ρ NMR Spectroscopy

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.

Characterizing complex dynamics in the transactivation response element apical loop and motional correlations with the bulge by NMR, molecular dynamics, and mutagenesis.

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.

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

We present a protocol for determining the relative orientation and dynamics of A-form helices in 13C/15N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson–Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.

Measurement of histidine pKa values and tautomer populations in invisible protein states.

Significance Electrostatic interactions in proteins play significant roles in conferring stability and in dictating function. Histidine residues are particularly important because their side chains can serve as both acids and bases over the physiological pH range and as both hydrogen bond donors and acceptors. Solution NMR spectroscopy is a powerful method for studying these residues in highly populated ground-state conformers. Here we develop a strategy for extending such studies to sparsely populated, short-lived protein states that can also play a significant role in defining protein function. An application to an invisible folding intermediate of the Im7 protein is provided, where site-specific pKa values of histidine residues have been determined that explain the strong pH-dependent stability differences between native and intermediate states. The histidine imidazole side chain plays a critical role in protein function and stability. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in ∼50% of all enzymes. NMR spectroscopy has become an important tool for studies of histidine side chains through the measurement of site-specific pKas and tautomer populations. To date, such studies have been confined to observable protein ground states; however, a complete understanding of the role of histidine electrostatics in protein function and stability requires that similar investigations be extended to rare, transiently formed conformers that populate the energy landscape, yet are often “invisible” in standard NMR spectra. Here we present NMR experiments and a simple strategy for studies of such conformationally excited states based on measurement of histidine 13Cγ, 13Cδ2 chemical shifts and 1Hε-13Cε one-bond scalar couplings. The methodology is first validated and then used to obtain pKa values and tautomer distributions for histidine residues of an invisible on-pathway folding intermediate of the colicin E7 immunity protein. Our results imply that the side chains of H40 and H47 are exposed in the intermediate state and undergo significant conformational rearrangements during folding to the native structure. Further, the pKa values explain the pH-dependent stability differences between native and intermediate states over the pH range 5.5–6.5 and they suggest that imidazole deprotonation is not a barrier to the folding of this protein.

Nonnative interactions in the FF domain folding pathway from an atomic resolution structure of a sparsely populated intermediate: an NMR relaxation dispersion study.

Several all-helical single-domain proteins have been shown to fold rapidly (microsecond time scale) to a compact intermediate state and subsequently rearrange more slowly to the native conformation. An understanding of this process has been hindered by difficulties in experimental studies of intermediates in cases where they are both low-populated and only transiently formed. One such example is provided by the on-pathway folding intermediate of the small four-helix bundle FF domain from HYPA/FBP11 that is populated at several percent with a millisecond lifetime at room temperature. Here we have studied the L24A mutant that has been shown previously to form nonnative interactions in the folding transition state. A suite of Carr-Purcell-Meiboom-Gill relaxation dispersion NMR experiments have been used to measure backbone chemical shifts and amide bond vector orientations of the invisible folding intermediate that form the input restraints in calculations of atomic resolution models of its structure. Despite the fact that the intermediate structure has many features that are similar to that of the native state, a set of nonnative contacts is observed that is even more extensive than noted previously for the wild-type (WT) folding intermediate. Such nonnative interactions, which must be broken prior to adoption of the native conformation, explain why the transition from the intermediate state to the native conformer (millisecond time scale) is significantly slower than from the unfolded ensemble to the intermediate and why the L24A mutant folds more slowly than the WT.

Quantifying millisecond time-scale exchange in proteins by CPMG relaxation dispersion NMR spectroscopy of side-chain carbonyl groups

A new pulse sequence is presented for the measurement of relaxation dispersion profiles quantifying millisecond time-scale exchange dynamics of side-chain carbonyl groups in uniformly 13C labeled proteins. The methodology has been tested using the 87-residue colicin E7 immunity protein, Im7, which is known to fold via a partially structured low populated intermediate that interconverts with the folded, ground state on the millisecond time-scale. Comparison of exchange parameters extracted for this folding ‘reaction’ using the present methodology with those obtained from more ‘traditional’ 15N and backbone carbonyl probes establishes the utility of the approach. The extracted excited state side-chain carbonyl chemical shifts indicate that the Asx/Glx side-chains are predominantly unstructured in the Im7 folding intermediate. However, several crucial salt-bridges that exist in the native structure appear to be already formed in the excited state, either in part or in full. This information, in concert with that obtained from existing backbone and side-chain methyl relaxation dispersion experiments, will ultimately facilitate a detailed description of the structure of the Im7 folding intermediate.

Probing slowly exchanging protein systems via 13Cα-CEST: monitoring folding of the Im7 protein

A 13Cα chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an ‘observable’ ground state and one or more ‘invisible’ excited states. Here a labeling strategy whereby [2-13C]-glucose is the sole carbon source is exploited, producing proteins with 13C at the Cα position, while the majority of residues remain unlabeled at CO or Cβ. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state 13Cα chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.

Variable helix elongation as a tool to modulate RNA alignment and motional couplings

The application of residual dipolar couplings (RDCs) in studies of RNA structure and dynamics can be complicated by the presence of couplings between collective helix motions and overall alignment and by the inability to modulate overall alignment of the molecule by changing the ordering medium. Here, we show for a 27-nt TAR RNA construct that variable levels of helix elongation can be used to alter both overall alignment and couplings to collective helix motions in a semi-predictable manner. In the absence of elongation, a four base-pair helix II capped by a UUCG apical loop exhibits a higher degree of order compared to a six base-pair helix I (theta(I)/theta(II)=0.56+/-0.1). The principal S(zz) direction is nearly parallel to the axis of helix II but deviates by approximately 40 degrees relative to the axis of helix I. Elongating helix I by three base-pairs equalizes the alignment of the two helices and pushes the RNA into the motional coupling limit such that the two helices have comparable degrees of order (theta(I)/theta(II)=0.92+/-0.04) and orientations relative to S(zz) ( approximately 17 degrees ). Increasing the length of elongation further to 22 base-pairs pushes the RNA into the motional decoupling limit in which helix I dominates alignment (theta(II)/theta(I)=0.45+/-0.05), with S(zz) orientated nearly parallel to its helix axis. Many of these trends can be rationalized using PALES simulations that employ a previously proposed three-state dynamic ensemble of TAR. Our results provide new insights into motional couplings, offer guidelines for assessing their extent, and suggest that variable degrees of helix elongation can allow access to independent sets of RDCs for characterizing RNA structural dynamics.