Angular variances for internal bond rotations of side chains in GXG-based tripeptides derived from (13)C-NMR relaxation measurements: Implications to protein folding

The study of backbone and side-chain internal motions in proteins and peptides is crucial to having a better understanding of protein/peptide "structure" and to characterizing unfolded and partially folded states of proteins and peptides. To achieve this, however, requires establishing a baseline for internal motions and motional restrictions for all residues in the fully, solvent-exposed "unfolded state." GXG-based tripeptides are the simpliest peptides where residue X is fully solvent exposed in the context of an actual peptide. In this study, a series of GXG-based tripeptides has been synthesized with X being varied to include all twenty common amino acid residues. Proton-coupled and -decoupled (13)C-nmr relaxation measurements have been performed on these twenty tripeptides and various motional models (Lipari-Szabo model free approach, rotational anisotropic diffusion, rotational fluctuations within a potential well, rotational jump model) have been used to analyze relaxation data for derivation of angular variances and motional correlation times for backbone and side-chain chi(1) and chi(2) bonds and methyl group rotations. At 298 K, backbone motional correlation times range from about 50 to 85 ps, whereas side-chain motional correlation times show a much broader spread from about 18 to 80 ps. Angular variances for backbone phi,psi bond rotations range from 11 degrees to 23 degrees and those for side chains vary from 5 degrees to 24 degrees for chi(1) bond rotations and from 5 degrees to 27 degrees for chi(2) bond rotations. Even in these peptide models of the "unfolded state," side-chain angular variances can be as restricted as those for backbone and beta-branched (valine, threonine, and isoleucine) and aromatic side chains display the most restricted motions probably due to steric hinderence with backbone atoms. Comparison with motional data on residues in partially folded, beta-sheet-forming peptides indicates that side-chain motions of at least hydrophobic residues are less restricted in the partially folded state, suggesting that an increase in side-chain conformational entropy may help drive early-stage protein folding. Copyright 1999 John Wiley & Sons, Inc.     

NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.     

Single-Molecule Motions of MHC Class II Rely on Bound Peptides

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.     

The dynamical response of hen egg white lysozyme to the binding of a carbohydrate ligand

It has become clear that the binding of small and large ligands to proteins can invoke significant changes in side chain and main chain motion in the fast picosecond to nanosecond timescale. Recently, the use of a "dynamical proxy" has indicated that changes in these motions often reflect significant changes in conformational entropy. These entropic contributions are sometimes of the same order as the total entropy of binding. Thus, it is important to understand the connections amongst motion between the manifold of states accessible to the native state of proteins, the corresponding entropy, and how this impacts the overall energetics of protein function. The interaction of proteins with carbohydrate ligands is central to a range of biological functions. Here, we examine a classic carbohydrate interaction with an enzyme: the binding of wild-type hen egg white lysozyme (HEWL) to the natural, competitive inhibitor chitotriose. Using NMR relaxation experiments, backbone amide and side chain methyl axial order parameters were obtained across apo and chitotriose-bound HEWL. Upon binding, changes in the apparent amplitude of picosecond to nanosecond main chain and side chain motions are seen across the protein. Indeed, binding of chitotriose renders a large contiguous fraction of HEWL effectively completely rigid. Changes in methyl flexibility are most pronounced closest to the binding site, but average to only a small overall change in the dynamics across the protein. The corresponding change in conformational entropy is unfavorable and estimated to be a significant fraction of the total binding entropy.     

Backbone dynamics of the human CC chemokine eotaxin: fast motions, slow motions, and implications for receptor binding.

Eotaxin is a member of the chemokine family of about 40 proteins that induce cell migration. Eotaxin binds the CC chemokine receptor CCR3 that is highly expressed by eosinophils, and it is considered important in the pathology of chronic respiratory disorders such as asthma. The high resolution structure of eotaxin is known. The 74 amino acid protein has two disulfide bridges and shows a typical chemokine fold comprised of a core of three antiparallel beta-strands and an overlying alpha-helix. In this paper, we report the backbone dynamics of eotaxin determined through 15N-T1, T2, and [1H]-15N nuclear Overhauser effect heteronuclear multidimensional NMR experiments. This is the first extensive study of the dynamics of a chemokine derived from 600, 500, and 300 MHz NMR field strengths. From the T1, T2, and NOE relaxation data, parameters that describe the internal motions of eotaxin were derived using the Lipari-Szabo model free analysis. The most ordered regions of the protein correspond to the known secondary structure elements. However, surrounding the core, the regions known to be functionally important in chemokines show a range of motions on varying timescales. These include extensive subnanosecond to picosecond motions in the N-terminus, C-terminus, and the N-loop succeeding the disulfides. Analysis of rotational diffusion anisotropy of eotaxin and chemical exchange terms at multiple fields also allowed the confident identification of slow conformational exchange through the "30s" loop, disulfides, and adjacent residues. In addition, we show that these motions may be attenuated in the dimeric form of a synthetic eotaxin. The structure and dynamical basis for eotaxin receptor binding is discussed in light of the dynamics data.     

Temperature-dependent spectral density analysis applied to monitoring backbone dynamics of major urinary protein-I complexed with the pheromone 2-sec-butyl-4,5-dihydrothiazole*

Backbone dynamics of mouse major urinary protein I (MUP-I) was studied by (15)N NMR relaxation. Data were collected at multiple temperatures for a complex of MUP-I with its natural pheromonal ligand, 2- sec -4,5-dihydrothiazole, and for the free protein. The measured relaxation rates were analyzed using the reduced spectral density mapping. Graphical analysis of the spectral density values provided an unbiased qualitative picture of the internal motions. Varying temperature greatly increased the range of analyzed spectral density values and therefore improved reliability of the analysis. Quantitative parameters describing the dynamics on picosecond to nanosecond time scale were obtained using a novel method of simultaneous data fitting at multiple temperatures. Both methods showed that the backbone flexibility on the fast time scale is slightly increased upon pheromone binding, in accordance with the previously reported results. Zero-frequency spectral density values revealed conformational changes on the microsecond to millisecond time scale. Measurements at different temperatures allowed to monitor temperature dependence of the motional parameters.     

Backbone dynamics of oxidised and reduced forms of human atrial natriuretic peptide

The backbone dynamics of the 28 residue 15N-labelled human atrial natriuretic peptide have been examined by 15N NMR methods. 15N R1, R2 and [1H]-15N NOE values were determined for the oxidised and reduced forms of the peptide (ANPox and ANPrd, respectively), and analysed using reduced spectral density mapping and an extended model-free approach. The two forms possessed correlation times for overall molecular motion of 4.7 ns and were highly flexible, with substantial contributions to relaxation processes from internal motions on picosecond to nanosecond time scales. Reduction of the Cys7-Cys23 disulphide bond to form ANPrd produced a very dynamic linear peptide with a mean overall order parameter of 0.2; the intramolecular cross-link in ANPox increased this to a mean value of 0.4. A simple model for segmental backbone motion accounted for the R2 values of both species using only two variable parameters, indicating that relaxation is dominated by interactions with sites <7 residues distant in the covalent network and that changes in the conformation of the disulphide bond lead to significant chemical exchange broadening in ANPox. The contributions of backbone dynamics to configurational entropy were determined and accounted for the different receptor binding affinities of cyclised and linear natriuretic peptides.     

Photoactivation Reduces Side-Chain Dynamics of a LOV Photoreceptor

We used neutron-scattering experiments to probe the conformational dynamics of the light, oxygen, voltage (LOV) photoreceptor PpSB1-LOV from Pseudomonas putida in both the dark and light states. Global protein diffusion and internal macromolecular dynamics were measured using incoherent neutron time-of-flight and backscattering spectroscopy on the picosecond to nanosecond timescales. Global protein diffusion of PpSB1-LOV is not influenced by photoactivation. Observation-time-dependent global diffusion coefficients were found, which converge on the nanosecond timescale toward diffusion coefficients determined by dynamic light scattering. Mean-square displacements of localized internal motions and effective force constants, <k′>, describing the resilience of the proteins were determined on the respective timescales. Photoactivation significantly modifies the flexibility and the resilience of PpSB1-LOV. On the fast, picosecond timescale, small changes in the mean-square displacement and <k′> are observed, which are enhanced on the slower, nanosecond timescale. Photoactivation results in a slightly larger resilience of the photoreceptor on the fast, picosecond timescale, whereas in the nanosecond range, a significantly less resilient structure of the light-state protein is observed. For a residue-resolved interpretation of the experimental neutron-scattering data, we analyzed molecular dynamics simulations of the PpSB1-LOV X-ray structure. Based on these data, it is tempting to speculate that light-induced changes in the protein result in altered side-chain mobility mostly for residues on the protruding Jα helix and on the LOV-LOV dimer interface. Our results provide strong experimental evidence that side-chain dynamics play a crucial role in photoactivation and signaling of PpSB1-LOV via modulation of conformational entropy.     

Analysis of the internal motion of free and ligand-bound human lysozyme by use of 15N NMR relaxation measurement: a comparison with those of hen lysozyme

Human lysozyme has a structure similar to that of hen lysozyme and differs in amino acid sequence by 51 out of 129 residues with one insertion at the position between 47 and 48 in hen lysozyme. The backbone dynamics of free or (NAG)3-bound human lysozyme has been determined by measurements of 15N nuclear relaxation. The relaxation data were analyzed using the Lipari-Szabo formalism and were compared with those of hen lysozyme, which was already reported (Mine S et al.. 1999, J Mol Biol 286:1547-1565). In this paper, it was found that the backbone dynamics of free human and hen lysozymes showed very similar behavior except for some residues, indicating that the difference in amino acid sequence did not affect the behavior of entire backbone dynamics, but the folded pattern was the major determinant of the internal motion of lysozymes. On the other hand, it was also found that the number of residues in (NAG)3-bound human and hen lysozymes showed an increase or decrease in the order parameters at or near active sites on the binding of (NAG)3, indicating the increase in picosecond to nanosecond. These results suggested that the immobilization of residues upon binding (NAG)3 resulted in an entropy penalty and that this penalty was compensated by mobilizing other residues. However, compared with the internal motions between both ligand-bound human and hen lysozymes, differences in dynamic behavior between them were found at substrate binding sites, reflecting a subtle difference in the substrate-binding mode or efficiency of activity between them.     

Backbone dynamics of SDF-1alpha determined by NMR: interpretation in the presence of monomer-dimer equilibrium

SDF-1alpha is a member of the chemokine family implicated in various reactions in the immune system. The interaction of SDF-1alpha with its receptor, CXCR4, is responsible for metastasis of a variety of cancers. SDF-1alpha is also known to play a role in HIV-1 pathogenesis. The structures of SDF-1alpha determined by NMR spectroscopy have been shown to be monomeric while X-ray structures are dimeric. Biochemical data and in vivo studies suggest that dimerization is likely to be important for the function of chemokines. We report here the dynamics of SDF-1alpha determined through measurement of main chain (15)N NMR relaxation data. The data were obtained at several concentrations of SDF-1alpha and used to determine a dimerization constant of approximately 5 mM for a monomer-dimer equilibrium. The dimerization constant was subsequently used to extrapolate values for the relaxation data corresponding to monomeric SDF-1alpha. The experimental relaxation data and the extrapolated data for monomeric SDF-1alpha were analyzed using the model free approach. The model free analysis indicated that SDF-1alpha is rigid on the nano- to picosecond timescale with flexible termini. Several residues involved in the dimer interface display slow micro- to millisecond timescale motions attributable to chemical exchange such as monomer-dimer equilibrium. NMR relaxation measurements are shown to be applicable for studying oligomerization processes such as the dimerization of SDF-1alpha.     

13C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein

The widespread importance of induced fit and order-disorder transition in RNA recognition by proteins and small molecules makes it imperative that RNA motional properties are characterized quantitatively. Until now, however, very few studies have been dedicated to the systematic characterization of RNA motion and to their changes upon protein or small-molecule binding. The U1A protein-RNA complexes provide some of the best-studied examples of the role of RNA motional changes upon protein binding. Here, we report (13)C NMR relaxation studies of base and ribose dynamics for the RNA internal loop target of human U1A protein located within the 3'-untranslated region (3'-UTR) of the mRNA coding for U1A itself. We also report the semi-quantitative analysis of both fast (nano- to picosecond) and intermediate (micro- to millisecond) motions for this paradigmatic RNA system. We measure (13)C T(1), T(1rho) and heteronuclear nuclear Overhauser effects (NOEs) for sugar and base nuclei, as well as the power dependence of T(1rho) at 500 MHz and 750 MHz, and analyze these results using the model-free formalism. The results provide a much clearer picture of the type of motions experienced by this RNA in the absence of the protein than was provided by the analysis of the structure based solely on NOEs and scalar couplings. They define a model where the RNA internal loop region "breathes" on a micro- to millisecond timescale with respect to the double-helical regions. Superimposed on this slower motion, the residues at the very tip of the loop undergo faster (nano- to picosecond) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts.     

Structure and dynamics of ASC2, a pyrin domain-only protein that regulates inflammatory signaling

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-kappaB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha2-alpha3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha2-alpha3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha2-alpha3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.     

Backbone dynamics of a winged helix protein and its DNA complex at different temperatures: changes of internal motions in genesis upon binding to DNA.

The dynamic properties of a winged helix protein, Genesis, and its DNA complex at different temperatures were studied. Due to the complexity of motions, the commonly used model-free formalism could not be used to reflect the dynamic properties. The reduced spectral density function mapping approach was proven to be a useful tool to describe the overall and internal motion of molecules on the picosecond to nanosecond time-scale, and conformational exchanges on the microsecond to millisecond time-scale. The local motions in DNA-free Genesis showed strong temperature dependence and the backbone dynamics of each secondary structural element responds to the temperature change differently, while the Genesis-DNA complex showed more stability with changing the temperatures. Furthermore, each DNA contact sequence of Genesis showed distinct dynamic perturbation after Genesis binds to DNA.     

Backbone dynamics of Tet repressor alpha8intersectionalpha9 loop

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi(1) x chi(2) potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.     

Backbone dynamics of receptor binding and antigenic regions of a Pseudomonas aeruginosa pilin monomer

Pilin is the major structural protein that forms type IV pili of various pathogenic bacteria, including Pseudomonas aeruginosa. Pilin is involved in attachment of the bacterium to host cells during infection, in the initiation of immune response, and serves as a receptor for a variety of bacteriophage. We have used (15)N nuclear magnetic resonance relaxation measurements to probe the backbone dynamics of an N-terminally truncated monomeric pilin from P. aeruginosa strain K122-4. (15)N-T(1), -T(2), and [(1)H]-(15)N nuclear Overhauser enhancement measurements were carried out at three magnetic field strengths. The measurements were interpreted using the Lipari-Szabo model-free analysis, which reveals the amplitude of spatial restriction for backbone N-NH bond vectors with respect to nano- to picosecond time-scale motions. Regions of well-defined secondary structure exhibited consistently low-amplitude spatial fluctuations, while the terminal and loop regions showed larger amplitude motions in the subnano- to picosecond time-scale. Interestingly, the C-terminal disulfide loop region that contains the receptor binding domain was found to be relatively rigid on the pico- to nanosecond time-scale but exhibited motion in the micro- to millisecond time-scale. It is notable that this disulfide loop displays a conserved antigenic epitope and mediates binding to the asialo-GM(1) cell surface receptor. The present study suggests that a rigid backbone scaffold mediates attachment to the host cell receptor, and also maintains the conformation of the conserved antigenic epitope for antibody recognition. In addition, slower millisecond time-scale motions are likely to be crucial for conferring a range of specificity for these interactions. Characterization of pilin dynamics will aid in developing a detailed understanding of infection, and will facilitate the design of more efficient anti-adhesin synthetic vaccines and therapeutics against pathogenic bacteria containing type IV pili.     

Domain-elongation NMR spectroscopy yields new insights into RNA dynamics and adaptive recognition

By simplifying the interpretation of nuclear magnetic resonance spin relaxation and residual dipolar couplings data, recent developments involving the elongation of RNA helices are providing new atomic insights into the dynamical properties that allow RNA structures to change functionally and adaptively. Domain elongation, in concert with spin relaxation measurements, has allowed the detailed characterization of a hierarchical network of local and collective motional modes occurring at nanosecond timescale that mirror the structural rearrangements that take place following adaptive recognition. The combination of domain elongation with residual dipolar coupling measurements has allowed the experimental three-dimensional visualization of very large amplitude rigid-body helix motions in HIV-1 transactivation response element (TAR) that trace out a highly choreographed trajectory in which the helices twist and bend in a correlated manner. The dynamic trajectory allows unbound TAR to sample many of its ligand bound conformations, indicating that adaptive recognition occurs by “conformational selection” rather than “induced fit.” These studies suggest that intrinsic flexibility plays essential roles directing RNA conformational changes along specific pathways.     

Characterization of protein dynamics from residual dipolar couplings using the three dimensional Gaussian axial fluctuation model

Residual dipolar couplings are potentially very powerful probes of slower protein motions, providing access to dynamic events occurring on functionally important timescales up to the millisecond. One recent approach uses the three dimensional Gaussian Axial Fluctuation model (3D GAF) to determine the major directional modes and associated amplitudes of motions along the peptide chain. In this study we have used standard and accelerated molecular dynamics simulations to determine the accuracy of 3D GAF-based approaches in characterizing the nature and extent of local molecular motions. We compare modes determined directly from the trajectories with motional parameterization derived from RDCs simulated from the same trajectories. Three approaches are tested, that either suppose a known three-dimensional structure, simultaneously determine backbone structure and dynamics, or determine dynamic modes in the absence of a structural model. The results demonstrate the robustness of the 3D GAF analysis even in the presence of large-scale motions, and illustrate the remarkably quantitative nature of the extracted amplitudes. These observations suggest that the approach can be generally used for the study of functionally interesting biomolecular motions.     

Propagation of experimental uncertainties using the Lipari-Szabo model-free analysis of protein dynamics

In this paper we make use of the graphical procedure previously described [Jin, D. et al. (1997) J. Am. Chem. Soc., 119, 6923–6924] to analyze NMR relaxation data using the Lipari-Szabo model-free formalism. The graphical approach is advantageous in that it allows the direct visualization of the experimental uncertainties in the motional parameter space. Some general rules describing the relationship between the precision of the relaxation measurements and the precision of the model-free parameters and how this relationship changes with the overall tumbling time (m) are summarized. The effect of the precision in the relaxation measurements on the detection of internal motions not close to the extreme narrowing limit is analyzed. We also show that multiple timescale internal motions may be obscured by experimental uncertainty, and that the collection of relaxation data at very high field strength can improve the ability to detect such deviations from the simple Lipari-Szabo model.     

Nanosecond Motions in Proteins Impose Bounds on the Timescale Distributions of Local Dynamics

We elucidate the physics of protein dynamical transition via 10-100-ns molecular dynamics simulations at temperatures spanning 160-300 K. By tracking the energy fluctuations, we show that the protein dynamical transition is marked by a crossover from nonstationary to stationary processes that underlie the dynamics of protein motions. A two-timescale function captures the nonexponential character of backbone structural relaxations. One timescale is attributed to the collective segmental motions and the other to local relaxations. The former is well defined by a single-exponential, nanosecond decay, operative at all temperatures. The latter is described by a set of processes that display a distribution of timescales. Although their average remains on the picosecond timescale, the distribution is markedly contracted at the onset of the transition. It is shown that the collective motions impose bounds on timescales spanned by local dynamical processes. The nonstationary character below the transition implicates the presence of a collection of substates whose interactions are restricted. At these temperatures, a wide distribution of local-motion timescales, extending beyond that of nanoseconds, is observed. At physiological temperatures, local motions are confined to timescales faster than nanoseconds. This relatively narrow window makes possible the appearance of multiple channels for the backbone dynamics to operate.     

Intrinsic flexibility of snRNA hairpin loops facilitates protein binding

Stem–loop II of U1 snRNA and Stem–loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops.